crispr technology for breast cancer: diagnostics...

Review

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CRISPR Technology for Breast Cancer: Diagnostics, Modeling, and Therapy

Rachel L. Mintz, Madeleine A. Gao, Kahmun Lo, Yeh-Hsing Lao, Mingqiang Li, and Kam W. Leong*

DOI: 10.1002/adbi.201800132

1. Introduction

1.1. Difficulties of Breast Cancer Prognosis

Breast cancer is the most frequently diag-nosed malignancy in women and the second leading cause of cancer-related death in affected females worldwide because of its tendency to metastasize to the brain, lungs, and liver.[1] Breast cancer represents a highly heterogenous family of malignancies with regard to molecular characteristics, treatment response, and clinical outcomes. Even after characteri-zation, breast cancer is a highly dynamic and complex disease. For the proper treatment of breast cancer, there requires a flexible and holistic approach for the diagnosis, modeling, and therapy. From a therapeutic point of review, breast can-cers are usually classified based on three parameters: 1) expression of the estrogen receptor (ER), which predicts response to hormone-based therapies and cyclin-dependent kinase inhibitors; 2) amplifi-cation of the human epidermal growth factor receptor-2 (HER2), which predicts response to anti-HER2 monoclonal anti-bodies; and 3) mutations in the BRCA1/BRCA2 genes, which predict response to

platinum compounds and poly adenosine diphosphate-ribose polymerase (PARP) inhibitors. The biological complexity of breast malignancies, however, extends beyond these operational definitions. Gene-expression studies, for example, have identi-fied at least four biological subtypes of breast cancer, character-ized by different transcriptional signatures: Luminal A, Luminal B, HER2-amplified, and Basal-like (Figure 1). According to this classification, tumors characterized by ER expression (ER+) are further subgrouped in two distinct categories, based on the dif-ferential expression of the progesterone receptor (PR): Luminal A (ER+/PR+) and Luminal B (ER+/PRlow). The Luminal B sub-type can include either the HER2-amplified or HER2− character-ization, thereby further complicating the proper identification and determining follow-up treatments.[2] The complexities and inhomogeneity, even within subtypes, demand an improve-ment on personalizing breast cancer identification and care. Tumors lacking both ER expression and HER2 amplification are often referred to as triple negative breast cancer (TNBC).

Molecularly, breast cancer represents a highly heterogenous family of neoplastic disorders, with substantial interpatient variations regarding genetic mutations, cell composition, transcriptional profiles, and treat-ment response. Consequently, there is an increasing demand for alter-native diagnostic approaches aimed at the molecular annotation of the disease on a patient-by-patient basis and the design of more personalized treatments. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) technology enables the development of such novel approaches. For instance, in diagnostics, the use of the RNA-specific C2c2 system allows ultrasensitive nucleic acid detection and could be used to characterize the mutational repertoire and transcriptional breast cancer signatures. In disease modeling, CRISPR/Cas9 technology can be applied to selectively engineer oncogenes and tumor-suppressor genes involved in disease pathogenesis. In treatment, CRISPR/Cas9 can be used to develop gene-therapy, while its catalytically-dead variant (dCas9) can be applied to reprogram the epigenetic landscape of malignant cells. As immunotherapy becomes increasingly prominent in cancer treatment, CRISPR/Cas9 can engineer the immune cells to redirect them against cancer cells and potentiate antitumor immune responses. In this review, CRISPR strategies for the advancement of breast cancer diagnostics, modeling, and treatment are highlighted, culminating in a perspective on developing a precision medicine-based approach against breast cancer.

R. L. Mintz, M. A. Gao, K. Lo, Y.-H. Lao, Prof. M. Li, Prof. K. W. LeongDepartment of Biomedical EngineeringColumbia UniversityNew York, NY 10027, USAE-mail: [email protected]. K. W. LeongDepartment of Systems BiologyColumbia University Medical CenterNew York, NY 10032, USAProf. M. LiGuangdong Provincial Key Laboratory of Liver DiseaseThe Third Affiliated Hospital of Sun Yat-Sen UniversityGuangzhou, Guangdong 510630, China

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adbi.201800132.

Targeted Therapeutics

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TNBCs usually include the majority of tumors with a basal-like gene-expression signature, but not all TNBCs express basal-like cell markers (Figure 1).[4] The TNBC subtype accounts for up to 30% of all breast cancers and is associated with a worse prognosis than other subtypes.[3,4] Overall, TNBCs are diffi-cult to treat because of i) the lack of targets for therapy; ii) the aggressive nature of TNBC’s and their insensitivity to hormone-based targeted therapies; and iii) the variable responses of TNBCs to chemotherapy.[4] Given the importance and complexi-ties of breast cancer characterization, disease modeling, and treatment, the clustered regularly interspaced short palindromic repeats (CRISPR) technology can be introduced as a useful tool in breast cancer management (Figure 2). CRISPR technologies offer unique and varied opportunities to tackle the complexities of breast cancer and further develop the goal of personalized precision medicine.

1.2. Overview of CRISPR Technology

Figure 3 illustrates a brief timeline of CRISPR/CRISPR-associ-ated (Cas) history.[5–7] The discovery of CRISPR repeats within

the bacterial genome in 1987 by Ishino et al. marked a revolu-tionary change in the field of genome editing.[8] However, many groups only began further research regarding CRISPR/Cas mechanisms in the early 2000s, after the identification of Cas genes, unique to prokaryotes with CRISPR loci.[9]

First proposed by Markarova et al. in 2006 and later con-firmed by Garneau et al. in 2010, the CRISPR/Cas systems originate from bacteria and Archaea as a defense mechanism within their adaptive immune system to protect themselves from foreign invaders, such as viruses and plasmids.[10–12] These foreign invaders are identified when a guide RNA (gRNA) made by the bacteria binds to its complementary for-eign DNA or RNA sequence. This adaptive immune response occurs in stages: adaptation, expression (crRNA maturation), and interference.[13] During the adaptation phase, the bacteria use Cas1 and Cas2 proteins, which are shared between all known CRISPR/Cas systems, to incorporate fragments of the foreign DNA called protospacers into the CRISPR array.[14] These protospacers exist between regions of direct repeats. Barrangou et al. determined that the protospacers along with the Cas genes allow for selective phage resistance.[15] In the expression phase, the bacteria transcribe the CRISPR array,

and after further processing, create CRISPR RNA (crRNA). The final interference phase involves the use of crRNA as guides for the Cas proteins to bind to the complementary foreign sequence site for cleavage.[16] In 2012 and 2013, Jinek et al. and Cong et al., respec-tively, adapted the CRISPR/Cas9 system for targeted gene editing by using predesigned crRNA sequences.[17,18] By customizing the CRISPR array, the CRISPR/Cas system can easily be manipulated for cleavage of artifi-cially selected genomic sites.

CRISPR/Cas systems’ gene editing capa-bility derives from their ability to cause breaks within double-stranded DNA and sub-sequent nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mam-malian cells. In a majority of cases, Cas9 tar-geted gene cutting is repaired by the NHEJ

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Figure 2. Overview of CRISPR technology applications for breast cancer.

Figure 1. Breast cancer subtype classification based on immunohistochemistry marker profiles.



mechanism. Without a repair template, cells rely on NHEJ to re-ligate double-stranded DNA breaks, resulting in either inser-tion or deletion mutations. These random insertions or dele-tions can result in frameshift mutations or even premature stop codons, thereby rendering the target gene ineffective. HDR occurs less frequently than NHEJ and only in actively dividing cells but can be used to generate more precise modifications at the target sequence for gene correction.[19]

In addition to their use for direct genome editing, CRISPR/Cas systems are also emerging as powerful tools for nucleic-acid detection techniques, specifically in the targeting of cancer mutations. In 2015, Shmakov et al. characterized two new Class 2 CRISPR systems: Cas12a (Cpf1) and Cas13a (C2c2).[20] In 2017, Gootenberg et al. demonstrated the use of CRISPR/C2c2 system for nucleic acid detection.[21] East-Seletsky et al., Abudayyeh et al., and Lee et al. have further continued the trend toward the use of CRISPR/Cas variations for detec-tion and diagnostic purposes.[22–24] Studies also show that the RNase and DNase capabilities of the CRISPR/Cas systems can be used to detect and diagnose tumor-specific biomarkers such as microRNA (miRNA) and circulating tumor DNA (ctDNA), which is the tumor-derived component of circu-lating cell-free DNA (cfDNA). Many recent advances in cancer therapeutics and detection demonstrate the wide applications

of CRISPR/Cas enzymes throughout the entire disease treat-ment and progression. CRISPR/Cas systems not only have the ability for precision gene editing, but also can be altered for enhanced accuracy and specificity with an off-switch to reduce potential off-target effects, particularly with the recent addition of anti-CRISPR systems to bind and inactivate CRISPR/Cas machinery. CRISPR/Cas’s unique mechanisms and advance-ments can be widely applied to aid in breast cancer diagnostics, modeling, and treatment in a manner more efficient and safer than current methods.[25]

In this review, we will focus on a selected subgroup of CRISPR/Cas systems of potential interest for breast cancer research applications including: Cas9, Cpf1, Cas13a (C2c2), and Cas13b. Pertinent genes can be knocked in and out to create more physiologically relevant breast cancer models for different subtypes of the disease. For breast cancer treatment, oncogenes and chemotherapeutic resistance genes can be disrupted, while mutated tumor suppressor genes can be repaired with CRISPR. These advances can help tackle the current difficulties with breast and potentially other types of cancers as well, thereby furthering the current era of precision medicine.

1.3. Mechanisms of CRISPR/Cas9, Cpf1, and C2c2 Systems

The CRISPR/Cas systems are categorized by class and type. Previous classifications separated the three main types of CRISPR/Cas systems by their particular signature protein: Type I uses Cas3 proteins, Type II uses Cas9, and Type III uses Cas10.[16] A subsequent classification of the CRISPR/Cas system into Class I and Class II by Makarova et al. was based on the different genes that encode separate and distinct CRISPR complexes.[16] The difference between these two classes is the following: Class I systems have multi-subunit crRNA-effector complexes, and Class II systems function solely with a single protein. Furthermore, Markarova et al. built upon the previous type classification, adding type IV and type V. The current clas-sification of CRISPR/Cas involves both Class, either I or II, and the Type ranging from I to VI.[16] For this review, we will focus on Cas9, Cpf1, C2c2, and Cas13b, which represent Type II, V, VI, and VI, respectively (Table 1) because of their potential uses in early cancer detection, diagnosis of molecular classification, modeling, and treatment.

Cas9 is an RNA-guided nuclease that induces a blunt-end double stranded break at a specified target genomic locus.[19] As a Class 2 Type II system, its mechanism is based on a single

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Figure 3. Selected milestones in the discovery and technological development of CRISPR/Cas gene-editing technologies, including recent discoveries with potential application in breast cancer diagnos-tics. For an in-depth discussion of the historical evolution of CRISPR/Cas9 technologies, please refer to several thematic reviews from the groups that have directly contributed to CRISPR discovery and development.[5–11,15,17,18,20,21,24,26,43]

Table 1. CRISPR classification of Cas9, C2c2, and Cpf1.

Class I Class II

Type I Cas6, Cas7, Cas5, Cas8, Cas3, Cas1,

Cas2, Cas4

Type II Cas9, Cas1, Cas2, Cas4

Type III Cas6, Cas7, Cas5, Cas10, Cas1, Cas2

Type IV Cas7, Cas5, Csf1

Type V Cpf1, Cas1, Cas2, Cas4

Type VI C2c2



Cas9 protein and a corresponding gRNA, which is composed of a crRNA and a necessary supplemental trans-activating crRNA (tracrRNA). The variations in protospacer adjacent motif (PAM) sequence of the Cas9 orthologs are shown in Table 2. The com-plementary tracrRNA aids in the processing of the crRNA array into a partial direct repeat and 20-nt guide sequence, which serves to direct Cas9 to the complementary 20-nt target site. Additionally, Streptococcus pyogenes (Sp) Cas9 requires a 5′-NGG PAM site immediately after the target DNA site, 3′ of the 30-nt target sequence. Each Cas9 ortholog has a unique PAM sequence which is an essential targeting component that the CRISPR/Cas systems recognize.[16,19] The functionality of the Cas9, which originates from the bacteria and Archaea defense mechanisms, has been translated into mammalian cells by hetero logous expression of a human codon-optimized Cas9 and the use of a gRNA. The Cas9 system can be easily custom-ized to target any DNA site by changing the gRNA’s 20-nt guide sequence as long as the target DNA immediately precedes a 5′-NGG PAM sequence.[19]

Cpf1 is a Class 2 Type V CRISPR effector and a crRNA-guided endonuclease that functions without a tracrRNA and uses a T-rich protospacer adjacent motif for cleavage.[14] The catalytic properties of Cpf1 are attributed to its RuvC-like domain as an active nuclease. The inhibition of this domain by mutation results in complete removal of catalytic activity. This suggests that the Cpf1 cleavage domain cuts both strands of target DNA.[14] Cpf1 orthologs with known efficient DNase activities are Lachnospiraceae bacterium (Lb), Acidaminococus sp. (As) and Francisella novicida (Fn). Table 2 juxtaposes Cpf1 in comparison to Cas9 and C2c2 preferential targeting sequences as well as common variants. Cpf1 targets and cleaves DNA, causing double-stranded breaks with sticky-ends. The main dis-tinctions between Cpf1 and Cas9 are threefold: crRNA matura-tion without the requirement for tracrRNA, cleavage of target DNA proceeded by a short T-rich PAM, and the induction of a staggered double-stranded DNA break with a 4-nt or 5-nt over-hang on the 5′ end. Zetsche et al. confirmed that the cleavage mechanisms of Cpf1 are only dependent on the Cpf1 protein

and crRNA derived from the CRISPR array, without the need for the tracrRNA.[14] They validated that the Cpf1 and crRNA complex efficiently cleaved the target plasmid. Additionally, they determined that FnCpf1 requires a 5′-TTN duplex form PAM site for effective DNA cleavage (Table 2). While the PAM site is amenable to certain nucleotide changes, Zetsche et al., found a significant decrease in PAM recognition if the middle thymine nucleotide was altered.[14] However, changes can be applied to the initial thymine without completely halting PAM recognition.

Cas13a (formerly known as C2c2) is another CRISPR/Cas system (Class 2 Type VI) discovered in 2016, similar to Cas9 and Cpf1.[22] Unique to C2c2 are its two higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains. Addi-tionally, C2c2 lacks the DNA nuclease domain found in C2c2, suggesting that it is functionally similar to monomeric ribo-nucleases and can potentially use gRNA for RNA targeting. Abudayyeh et al. isolated the Leptotrichia shahii (Lsh) Type IV locus containing Cas1, Cas2, C2c2, as well as the CRISPR array, and constructed a low-copy LshC2c2 plasmid for later use in E. coli.[22] They studied the functionality of C2c2 as a HEPN endor-ibonuclease. C2c2 functions by targeting specific 28-nt sites complementary to the mature crRNA, which contains a 28-nt spacer and a 28-nt direct repeat (DR) sequence (Table 2).[22] These ssRNA target sites are composed of a protospacer with a preceding non-G PFS, which is similar to the PAM sequence. Interestingly, despite the specification of the ssRNA target sites, Cas13a cleaves the target RNA at variable distances from the crRNA binding site, which is dependent on the PFS. The DR sequence of the crRNA contains a single stem loop, creating a secondary structure that is essential for the LshC2c2 ssRNA cleavage. Direct repeats shorter than 24-nt have been shown to greatly hinder the cleavage potential of C2c2 due to the importance of the crRNA stem loop in complex formation and cleavage activity. The C2c2–crRNA complex selectively binds and cleaves only exposed ssRNA with a preference for uracil protospacer flanking sites. This catalytic activity has been attrib-uted to the two cooperative HEPN domains, which contain con-served arginine and histidine residues, similar to those of the HEPN endoribonucleases.[22]

Compared with Cas9 and Cpf1, C2c2 has the unique ability to remain active after cleavage of the target site.[22] Once C2c2 binds to its target ssRNA, it cleaves the target RNA and remains active, collaterally cleaving other nonspecific ssRNA sequences. However, in the absence of the target RNA, C2c2 will not be activated to cleave nonspecific ssRNA. Therefore, the catalytic nature of C2c2 can be regulated by the addition of target RNA. This two-step mechanism can be attributed to the two HEPN domains that separately recognize RNA and induce cleavage. In bacteria, the collateral cleavage of unspecified ssRNA results in cytotoxicity and inhibition of cellular growth, inducing a dor-mant phase, during which the bacteria can slow infection and strengthen its adaptive immunity.[22,23] In mammalian cells, C2c2 has been successfully used for RNA interference without any unanticipated collateral effects evidenced in bacterial systems.[26]

The mechanisms of Cas9, Cpf1, and C2c2 indicate the poten-tial of these CRISPR/Cas systems in gene target recognition for cancer detection. Indeed, several studies demonstrate the

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Table 2. CRISPR/Cas variants with respective PAM, also known as protospacer-flanking site (PFS), sequences and preferred length of their nucleotide guide sequences.

CRISPR/Cas

Common Variants PAM (PFS) Preferred length of nucleotide guide

sequence

Cas9 Streptococcus pyogenes (Sp) 5′-NGG 20-nt

Staphylococcus aureus (Sa) 5′-NNGRRT or

NNGRR

21-nt to 23-nt

C2c2 Leptotrichia buccalis (Lbu)

Listeria seeligeri (Lse)

Leptotrichia shashii (Lsh) 5′-C, U, or A 28-nt

Leptitrichia wadei (Lwa) 28-nt

Cpf1 Francisella novicida U112 (Fn) 5′-TTN

Lachnospiraceae bacterium

ND2006 (Lb)5′-TTTN

Acidaminococus sp. BV3L6 (As) 5′-TTTN



functional possibility of altering these systems for nucleic acid detection rather than just purely for gene editing.[21,24,26]

1.4. Overview of This Review

The unique properties of CRISPR variants may be exploited at all stages of breast cancer pathogenesis to potentially foster improved patient outcomes. This review will specifically ana-lyze the applications of CRISPR technology at three stages of breast cancer intervention: detection/diagnosis, modeling, and therapy. The overall potential for applying precision medicine to breast cancer with CRISPR will be discussed along with experi-mental findings and concluding remarks on future research directions.

2. CRISPR Technology for Breast Cancer Diagnostics and Detection

Current methods for breast cancer detection involve mam-mography screening, magnetic resonance imaging (MRI), or ultrasound.[27–29] Mammography is the accepted gold-standard for breast cancer screening. Despite its widespread application, false-positives in mammography remain high, especially for those at higher genetic risk for breast cancer or women with heterogeneously dense breasts.[29] Additionally, mammograms can only be used for local diagnosis, but not for the detection of metastatic breast cancer.[30] Ultrasound is another method used in breast cancer detection. It uses high-frequency transducers with sonographic technology to visualize changes in breast tissue. However, it cannot reliably identify microcalcifications without supplemental information from other imaging modali-ties, such as mammograms.[27] Therefore, on its own, ultra-sound screening is not an accurate method for breast cancer detection. Alternatively, MRI for breast cancer detection has a high sensitivity of over 94%.[28] However, it is not generally used for breast cancer detection given its limited availability, high cost, and the later difficulties in locating and biopsying tis-sues that are uniquely distinguished by the MRI.[28] Therefore, there is a need for a more reliable method to detect and diag-nose breast cancer. A new diagnostic tool for breast cancer can potentially be found within the realm of CRISPR/Cas.

2.1. Nucleic Acid-Based Biomarkers for Breast Cancer

Due to a lack of diagnostic precision in current screening models, several groups have begun to examine circulating microRNAs (miRNAs) and ctDNA as possible biomarkers for the noninvasive monitoring of breast cancer progression.[31–33] Both miRNAs and ctDNA have important advantages when compared with other classes of biomarkers. For instance, other biomarkers such as circulating protein antigens have low sensi-tivity and cannot be amplified to provide accurate diagnosis.[34] Alternatively, miRNA and ctDNA can both be easily obtained in blood samples from patients for further amplification. In addi-tion, miRNAs are stable in plasma and serum and thus do not require any special blood processing conditions.[34] The unique

stability of miRNAs in liquid biospecimens represents an attrac-tive feature for diagnostic purposes. The miRNAs comprise a large family of noncoding RNAs ≈22-nt long with negative regu-latory functions.[30] They serve as gene regulators for the degra-dation or inhibition of their target mRNAs and can function as tumor suppressors or oncogenes; these miRNAs are commonly referred to as “oncomiRs.” The expression profiling of miRNA already appears to serve as a more precise way of classifying cancer subtypes compared with other methods such as pro-tein-coding (i.e., mRNA) gene profiles.[35] Specifically in breast cancer, miRNAs, such as miR-21and miR-105, often found in early onset and metastatic cancers, appear to play a role in cell proliferation, metastasis, as well as disease progression.[30] Each progressive stage of breast cancer, from initial tumor mutation, proliferation, invasion to metastasis, has its unique combination of miRNA expression levels. The miRNA expres-sion levels are closely linked with cancer progression because of their roles in regulating either tumor suppression or other oncogenic mechanisms and can be detected in blood serum or plasma.[34] Additionally, research shows that different circulating miRNAs such as miR-17 and miR-155 can correspond to spe-cific breast cancer subtypes and metastatic potential.[30] Ma et al. identified miR-10b as the first miRNA to be highly expressed particularly in metastatic breast cancer.[36] Other studies on miRNA expression levels further suggest miRNAs as regulators of the metastatic process. For instance, studies have shown that miRNA-105, miRNA-21, and miRNA-373 are upregulated while miRNA-441, miRNA-452, and miRNA-17 are down regulated in metastatic breast tumors. Other miRNAs such as miRNA-210 and miRNA-328 are thought to be predictors of tumor recur-rence risks.[30] Given the well-identified and categorical nature of certain miRNA expression profiles for breast cancer metastasis, miRNA biomarkers may be used for further identification and diagnosis of breast cancers. However, the initial research dem-onstrating the associations between specific miRNA expression levels and breast cancer stages needs to be validated, because of the lack of a clear-cut interdependence between the two, specifi-cally with regard to different molecular breast cancer subtypes.

In addition to miRNAs, research groups have also focused on ctDNA as a biomarker for breast cancer. The ctDNA is a class of cfDNA that is released from tumors into the blood-stream. Identification and quantification of ctDNA has been used to detect and monitor breast cancer progression and pre-dict drug resistance. Furthermore, Guttery et al. determined that patients with advanced stages of breast cancer may acquire cfDNA mutations in estrogen receptor 1 (ESR1) and phosphati-dylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) genes, both of which are associated with disease progression.[37] These mutations can then be detected using CRISPR. Approaches using miRNA and ctDNA to detect breast cancer have several advantages, and therefore the use of such nucleic acid-based biomarkers hold potential for applications in breast cancer diagnostic devices.

2.2. Current Nucleic Acid-Based Diagnostic Methods

Because of the low abundance of miRNA and cfDNA, the cur-rent development of nucleic-based diagnostic methods requires

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signal amplification for accurate detection. After the detection of early-stage breast cancer, a sentinel lymph node (SLN) biopsy is often obtained to determine the risk of breast cancer metas-tasis to regional lymph nodes.[38] One-step nucleic acid amplifi-cation (OSNA) assay is used to analyze the SLN biopsy sample. The assay procedure starts by solubilizing the biopsy sample and then amplifying the target CK19 RNA via reverse transcrip-tion loop-mediated isothermal amplification. The assay then determines the level of CK19 RNA present and gives one of three results: (−) negative if <2.5 × 102 copies µL−1, (+) positive if ≥2.5 × 102 and <5.0 × 103 copies µL−1, and (++) positive if ≥5.0 × 103 copies µL−1.[38] The OSNA assay is more useful than traditional pathological examinations in clinical settings, as it allows for intraoperative examination of the SLN.[38] However, this assay still relies on a lymph node biopsy and only results in therapeutic value for positive SLNs. The total removal of the SLN for staining and OSNA carries the risk of lymphedema due to a compromised lymphatic system. Although RNA detection using OSNA requires only a partial removal,[39] there remains a need for better ways of quantifying CK19 RNA levels without the risks that come with the removal of lymph nodes.

Duan et al. developed a strategy that uses hairpin-medi-ated quadratic enzymatic amplification (HQEA) combined with molecular beacon (MB) probes to quantify miRNA.[40] The HQEA reaction involves several components: a primer, Bst DNA polymerase, Nb.BbvCI nicking endonuclease, and Lambda exonuclease. Hybridization of the target miRNA with a complementary sequence in the loop region of the MB probe initiates detection and thereby triggers a conformational change of the MB stem and results in the emission of a fluorescent

signal from the open MB stem. The primer then anneals with the open stem, displacing the miRNA when the Bst polymerase starts the synthesis of a DNA duplex according to the MB probe. Thus, the miRNA is free to activate another MB probe and initiate a new cycle (Figure 4A). Additionally, the newly synthesized duplex beacon contains a recognition site for the nicking endonuclease, which cleaves the 5′ end of the beacon and reveals a recognition site of lambda exonuclease. The lambda exonuclease dissociates the rest of the MB probe from the remaining ssDNA. Similarly, the ssDNA can then bind to another MB probe and initiate a new cycle. The recycling of the ssDNA and the miRNA allows for a quadratic enhancement of the fluorescent signal with a sensitivity of 10 × 10−15 m miRNA at 37 °C.[40] Sensitivity is increased to 1 × 10−18 m miRNA when amplification is performed at 4 °C because of the sup-pressed activity of residual lambda exonuclease activity against unbound MB, decreasing background fluorescent signals. Duan et al. demonstrate its utility by accurately detecting the presence of miR-21 in crude extracts of breast cancer tissues. When they challenged their assay by increasing the ratio of normal samples to cancer samples from 1:1 up to 10:1, there were very little observed changes in fluorescence from the first study. This demonstrates the reliability of assay results. How-ever, their proof-of-concept testing uses isolated RNAs from breast cancer tissues, which does not resolve the need for a less invasive method.[40]

Another approach is to quantify mutated miRNA levels in blood serum. Mangolini et al. quantified several distinct circu-lating miRNAs in collected serum from breast cancer patients using digital droplet PCR (ddPCR) technology.[34] ddPCR is a

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Figure 4. CRISPR/Cas and other current for breast cancer diagnostics. A) Schematic combination diagram of HQEA with MB probes within the recycling process. B) Schematic of SHERLOCK using target cfDNA and miRNA for detection. Adapted with permission.[40] Copyright 2013, American Chemical Society. C) Summary of current advantages and limitations of CRISPR/Cas-based diagnostics.



technique that integrates end-point PCR and limiting dilution to first amplify the target nucleic acid and then give an absolute quantification of the target molecule. Based on their results, they identified an association between upregulated miR-10b-5p, the forward 5′- strand, levels with a higher tumor grading and lymph node metastases, both of which are significant fac-tors in patient prognosis.[34] However, it should be noted that sensitivity levels vary among the different ddPCR instruments currently available on the market. Additionally, the preparation steps involved in prepping the template may affect amplifi-cation steps, and consequently, quantification results from ddPCR.[41]

The detection of cfNAs, both cfDNA and cfRNA, in serum is also possible with an assay designed by Das et al. that uses probed electrochemical sensors with peptide nucleic acids (PNAs) to directly analyze cfDNA levels in collected serum sam-ples from cancer patients.[42] The electrochemical sensors are fabricated on a microchip and functionalized with PNA probes specific to the target cfDNA. Before applying patient serum sample onto the microchip, the cfDNAs in the serum are first isolated and then purified by treating with PNAs specific to wild-type DNA. The PNAs function as a clamp for nontarget DNA to eliminate any cross-reactivity with the probes on the sensor. Remaining target cfDNA in the serum sample will then hybridize with the immobilized PNA probes on the sensors and be detected via an electrochemical signal.[42] Using a universal probe mixture, they were able to screen for multiple mutated cfDNAs simultaneously. To determine the detection limits of the assay, they tested a range of RNA concentrations and dem-onstrated that the detection limit of their assay is 1 fg µL−1 of isolated RNA.[42] Furthermore, they have shown that it was suc-cessful in identifying KRAS and BRAF mutations in untreated serum samples of fourteen lung cancer, and nine breast cancer patients.[42]

2.3. CRISPR/Cas-Based Diagnostic Methods

One possible approach for detecting miRNA makes use of the targeted RNA activity of Cas13a/C2c2. It has been demon-strated that catalytically inactive LwaC2c2 maintains its ability to bind target RNA with the addition of appropriate guides, and thus, can be used as a detection tool.[26] Abudayyeh et al. quanti-fied the RNA bound to the “dead” LwaC2c2 with RNA immu-noprecipitation and qPCR.[26] They demonstrated a 2.1-fold to 11.2-fold enrichment of the targeted Gluc and ACTB transcripts.

The RNA-guided collateral cleavage is another feature of C2c2 that can be exploited to detect specific RNAs as shown by East-Seletsky et al.[43] They devised a method that harnesses this func-tion of LbuC2c2 to cleave fluorophore quencher-labeled reporter RNAs after specific cleavage of the target RNA. The trans-cleavage of the reporter RNA results in an enhanced accumula-tion of fluorescent signal upon RNA target activation. In proof of concept testing, they achieved a 104-fold increase in observed fluorescence signal with the addition of as little as 10 × 10−12 m target RNA.[23] In a following paper, East-Seletsky et al. further broadened the potential application of their protocol by using two orthogonal C2c2 homologs, LbuC2c2 and Lachnospiraceae bacterium (LbaC2c2), to simultaneously detect multiple target

RNAs. However, while both homologs were able to generate similar levels of fluorescent signal, LbaC2c2 required at least 1 × 10−9 m of target RNA because of its decreased sensitivity.[43] This may pose an issue as miRNA levels can be extremely low.[40] Thus, advancements still need to be made for this tech-nique to be applicable to multiplex RNA targeting.

Further building on this principle, Gootenberg et al. cre-ated a platform for targeted nucleic acid detection known as SHERLOCK (Specific High-Sensitivity Enzyme Reporter UnLOCKING).[21] The platform combines isothermal ampli-fication with C2c2-mediated collateral cleavage of a fluoro-phore-labeled reporter RNA to achieve detection sensitivity of attomolar concentrations (Figure 4B). Target DNA or RNA is amplified via recombinase polymerase amplification (a reverse transcriptase enzyme is added to amplify RNA). Subsequently, T7 RNA polymerase is added to transcribe the amplified DNA to RNA. The target RNA is detected using LwaC2c2, which is activated to collaterally cleave the fluorescent reporter RNA for signal detection. Gootenberg et al. then demonstrated that SHERLOCK is able to discern single nucleotide polymorphism-containing alleles at levels as low as 0.1% of nontargeted DNA.[21] They also detected two cancerous mutations, EGFR L858R and BRAF V600E, in mock cfDNA samples at levels of 0.1%. Thus, in comparison to current methods of detection that use ddPCR, SHERLOCK has a much higher sensitivity for low cfDNA levels.[21]

Yet another approach for targeting nucleic acid detection is cleaving nontarget ctDNA with CRISPR/Cas before amplifica-tion of target ctDNA using PCR. This method, called CUT-PCR (CRISPR-mediated, Ultrasensitive detection of Target DNA-PCR), uses both Cas9 and Cpf1 to cut nontarget DNA, elimi-nating background DNA signals in an unbiased manner.[24] Wild-type DNA contains the correct PAM sites, allowing Cas9 or Cpf1 to induce double-stranded breaks. The targeted ctDNA sequences have mutations in the PAM sites and will therefore not be recognized and cleaved by the CRISPR endonucleases. Thus, only the uncleaved target ctDNA can anneal with the PCR primer. Since Cas9 and Cpf1 recognize different PAM sites, CUT-PCR is applicable to detecting different oncogenic mutations. In fact, about 80% of the oncogenic mutations cur-rently in the catalogue of somatic mutations in cancer database may be detected via CUT-PCR.[24]

Lee et al. were able to detect rare oncogenic-specific muta-tions in mixtures prepared with plasmids containing wild-type or mutant KRAS.[44] After treating the mixture with the appro-priate combination of CRISPR endonuclease and the specific guide RNA, the target sequence was amplified using PCR. They further examined the sensitivity of this method. For mix-tures containing ≥0.01% of the mutated KRAS plasmids and treated with one cycle of CUT-PCR, there was at least a sixfold increase in the detected target sequence by deep sequencing compared with an internal control PCR product. This indicates that the sensitivity of CUT-PCR-based deep sequencing is at least 0.01%. They were also able to identify other oncogenic mutations in GNAQ, CTNNB1, and EGFR. Following this, Lee et al. demonstrated that their technique could detect several single substitution KRAS mutations in cfDNA extracted from blood plasma samples of eight colorectal cancer patients.[44] After performing multiple rounds of CUT-PCR, they found a

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significant increase in mutant allele frequency in at least half of the patients. For instance, results showed a 164-fold to 160-fold increase in allele frequency in five patients with the KRAS c.35G>A mutation, and the remaining three patients were found to have other KRAS mutations such as KRAS c.34G>C. However, CUT-PCR is presently hindered by the fact that there are only 12 possible oncogenic mutations that are identifiable by the two CRISPR endonucleases (NGG>NGH or NGG>NHG where H is A, C, or T, and TTN>TVN or TTN>VTN where V is A, C, or G). Thus, the discovery of new CRISPR endonuclease with different PAM sites is necessary for the identification of other somatic oncogenic mutations.[24] A holistic summary of the current advantages and limitations of CRISPR/Cas-based diagnostics can be seen in Figure 4C.

2.4. Perspectives on CRISPR/Cas Diagnostics

One of the main motivations behind developing CRISPR/Cas-based diagnostics for breast cancer is to reduce the need for invasive methods such as tissue biopsies, which are currently needed to determine whether tissue abnormalities seen in mammograms are cancerous. However, in some cases, a biopsy can miss the cancerous tissue if the needle fails to take the sample from the correct area, and repeat biopsies are expensive and may cause trauma to the patient.[37] Even if healthcare pro-fessionals obtain a correct biopsy sample, false negative results can occur because sample analysis is dependent on the patholo-gist’s interpretations.[45] There is also limited spatial coverage of a tissue biopsy, and recent research shows that cancerous tumors have a high degree of genetic heterogeneity.[46] Thus, tissue biopsies may not provide enough information to defini-tively identify what type of breast cancer a patient may have, which is essential for a physician to determine the optimal treatment. Therefore, CRISPR/Cas9 may serve as an alternative for breast cancer diagnosis that is highly sensitive and yet mini-mally invasive.

A less invasive way of obtaining the necessary nucleic acids for genotyping is through liquid biopsies as they only require blood samples, which contain miRNA and cfDNA in the serum, from the patients. Liquid biopsies can also address the need for repeat biopsies of a breast cancer patient for re-evaluation of tumor type.[47,48] Additionally, liquid biopsies can be used to monitor cancer development and therapeutic efficacy of patients at high risk for relapse.[48] Breast cancer tumors have been shown to change genotype in response to estrogen with-drawal therapy and antiestrogens.[37] These acquired muta-tions can be potentially detected and monitored by CRISPR/Cas-based diagnostic methods. Moreover, previous research has associated high mutation frequencies of PIK3CA and low mutation frequencies in TP53 and MYC with apocrine variants of TNBCs, indicating that specific genetic mutations might associate with specific histological variants of the disease.[49] This finding highlights another possible benefit of CRISPR/Cas-based diagnostics in identifying tumor type without the need for a tissue biopsy.

CRISPR/Cas-based diagnostics can be combined with the technology of microfluidic chips. This combination may make the “point-of-care” diagnostics for breast cancer possible,

streamlining the process of diagnosis with a higher sensitivity. Current PCR microchips are already capable of rapid evalua-tion of miRNA expression levels in prostate cancer patients.[50] Considerable advances have also been made in making micro-fluidic-based capillary array electrophoresis (µCAE) chips for parallel sequencing of oncogenes. For instance, a spatial tem-perature gradient µCAE chip platform was introduced for the detection of KRAS mutations in tissue biopsy samples from colorectal cancer patients.[51] The sensitivity of these microfluidic platforms may be further enhanced by the inte-gration of SHERLOCK and CUT-PCR to amplify signal detec-tion of target RNA and DNA. The combination of CRISPR/Cas systems with microfluidic chips may give rise to powerful lab-on-a-chip systems that will allow for more rapid and accurate diagnosis of breast cancer.

Nevertheless, there are several limitations presently con-straining the clinical usage of CRISPR/Cas-based diagnostics. At present, ctDNA analysis is only used to select patients with nonsmall cell lung cancer patients for EGFR-directed treat-ment and in the event of a negative result, a tissue biopsy is still needed for confirmation of results.[46] Currently, clinical trials for detection of ctDNA screening from blood tests are underway to determine disease presence in patients undergoing treatment for TNBC (NCT03145961) as well as the evaluation of ctDNA detec-tion as an indicator for early breast cancer diagnosis, therapy effi-ciency, and postoperative observation (NCT02797652). Moreover, in order to expand its application to breast cancer diagnostics, ctDNA-based screening methods such as the ones outlined in this review need to confirm that variants detected in cfDNA are indeed cancerous mutations by evaluating the specificity of such assays in a large healthy population as representative of a nega-tive control. Additionally, a longitudinal study of these healthy subjects is required to distinguish between false positive and true positive results.[52] There are also issues with the standardi-zation and reproducibility of results from miRNA assays. Dis-crepancies between data on circulating miRNA among several studies are due to differences in experiment setups have been reported. There is supplemental evidence that miRNA levels can be affected by sample collection methods.[34] Standardization of data processing and normalization analyses is lacking, resulting in batch effects.[53]

Although more work needs to be done before CRISPR/Cas-based diagnostics can be fully realized in clinical settings, these technologies promise a faster and more accurate diagnosis of breast cancer. They can offer great advantages over present diagnostics due to the possibility of detecting breast cancer at earlier stages, thereby improving patient prognosis.[46] They may also provide physicians with a more comprehensive pic-ture of the type of breast cancer a patient may have, enabling them to plan a more effective treatment. Furthermore, if combined with present technologies like microfluidic chips, CRISPR/Cas-based diagnostics can be a powerful tool to pro-vide point-of-care testing.

3. CRISPR Technology for Breast Cancer Modeling

After the characterization and diagnostics of individual breast cancer subtypes, the ability to model these characteristics

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and unique mechanisms allows for further understanding and potentially more rational selection of treatment methods. Engineering physiologically relevant cell and animal models is imperative to gain a realistic understanding of the mecha-nisms underlying each breast cancer subtype. Breast cancer models have been expanded to include cell lines established from human tumors, xenografts derived from cell lines or explants, and transgenic genetically engineered mice.[54] The technical and clinical advantages and disadvantages of xenografts and genetically engineered mouse models as well as combinational breast cancer models have recently been reviewed by Holen et al.[55] Studies are also exploring the use of microfluidic devices for 3D breast tumor models to better mimic the physiological microenvironment in vitro.[56] How-ever, current breast cancer models are limited in that many do not account for the dynamic transformation of the cancer genome to include the spatiotemporal mutations and genomic rearrangements that inevitably arise as the breast cancer pro-gresses through clonal evolution.[57,58] Advances in CRISPR gene editing can not only be used to characterize and diagnose breast cancer, but also precisely mimic breast cancer progres-sion at discrete intervals, thus enabling the development of more accurate preclinical models for breast cancer research.

3.1. Genetic Rearrangement Model Generation with Cas9

To faithfully model breast cancer, genetic rearrangements can be included. Genetic rearrangements are mutations that arise in the genome due to DNA breaks followed by error-prone repair. These rearrangements include but are not limited to translocations, insertions, and deletions. Rearrangements are known to create driver oncogenes and also disable certain tumor suppressors, culminating in cancer.[59] Previously, the laborious generation of mouse models to study chromosomal rearrangements resulted in the development of nonphysi-ological levels of oncogene gene fusions.[60] With the advent of CRISPR/Cas technology, more physiologically relevant breast cancer models that measure disease pathogenesis based upon genetic rearrangements can be efficiently generated.[61] Given these advantages, CRISPR may be a fruitful avenue to pursue for breast cancer modeling if specific breast cancer rearrange-ments can be pinpointed for modification.

Genomic instability, the increased rate of genomic altera-tion, arises in early carcinogenesis. Although the exact mecha-nisms leading to genomic instability are largely unknown, it is hypothesized that telomere dysfunction may play a role in the context of breast cancer genomic instability.[62] Due to the lim-ited standard cytogenetic analyses of cultured cell lines coupled with the heterogeneous nature of breast cancer, signature rear-rangements were not originally pinpointed in breast cancer.[59] Now, advanced next-generation sequencing methods used in conjunction with comparative genomic hybridization tech-niques have resulted in a better understanding of the genetic rearrangements occurring in breast cancer. In applying these technologies, three separate patterns of copy-number altera-tions became evident depending upon the breast cancer sub-type, providing further evidence that breast cancer cannot be treated as one disease. The three patterns were as follows:

simple, amplifier, and segmented. The simple pattern is associ-ated with ER+ breast cancer and is characterized by a gain of 1q/16p and a loss of 16q. The amplifier pattern is so called due to the high degree of clustered chromosomal DNA amplifica-tion typically of 8p12 (FGFR1), 8q24 (MYC), 11q13 (CCND1), 12q15 (MDM2), 17q12 (ERBB2), and 20q13 (ZNF217) and is indicative of luminal B and ERBB2 breast tumors. The seg-mented profile is complicated by multiple low-amplitude gains and losses of short chromosomal regions that are most common in TNBC. Identifying these unique patterns of genetic rearrangement may lead to improved cancer classifications, establishing distinct genetic highly specific markers.

These genetic rearrangements may result in the forma-tion of fusion genes with oncogenic potential that may also be important to consider in breast cancer models generated with CRISPR. A more comprehensive characterization of gene fusions in different breast cancer subtypes and their corre-sponding therapeutic significance based upon clinical cohorts is reviewed elsewhere,[59] and may provide more insight into the CRISPR breast cancer genetic targets for modeling. One such clinically recurrent gene fusion in breast carcinoma is the ETV6-NTRK3 fusion. Research has demonstrated that when ETV6-NTRK3 is transduced via retrovirus to mammary epi-thelial cells, tumors readily formed, confirming the relation-ship between the gene fusion and cancer development.[63] The MAG13-AKT3 fusion has been reported in TNBC, leading to the continuous activation of AKT3 kinase, a regulator of cell survival and tumor formation.[64] This characteristic fusion should be considered in TNBC specific models. Without prior knowledge of potential genetic rearrangements, breast cancer genetic targets can still be determined through careful exami-nation of human mammary epithelial cells that escape senes-cence in culture. Subsequent next-generation sequencing methods can elucidate any distinct gene fusions or rearrange-ments present.[65] Cas9 can then be used to assess or verify the functionality of the candidate genes identified.

This approach has already been applied in multiplex to iden-tify the chromosomal translocations that contribute to lung cancer and can potentially be extended to breast cancer by altering the molecular targets. A lentivirus carrying CRISPR to delete the genes of interest and the Cre recombinase was delivered through the trachea into Cre/loxP transgenic mouse lung cancer models in order to study the gene editing effects of tumor suppressor genes such as Nkx2-1 and PTEN.[66] Ten weeks after infection, histology surveyor assays and deep sequencing of the targeted alleles revealed that the tumors of animals expressing gRNAs targeting PTEN or Nkx2-1 displayed marked histopathological differences compared with those that were not given the targeted gRNAs. Tumors with total loss of PTEN were significantly larger than the wild-type animals, perhaps indicating a selective advantage. Disruption of PTEN also increased the tumor burden or the ratio of tumor area to lung area, while disruption of Nkx2-1 only increased the tumor burden in loxP-Stop-loxP KrasG12D/+ mice. Since off-targeting is a possibility with gene-editing, the three most likely off-target loci for Cas9 gene editing gRNA PTEN and gRNA Nkx2-1 were evaluated for which there were negligible off-target effects, indi-cating that the results reported for the gRNAs were not a conse-quence of editing another target.

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Multiple different lung adenocarcinoma subtypes were modeled in this study, which is promising for the modelling of breast cancer, which is also characterized by numerous sub-types. This study’s modelling method may allow the detailed examination of subtype-specific molecular mechanisms dic-tating breast cancer development. Cas9 can likely be applied to replace current Cre/LoxP breast cancer transgenic mouse models including existing Cre/LoxP models with corresponding transgenes to elucidate the role of BRCA1/2 genes in TNBC.[67] Cas9 offers the capacity to single out precise genetic events in the pathogenesis of breast cancer for more specific, isolated modelling and in-depth discrete analysis.

Once specific genes have been determined to play a role in breast tumor progression as described above, Cas9 can be used to model chromosomal rearrangements in vivo in mice. Cas9 lentiviral vectors were engineered to induce the inver-sion of Eml4 and Alk loci that are found in non-small-cell lung cancers (Figure 5A,B).[61] CRISPR-induced two double-strand breaks (DSBs) concomitantly to trigger the rearrangement of ≈11 base pairs. The gRNAs targeting the Eml4 and Alk sites were cloned into the Cas9 plasmid and delivered to the lungs of adult mice intratracheally through an adenovirus shuttle vector. At ≈6–8 weeks post infection, tumors were detectable by microcomputed tomography. Fluorescent in situ hybridiza-tion analysis determined that the Em14–Alk inversion was pre-sent in all the tumors that received the Cas9 lentivirus. This methodology has since been confirmed in another study that explored CRISPR-induced rearrangements on tumor devel-opment in context of p53 status.[68] Mice were injected with a Cas9 lentivirus expressing EmI4 and Alk gRNAs that were heterozygous (p53+/−) or homozygous (p53−/−) for the p53 dele-tion, which was hypothesized to expedite tumor formation. Two months post injection the mice were sacrificed, revealing that 100% of p53−/− (6/6) and p53+/− mice (4/4) and none of the control mice exhibited lung tumors. Laser capture micro-dissection was used to isolate the genomic DNA from the tumors for PCR analysis to determine if the tumors contained

the Eml4–Alk rearrangement. The Eml4–Alk rearrangement was present in the DNA extracted from tumors but absent from the DNA extracted from adjacent nontumorous lung tissue, indicating the efficacy of the CRISPR/Cas9 lentivirus expressing the Eml4 and Alk gRNAs, as well as the role of the rearrangement in tumor development. These studies establish the adaptability of the CRISPR system for engineering chro-mosomal rearrangements.

3.2. In Vivo Breast Cancer Models Generated with Cas9

Targeted breast cancer mutations characteristic of each sub-type can be modeled in vivo. Annunziato et al. modeled inva-sive lobular breast carcinoma (ILC), the second most common form of breast cancer distinguished by the lack of E-cadherin, in female mice.[69] Cas9 was delivered through intraductal injec-tion by the lentiviral vector expressing Cre recombinase and/or CRISPR/Cas9 with a gRNA targeting PTEN in mice with variable E-cadherin genotypes. PTEN is a negative regulator of AKT, which when inhibited during a phase II clinical trial (NCT02162719) has been demonstrated to extend the lives of individuals with metastatic TNBC.[70] As shown in Figure 5C, there is strong expression of Cas9 in mouse mammary epithe-lial cells homozygous negative for E-Cadherin treated with Cas9 with Pol II shown as a loading control. The resultant PTEN-editing by the injection of lentiviral vectors for Cre recombi-nase and PTEN-targeting gRNA in mouse mammary epithelial cells is shown in Figure 5D, while the controls treated with PTEN gRNA alone did not induce any gene editing. As demon-strated in Figure 5E, the tumors tested all evidenced type ILC histology in that there were abundant fibroblasts and collagen deposits present. Therefore, the lentivirus induced successful ILC formation characteristic of this distinct breast cancer subtype, serving as a viable model. This modeling method is advantageous because it is conducive to spatiotemporal con-trol of breast cancer, permitting the study of any disease event

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Figure 5. Cas9 for breast cancer modelling. A) Schematic of the loci before and after inversion with the location of primers labeled A, B, C, and D. B) Fusion transcript confirmation. Reproduced with permission.[61] Copyright 2014, Nature Publishing Group. C) Expression of Cas9 following in vitro transduction visualized post-immunoblotting with anti-Flag and anti-Cas9 antibodies. D) Cas9 efficiency post transduction with LentiGuide-gPTEN. E) Immunohistochemical analysis of LentiGuide-gPTEN. Reproduced with permission.[69] Copyright 2016, Cold Spring Harbor Press. F) One-step method to generate multiple gRNA delivery plasmid. Reproduced under the terms and conditions of the Creative Commons Attribution license 4.0.[77] Copyright 2016, the authors, published by Oxford University Press. G) Schematic of lentivirus Cpf1 construct. Reproduced with permission.[78] Copyright 2017, Nature Publishing Group.



including initiation. Additionally, Cas9 tumor induction can be confined to a single gland. Alternatively, in transgenic animals, it is more difficult to localize the mammary cancer, which may make it difficult to thoroughly examine the tumor in a prem-etastasis model. This model can then be extended to monitor metastatic developments after the tumor induction is removed.

Using the Cas9 knockout method, other breast cancer sub-types including metastatic TNBC can be modeled to elucidate its respective cancer progression. To engineer a TNBC model, a Cas9 plasmid was engineered with a corresponding targeting gRNA to disrupt the embryonic stem cell marker Cripto-1 gene in mice.[71] The Cripto-1 gene encodes an epidermal growth factor protein that serves as a receptor for the tranforming growth factor (TGF) signaling pathway and facilitates the matu-ration of Notch receptors.[72] Notch proteins are likely involved in the maintenance of breast cancer cells, and mutated Notch proteins are often found in TNBC, indicating the oncogenic potential of the Cripto-1 gene.[73] When the Cripto-1 gene was knocked out in mouse JygMC cells that molecularly resemble TNBC, there was a 35% decrease in tumor cell growth in the homozygous knockout clones and a 45% reduction in primary tumor volume as well as a decrease in the number of metastatic nodules as compared with the parental cells. This TNBC mouse model is distinct because it uniquely displays spontaneous epi-thelial-mesenchymal plasticity, a development central to stud-ying breast cancer metastasis.[74] In another study, CRISPR was applied to derive a TNBC breast cancer murine model centered around the E3-Ubiquitin Ligase UBR5, a key cell signaling and proliferation regulator.[75] UBR5 gene amplification is common in TNBC, and patients with unusual UBR5 expression tend to have poor survival rates.[76] The in vivo deletion of UBR5 with Cas9 through Lipofectamine transfection reduced TNBC tumor growth and removed lung metastatic colonization by regulating E-cadherin, substantiating the claim that TNBC models should potentially include UBR5 overexpression.

As breast cancer is a multigenic disease, multiplex modeling in which many genes are concomitantly manipulated with Cas9 may provide an improved and comprehensive breast cancer model. Cao et al. developed stable, Cas9-inducible breast cancer cell line models (MCF7, SKBR3, MCF10A, BT474, and NT2) using a lentiviral vector.[77] Then, three gRNAs each targeting different breast oncogene demethylases, including KDM5A, KDM5B, and KDM5C, were introduced. The targeted protein expression was decreased by 10–15% depending upon the cell line. To increase multiplex efficiency and provide a more thorough demethylase-depleted cell model, all three gRNAs were cloned into a single Lentiviral construct (Figure 5F) and introduced in the induced Cas9 cell lines. Cells exposed to all three gRNAs showed simultaneous depletion of the targeted oncogenes, indicating the potential for efficient Cas9 multi-plex modeling in breast cancer. A more streamlined approach to multiplex editing has been made possible with the CRISPR variant Cpf1 (Figure 5G), as Cpf1 only requires one RNA Polymerase III promoter to express multiple small crRNAs.[78] When HEK293T cells were transduced with Cpf1 targeting three genes that regulate breast cancer including DNA meth-yltransferases (DNMT), VEGFA, or GRIN2b, the resultant average efficiency determined for each targeted gene was greater than 40%, indicating that Cpf1 may be a more efficient

method for multiplex targeting in breast cancer to create models that represent multiple facets of the disease.[79–81]

3.3. Breast Cancer Drug Screening, Resistance, and Discovery with CRISPR

The aforementioned Cas9 models can serve as strong plat-forms for breast cancer drug screening and discovery. Cur-rent screening methods are limited due to the difficulty in activating or deactivating both copies of a gene in diploid mammalian cells.[82] While targeting mRNA through RNA interference (RNAi) could be used to solve this issue, RNAi can only partially modulate the target gene expression, leading to false negative and false positive screening results. False posi-tives are known to arise from reagent-specific off-target effects, and false negatives result from experimental noise inherent to large-scale studies.[83] For example, in a meta-analysis of genome-wide Drosophila RNAi screens, the rate of false nega-tives and false positives was 10% and 1%, respectively.[84]

Recently, the use of CRISPR for large-scale and efficient screening has been explored as an alternative solution to RNAi. One study directly compared screening with CRISPR to RNAi, and revealed that CRISPR screening identified 2–5 times more essential genes than RNAi screens, which was attributed to more complete CRISPR activation.[85] In these systems, cancer cells can be edited with CRISPR to induce one or more specific gene mutations, which can be monitored to establish the sen-sitively, scope, and resistance to potential cancer therapeutics for preclinical validation. Genome-wide Cas9 knockout screen-ings of oncogenes have culminated in the identification of genes required for cancer cell proliferation.[86,87] Positive Cas9 selection techniques have also led to the discovery of genes that are involved in metastasis and genes that confer drug resist-ance.[84,88] Cas9 whole-genome dropout screens have been used to distinguish between oncogenic drivers and expected survival genes in cancer cell lines with known mutations and identify genes whose Cas9 knockout results in cell death.[89] Compre-hensive Cas9 regulatory region screening in which Cas9 cleaves and disrupts the function of specific DNA sequences in criti-cally active regions is also underway.[90]

Epigenetic screening for HER2 enriched breast cancer, which has the second poorest outcome of breast cancer subtypes after TNBC, was conducted to discern crucial gene regulatory ele-ments in this form of breast cancer in a method called “Cas9 based epigenome regulatory element screening (CERES).”[91] DNase I hypersensitivity sequences (DHSs), transcription factor measurements of chromatin accessibility that are likely regulatory elements, were generated for the SKBR3 HER2-amplified breast cancer cell line. The protospacer sequence within each DHS surrounding the target gene was identified and rated by possible off-target alignments, forming the gRNA library (Figure 6A). Once the library was synthesized, it was cloned into a Lentiviral vector for delivery to target cells. Then, a cancer-associated cell line, A431 epidermoid carcinoma cells, which express elevated levels of HER2, were transduced with dCas9 lentivirus, and the stable cells were then transduced with the gRNA library. Flow cytometry was employed to sort the cells based upon levels of HER2 expression levels. Several DNase

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hypersensitivity sites contained gRNAs that differed signifi-cantly from the groups displaying the lowest and highest HER2 expression (Figure 6B,C). The positive correlation between the identified gRNAs and HER2 expression was confirmed through real-time polymerase chain reaction in the A431 cells treated with dCas9, using HEK293T cells as a basis for comparison (Figure 6D). CERES presents a powerful screening method that can be further applied to assess HER2 inhibitors, drug resist-ance, or cancer cell growth. Additional screening of noncoding regions may shed light on new regions for targeted breast cancer therapy. Rearrangements found in introns are common in breast cancer and may hold many characteristic genetic changes that are overlooked by standard sequencing approaches but can serve as additional genetic targets for those breast cancer subtypes such as TNBC that lack molecular targets.[59]

The use of CRISPR for TNBC drug discovery may prove to be an effective strategy to overcome the dearth of mole-cular targets. The maternal embryonic leucine zipper kinase (MELK) has been reported as a particularly effective target for the treatment of TNBC, in which MELK is overexpressed.[92] Consequently, multiple drugs that inhibit MELK activity have been synthesized as a means for therapy.[93] Although the MELK inhibitor OTS167 has been studied in several clinical trials (NCT01910545, NCT02768519, NCT02795520, and NCT02926690), Lin et al. discovered that for TNBC, the mutagenesis of MELK with Cas9 did not affect the growth of TNBC cell lines.[94] Furthermore, when CRISPR induced a null mutation at the reported drug target, there was no evident drug resistance. Altogether, these results suggest that the reported MELK activity found in vivo was likely attributed to one or more off-target effects. In other words, knocking out MELK in cells with Cas9, the purported function of MELK inhibitors, did not affect proliferation, yet the inhibitors quizzically accomplish this goal in vivo. This study highlights the importance of using Cas9 as a modality to better pinpoint precise cancer dependen-cies for targeted drug discovery prior to human clinical trials.

Drug screening and discovery are further complicated by cancer combinational therapy in which multiple drugs concomitantly target different molecular pathways. Employing current discovery methods is challenging due to the extensive cost and time of laborious testing pairwise drug combinations on different cell lines. Wong et al. created the strategic com-binatorial genetics en masse (CombiGEM)-CRISPR to conduct efficient pooled screening of pairwise genetic knockouts against genes coding for epigenetic regulators.[95] The team developed a barcoded combinatorial gRNA library that can be delivered via lentivirus and tracked through the barcode sequences. Using this innovative system, it was discovered that combinato-rial inhibition of lysine demethylase, KDM6B, and epigenetic reader that contributes to epigenetic longevity, BRD4, suc-cessfully halted breast (MDA-MB-231) and ovarian (OVCAR8) cancer cell growth. This methodology can be then extended in the future to test for successful combinations of drugs in addi-tion to epigenetic regulators in the context of individual breast cancer subtypes.

Chemotherapy can cause genetic mutations that result in the development of chemoresistance, and ultimately enable tumor progression. CRISPR technologies can be harnessed to effi-ciently screen for genes involved in regulating such acquired forms of drug resistance in addition to the initial drug dis-covery screening.[96,97] Currently, gene mutations are measured in the tumor tissue directly collected from cancer patients to select therapeutic strategies and gauge the potential for drug resistance, though there exists heterogeneity within different locations of the same tumor between cells, such as intratumor heterogeneity. CRISPR can overcome this drawback because it allows for the analysis of single-cells in parallel through pooled screening to comprehend the emerging tumor drug resistance. The previously discussed lung cancer model generated by the inversion of the echinoderm microtubule-associated protein-like (EML)–anaplastic lymphoma kinase (ALK) system con-firmed that a Cas9 model could also be used to explore drug

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Figure 6. Cas9 for breast cancer drug screening. A) Generation of gRNA library for DHSs introduced into cells expressing dCas9krab with the mCherry reporter. B) HER2 mRNA fold change in response to gRNA from the library. C) HER2 mRNA log2 fold change in response to gRNA treatment versus log2 fold change of the gRNA in the HER2 HEK293T cas9p300 screen. D) Log2 fold change of gRNA abundance in dcas9krab screen versus the cas9p300 screen in A431 cells. Reproduced with permission.[91] Copyright 2017, Nature Publishing Group.



resistance.[61] Once the tumors were formed as described ear-lier, the mice were treated with the drug Crizotinib, a dual ALK/MET proto-oncogene receptor tyrosine kinase inhibitor. Micro-computed tomography scans revealed that the mice treated with the inhibitor had either complete (6/7) or partial tumor regression (1/7), and there was no observed tumor regres-sion in the control animals.[61] As different chemotherapeutic agents are often used throughout therapy, this method can be extended to explore the development of therapeutic resist-ance to these chemotherapeutic cocktails in progressive breast cancer models.

4. CRISPR Technology for Breast Cancer Therapy

The advancements of CRISPR technologies already show potential in characterization, diagnostics, and modeling for breast cancer. For ultimate impact on precision medicine, CRISPR technologies must partner with therapeutic advances to improve breast cancer treatment. Neoadjuvant chemotherapy has become the standard course of treatment for patients with localized and metastatic breast cancer. However, it is difficult to assess the precise location and extent of the primary breast tumor prior, during, and post neoadjuvant chemotherapy. Breast cancer genomic profiling and molecular subtyping can further individualize treatment based on sensitivity to existing therapeutic agents and known patterns of metastasis. Although targeted therapeutics have been developed for ER+ and HER2+ breast cancer, TNBC remains unresponsive to similar tar-geted therapeutics. CRISPR technology offers the potential to intervene at precise genetic loci to halt cancer progression by disrupting or repressing oncogenes, enhancing the efficacy of chemotherapeutics through the disruption of genes that confer resistance, and enhancing the specificity of T-cells for emerging immunotherapy treatment modalities.

4.1. Cas9-Induced Breast Cancer Gene Editing

The most direct application of CRISPR to breast cancer is through targeted, permanent oncogene disruption via Cas9 to inhibit cancer cell proliferation.[98] Genetic screening projects, combined with advances in computational methods, have allowed for the identification of driver genes that contribute to breast cancer. Rajendran and Deng identified 195 driver genes and concluded 63 were specific candidates for breast tumori-genesis including such oncogenes as ATK1, GATA3s, PIK3CA, and MAP3K1.[99] Cas9 can target and directly disrupt the func-tion of these oncogenes as well as any downstream pathways that may contribute to tumor growth. Included among these driver genes are also tumor suppressor genes that are likely subject to mutation such as the BRCA1/2 genes.[100] However, in the case of mutated tumor suppressor genes, Cas9 disrup-tion would not be an effective strategy to prevent tumor cell proliferation. Instead, Cas9 gene editing to correct the mutation and restore physiological tumor suppressor function would be the best course of action to suppress tumor growth.

Several studies have explored the therapeutic efficacy when Cas9 knockouts various genes that serve as potential

therapeutic loci in breast cancer. In a proof-of-concept study, Cas9 was delivered with the correct corresponding gRNA and successfully disrupted the SHCBP1 gene (Figure 7A), which is over-expressed in breast cancer and associated with the pro-liferation of malignant breast cancer cells in both MCF-7 and MDA-MB-231 cell lines.[101] As a result of the gene disrup-tion, the viability of the cancer cells was significantly reduced (Figure 7B,C). Follow-up mechanistic studies showed that the knockout might be mediated by increased cyclin-dependent kinase inhibitor p21 with decreased cyclin B1 and CDK1, indi-cating that the effects of gene knockout on signaling path-ways may be central to determine the nature of the resultant therapeutic efficacy rather than the isolated genetic disruption itself. In another study, knockdown of Brahma (BRM) and Brahma-related Gene 1 (BRG1) ATPases (Figure 7D), which are both overexpressed in breast cancer, decreased tumor formation in vivo.[102] When cell proliferation was monitored in MDA-MB-231 post Cas9 knockout over the course of nine days, the BRG1 and BRM knockout cells did not initially proliferate though after long-term passage the cells began to grow again (Figure 7E). While the Cas9 system facilitates multiplexed gene editing, it behooves the researchers to understand how the dis-ruption of multiple oncogenes will influence different signaling pathways and overall tumor cell viability.

Instead of knocking out oncogenes, it is also possible to knockout other genetic targets including miR implicated in breast cancer. For instance, Cas9 could be used to target genes involved in regulating multidrug chemoresistance. This approach has already been applied in EFGR mutant lung cancer and osteosarcomas, though not in TNBC.[103,104] CRISPR/Cas9 was engineered with a gRNA to target the md1 gene that codes for a drug efflux pump responsible for a prominent mecha-nism in cells that become drug resistant.[105] Three Cas9/gRNA delivery methods were explored (Cas9/gRNA plasmid delivered by the protein transduction domain TAT, Cas9 Protein/gRNA complex delivered by Lipofectamine, and Cas9/gRNA complex delivered by cell penetration peptide) and yielded similar gene and protein disruption between 50–60% in MCF7 breast cancer cells. The breast cancer cells were then treated with doxorubicin to assess the resultant drug sensitivity. Flow cytometry revealed that the drug uptake level was lower in the gene-edited cells than the wild-type MCF7 cells. Doxorubicin also caused significant toxicity in the gene-edited breast cancer cells compared with the untreated MCF7 cells. The drug resistance was almost reversed at lower doxorubicin concentrations, highlighting the power of this approach to overcome chemotherapeutic resistance. A similar strategy can also be employed to target miR involved in regulating drug resistance. Cas9 can be used to disrupt miR-21, which is known to confer resistance to chemotherapy in HER2+ breast cancer patients and predict TNBC patient survival.[106,107] The results of this study indicated a significant reduction in cancer cell proliferation after Cas9 induced miR-21 disruption. Noncoding RNAs involved in breast cancer signaling pathways and associated with poor prognosis have been identified by Cas9 synergistic activation mediator systems as well as bioinfor-matics systems using genome atlases and may serve as viable disruption targets as well.[108,109] Thus, other loci can be inves-tigated further with Cas9 specifically for breast cancer subtypes that lack sufficient molecular targets.

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4.2. Epigenetic Modulation with dCas9

Reversible DNA methylation and histone acetylation by epige-netic DNA DNMT and histone deacetylate (HDAC) inhibitors may be successful methods to target breast cancer, though these approaches have not proven to be effective in all breast cancer subtypes.[110] More comprehensive analyses of the pro-gression of DNMT and HDAC inhibitors in overall cancer therapy can be found in the other reviews. Therefore only a few highlights pertinent to breast cancer will be mentioned here. While HDAC inhibition is the first successful form of epige-netic cancer therapy, they consistently fail to treat solid tumors and are ineffective against TNBC.[111,112] Specifically, in a phase II clinical trial of metastatic breast cancer (NCT00132002), the HDAC inhibitor, Vorinostat, did not satisfy the necessary response evaluation criteria in solid tumors to be considered an effective breast cancer monotherapy.[113] Zeng et al. investigated molecular explanations for the ineffectiveness of HDAC inhibi-tors in breast cancer and concluded that the LIFR-JAK1-STAT3 signaling pathway is the likely cause of the observed failure.[111] The pathway is as follows: HDAC inhibition elevates LIFR gene promoter histone acetylation leading to BRD4-mediated activa-tion of LIFR, which then activates the JAK1-STAT3 pathway. Based on the proposed mechanism, the researchers inhibited JAK1 and BRD4 with siRNA, noting that the efficacy of HDAC inhibitors was enhanced even in TNBC.

Given the inefficacy of epigenetic regulators alone, other forms of combinational therapy including epigenetic inhibi-tors, chemotherapy, and other targeted therapy such as PARP inhibitors have emerged in breast cancer clinical trials. Lysine-specific demethylase 1 (LSD1) and HDACs together modu-late the growth of breast cancer cells.[114] The HDAC inhibitor romidepsin has been combined with paclitaxel to treat inflam-matory breast cancer.[115] Similarly, Hydralazine, a known

DNMT inhibitor, was coupled with doxorubicin in a phase II clinical trial and yielded a complete clinical response in 31% of patients (NCT00395655).[116] Interestingly, HDAC inhibition can produce a sense of “BRCAness” in TNBC cells, meaning that they become sensitive to PARP inhibition.[117] The mecha-nistic relationship between HDAC inhibition and “BRCAness” is not completely understood. However, it is believed that HDAC inhibition downregulates DSB repair, causing PARP trapping.[118] Exploiting this relationship, olaparib was shown to elevate the effects of the HDAC inhibitor, Vorinostat, or suberanilohydroxamic acid in TNBC.[119] Another study dis-covered that low doses of DNMT inhibitors combined with PARP inhibitors in vivo improved PARP inhibitor efficacy by increasing PARP chromatin binding, extending DSB retention, and inducing cytotoxicity.[120]

The dCas9 variant can be employed as a precise and efficient epigenetic treatment tool for multiple genes perhaps en lieu of combinational therapy by fusing with specific regulatory effector domains.[121–123] For instance, LSD1, which has been shown to modulate breast cancer progression, was fused with a dCas9 ortholog to achieve genetic repression of specific enhancers.[124] In other studies, the dCas9 fusions were shown to be highly specific, inducing a 50% increase at target loci with either few or no evident off-target effects with dCas9 in vivo post 24 h of addition.[125,126] A notable advantage of dCas9 compared with current methods is that it can be applied to simultaneous activation of tumor sup-pressor genes and repression of oncogenes to counter individual-istic epigenetic breast cancer profiles by using gRNAs of different lengths that are distinct for each specific target locus.[127–130]

The epigenetic effects induced by dCas9 can be further amplified and specified through synthetic biology genetic circuits.[131] For example, dCas9 and gRNAs were designed to construct logic gates and connect them to native E.coli regulatory networks. The initial gRNA NOT gate includes a

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Figure 7. Cas9 for breast cancer treatment. A) Western blot analysis of SHCBP1 protein expression in MDA-MB-231 and MCF-7 cells infected with eGFP gRNA and SHCBP1 gRNA lentivirus. SHCBP1 knockout by gRNA lentivirus significantly inhibited the growth rate of B) MDA-MB-231 and C) MCF-7 cells, as shown by cell count for 5 d MTT assay. Reproduced with permission.[101] Copyright 2016, Elsevier. D) Western blot demonstrates the efficacy of CRISPR/cas9 mediated knockout of BRG1 and BRM in MDA-MB-231 cells. E) Dynamic cell proliferation given different gRNAs. Reproduced with permission.[102] Copyright 2015, Wiley-VCH.



start promoter to provide direction and a guide region to target dCas9 to the cognate bacterial promoter culminating in a ter-minator to halt transcription (Figure 8A). The promoter, gRNA and terminator can be engineered from ssDNAs, which contain restriction enzyme sites. The oligonucleotides are annealed and extended until the double-stranded entities are combined into a circuit plasmid in a “Golden Gate assembly” (Figure 8B). In Figure 8C, the connection between the synthetic dCas9 circuit and the native E.coli network is depicted, in which a gRNA targets the host E.coli transcription factor called malT. This final step converts the circuit output to a cellular phenotypical result including resistance or chemotaxis. This methodology can be generalized to allow for a variety of engineered responses defined by the circuity in living cells that can be applied to breast cancer. Nevertheless, gene circuits are limited by several factors, including the number of orthogonal, yet nonoverlap-ping or parallel transcription factors available. CRISPR, how-ever, circumvents this barrier due to its boundless orthogonality revealed through bioinformatics screening or the synthetic engineering of alternative variants.[132] The application of dCas9 can become more intricate by using synthetic transcriptional logic gates to perform computation in breast cancer cells, which would be helpful in turning on and off different genes at

different times in malignant cells.[133] Consequently, as breast cancer progresses through different stages, therapy can corre-spondingly change to provide an individualized treatment that coincides with the idiosyncratic stage of the cancer.

4.3. RNA Interference with dCas9 and C2c2

Prior to the advent of CRISPR, RNAi was the dominant genetic editing tool. RNAi can post-transcriptionally modulate genetic expression through double-stranded RNA mediated homology. Essentially, small interfering RNAs (siRNAs) are endogenously or exogenously generated to specifically target regions homol-ogous to the intended RNA target, similar to the way that gRNA is designed to bind to the double-stranded DNA target sequencing with Cas9. Transient gene knockdown can also be achieved by applying catalytically inactive CRISPR dCas9 through CRISPR interference (CRISPRi). CRISPRi works on the level of DNA by down-regulating gene transcription, which is fundamentally different from the post-transcriptional modi-fication induced by RNAi. CRISPRi has been shown to repres-sion genes by 1000-fold without any off-target effects and is capable of repressing multiple genes simultaneously.[134] Based

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Figure 8. dCas9 for breast cancer epigenetic treatment. A) CRISPR/dCas9 NOT gates comprised of an input promoter that transcribes gRNA, and a syn-thetic output promoter. When the dCas9 handle of the gRNA (dark green) complexes with dCas9 (blue), the gRNA binds the operator (light green) and a sigma factor binding site (gray), causing transcription repression initiation at the output promoter. B) CRISPR/Cas genetic circuits are constructed from pairs of ssDNA oligonucleotides ≤200 nt long. C) dCas9 (blue) mediates repression of synthetic promoters by programmable gRNAs (solid colored rectangles). Reproduced under the terms and conditions of the Creative Commons Attribution license 4.0.[133] Copyright 2014, the authors, published by European Molecular Biology Organization.



on a thorough analysis of the techniques and applications of RNAi and Cas9, it is theorized that while RNAi will still be used for clinical applications, CRISPR technology may ultimately supersede RNAi in the long term for certain research tool applications.[135]

Furthermore, C2c2 or Cas13a can complement CRISPRi by directly targeting RNA instead of DNA and is confirmed as a valid platform for the therapeutic manipulation of RNA in mammalian cells.[26] A preliminary screening interference assay in E. coli consisting of fifteen potential C2c2 orthologs showed that LwaC2c2, had greater RNA targeting specificity than RNAi, with a comparable knockdown efficiency. The LwaC2c2 was delivered into mammalian cell lines with a GFP tag and either a nuclear export sequence or a nuclear locali-zation signal (NLS). LwaC2c2-GFP-NLS could achieve a high knockdown efficiency of ≈75%, which was comparable to the RNAi control that induced 78% knockdown. Knockdown was tested with several genes including KRAS, for which the knock-down was ≈58%. Once the system was further optimized with tiling screens, the maximum KRAS knockdown efficiency was 85%. The knockdown efficiencies of the top three guides for KRAS tiling screens were compared with the efficiency of the RNAi-based knockdowns, for which LwaC2c2 was significantly better for two of the six guides. Knockdown was removed by simply mutating the catalytic domain of LwaC2c2, indicting the gene knockdown is a direct result of C2c2 and not some other potential reason.

If the knockdown efficiency of KRAS can be further improved with this novel approach, this treatment modality may be viable for TNBC. The role of KRAS has been well doc-umented in pancreatic, colon, and lung carcinoma, but it has not been a focus of study in breast cancer until recently when the role of KRAS was explored in TNBC.[136] The study revealed that KRAS activity was higher in TNBC compared with luminal breast cancer, as indicated by the interaction with RAF-1, since the active form of KRAS can interact with RAF-1. In vivo data revealed that KRAS was essential for the metastatic ability of the mesenchymal-based TNBC. Specifically, MDA-MB-231 cells were transfected with siRNA- targeting KRAS or scrambled control siRNA, and then injected into mice. After five weeks, lung metastasis was blocked by the downregulation of KRAS in MDA-MB-231 triple-negative breast cancer cells, where the metastatic frequency was quantified by the number of foci found on the lung surface.

In addition to evaluating the single knockdown with LwaC2c2, multiplex knockdown with the combination of five guides against KRAS, PPIB, CXCR4, TINCR, and PCAT along a single promoter was also assessed. The efficiencies of multi-plex, pooled single-guide delivery, and singe guide delivery were comparable. Additionally, the multiplexed delivery of the tar-geted guides for KRAS, PPIB, and CXCR4 with a nontargeting guide were also assessed. When a targeted guide was absent, only the targeted transcripts were affected, illustrating the specificity of the multiplex system. Knockdown was sensitive to single and double mismatches introduced into guide targets. Transcriptome-wide mRNA sequencing revealed that there were significant off-target effects induced by shRNA, but not induced in the LwaC2c2 conditions, given comparable levels of the knockdown transcripts. Therefore, LwaC2c2 appears

to be better suited than shRNA for breast cancer therapeutic applications based due to its lack of the undesirable off-target effect, coupled with the high efficiency.

4.4. Gene and RNA Correction and Replacement with Cas9 and C2c2

To permanently fix breast cancer point mutations, single base repair in lieu of double-stranded breaks induced by Cas9 may be a more precise solution. Fusions of Cas9 and a cytidine deaminase enzyme were engineered to specifically convert a cytosine (C) to a thymine (T) or a guanine (G) into an adenine (A), and the system was optimized through several iterations to achieve maximal efficiency.[137] While this application may seem limited in scope, the dominant-negative p53 mutation associ-ated with numerous cancers including breast cancer can be cor-rected by a C to T conversion. Breast cancer cells homozygous for the p53 mutation were nucleofected with DNA encoding the third generation base editor and gRNA programmed to correct the mutation. This resulted in a 3.3–7.6% correction efficiency with less than 0.7% off-target effects. Additionally, there was no correction evident in the breast cancer cells treated with wild-type Cas9. The most common single base-pair change that accounts for half of known disease causing single nucleotide polymorphisms is the switch from G–C to T–A, but there is no known enzyme that can directly and independently undo this conversion. More recently, the same group engineered a transfer RNA adenosine deaminase to act on DNA when fused to a catalytically inactive Cas9 mutant to successfully make these base conversions. The system was evolved seven times with various alterations to convert the base pairs with 50% effi-ciency in human bone cancer cells with nearly 100% purity.[138] Together, these two base-editing systems can add to the abilities of CRISPR to precisely module breast cancer genetics.

Parallel RNA editing methods have been developed with C2c2 that may allow for the alteration of individual nucleosides. RNA Editing for Programmable A to I Replacement (REPAIR) was developed with catalytically inactive C2c2 (dC2c2) to direct adenosine to inosine deaminase activity by ADAR 2 to transcripts in mammalian cells.[139] A subset of the C2c2 (21 orthologs), Cas13b (15 orthologs), and Cas13c (7 orthologs) protein families were analyzed for RNA knockdown activity with two different guide RNAs for each ortholog, and it was concluded that Prevotella sp. P5-125 (PspCas13b) had the highest knockdown efficiency of ≈63%. The knockdown efficiency of PspCas13b was compared with LwaC2c2 through position-matched guides tiling, and PspCas13b had a higher efficiency of about 92% compared with 40.1% efficiency of LwaC2c2. The targeting specificity of PspCas13b and LwaC2c2 were compa-rable in HEK293FT cells transfected with either variant, a fixed gRNA targeting sequence, and a mismatched target library spe-cific for either system. Both variants had similar sensitivity to single and double mismatches.

To generate the catalytically inactive PspCas13b, the cata-lytic residues in the HEPN were mutated, and a mismatched cytidine opposite the target adenosine was introduced. To enhance deamination rates, the dcas13b was fused with the deaminase domains of human ADAR1 or ADAR2 containing

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hyperactivation mutations to enhance catalytic activity. The RNA knockdown efficiency with PspCas13b REPAIR was 23%, but there were prevalent off-target effects. These off-target effects were present for other genetic targets as well, such as PPIB. Due to off-target effects of the first version of REPAIR, REPAIR2 was generated in attempt to improve gene disruption specificity by mutating residues of ADAR2DD. In this process, 17 single mutants were tested with both targeting and nontargeting guides. The mutant ADAR2DD (E488Q/T375G) had the highest percent editing of the four mutants with the lowest number of off-target effects. This variant, dubbed REPAIRv2, was also tested on KRAS, which is pertinent in TNBC treatment. The efficiency was comparable to that of REPAIRv1 (≈27%), but there were no detectable off-target edits. Overall, REPAIRv2 had ≈900-fold increase in specificity compared with REPAIRv1.

The C2c2 binding system may offer advantages for breast cancer treatment beyond independent Cas9 gene disruption. While DNA base editors are limited because the Cas9 needs a PAM at the editing site to function, C2c2 has limited con-straints or motif preferences for RNA interference. C2c2 can target any adenosine in the transcriptome with the REPAIR system. The authors attribute this versatility to the increased concentration of REPAIR at the target site as a result of dCas13b binding.[139] Therefore, REPAIR may be a more flex-ible system compared with traditional DNA base editors. The REPAIR system also does not rely upon endogenous repair pathways such as NHEJ to achieve the desired editing. Addi-tionally, RNA editing is transient, allowing for temporal control over editing outcomes, which may be advantageous when only a short treatment approach is needed for therapeutic efficacy without permanently changing the genome or introducing per-manent off-target effects that are difficult to reverse. REPAIR can modify the sequence of expressed risk genetic variants to decrease the chance of patients developing cancer.

4.5. Immune System Reprogramming with Cas9

Immunotherapy continues to advance precise breast cancer treatment, which can further be enhanced by Cas9.[140] Breast cancer has longtime been considered poorly immunogenic compared with other cancers such as melanoma, perhaps due to the heterogeneous nature of the breast cancer tumor micro-environment.[141] However, some reports suggest that certain breast cancer subtypes have a strong infiltration of immune cells and may respond positively to immunotherapy. Uncov-ering immunotherapeutic applications for breast cancer is an active field of investigation that may become easier with Cas9.[142,143]

Cas9 can improve immunotherapy along multiple fronts to achieve enhanced clinical efficacy.[144] T-cells can be genetically engineered with Cas9 to express a chimeric antigen receptor (CAR) to recognize an antigen specific to breast cancer. CAR T-cells (CART) immunotherapy consisting of an extracel-lular single-chain variable fragment specific to an antigen and an intracellular chimeric signaling domain that drives T-cell induced killing has already been FDA approved for the treat-ment of blood cancer and shows promise for the treatment of B-cell acute lymphoblastic leukemia and non-Hodgkin’s

lymphoma.[145] In fact, CART targeting the breast cancer HER2 antigen is currently in clinical trial (NCT02713984).

Clinical trials frequently use patient-derived or autologous T-cells, which are labor-intensive and expensive to manufacture, hampering the clinical development of CART. In the autolo-gous approach, a patient’s own cells are engineered ex vivo, cul-tured in vitro to amplify the number of cells, and infused back into the same patient in order to maintain human leukocyte antigen (HLA) compatibility.[146] In the case of allogenic T-cells, cells from a donor are engineered to treat a different patient. The efficacy of the treatment can be limited by multiple prob-lems: a) allogeneic T-cells could be recognized as foreign by the recipient’s immune system and rejected; and b) the recipient’s normal tissues could be recognized as foreign by the allogeneic T-cells and cause graft-versus-host-disease. To avoid these prob-lems, methods exist to inactivate or replace genes involved in MHC recognition with Cas9.[147]

While using autologous T-cells may be the most precise immunotherapy treatment method, it is laborious, expensive, and variable in efficacy, motivating the creation of “universal T-cells” and providing a standardized therapy. In one study, multiplex CRISPR gene editing was used to develop universal T-cells, which lack HLA Class 1 and T-cell receptors (TCR) to avoid immune rejection and concomitantly disrupt other genes to enhance the overall treatment efficacy.[146] Specifically, Cas9 mRNA and gRNAs targeting β-2 microglobulin (B2M) and PD1 were delivered to primary human T-cells together by RNA electroporation. Two iterations of gRNA electroporation resulted in more than 90% targeting efficiency at the protein level for each gene disruption. To disrupt the TCR and B2M genes, CAR and CRISPR were delivered by lentivirus. The triple gene disruption (TCRβ region, B2M, and PD1) effi-ciency was ≈65%, and perhaps can be made more efficient with alternative delivery strategies. However, alternative nonviral methods such as nucleofection are correlated with increased toxicity to T-cells.[128] Therefore, ways to improve gene disrup-tion efficiency without sacrificing T-cell viability at the delivery level should be explored. Overall, this combinational gene dis-ruption demonstrated antitumor effects in leukemia tumor mouse models that were on par with untampered T-cells. Rare off-target effects were observed when targeting the α and β constant regions of the TCR. Targeting the T-cell receptor α constant may be a promising approach for multiplex editing because it results in uniform CAR-expressing T-cells. Further-more, the resultant Cas9-edited CART display enhance potency and delayed exhaustion of effector T-cells compared with CART cells generated conventionally.[148] Therefore, it may be possible that Cas9 can directly and simultaneously disrupt immune receptors and insert the CAR ligand at the disruption site for an enhanced therapeutic response, highlighting the promise of Cas9 in the advancement of CART therapy.

In some cancers, such as acute myeloid leukemia, a lack of cancer-restricted markers is a major problem associated with CART therapy that Cas9 may be able to solve. One noteworthy acute myeloid leukemia diagnostic and therapeutic marker, CD33, is expressed on all normal myeloid cells downstream of the common myeloid progenitor. CD33 is traditionally difficult to target therapeutically with CART immunotherapy because of the resultant, previously unavoidable systemic toxicity to

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the normal myeloid cells. Kim et al. used Cas9 to delete CD33 from normal hematopoietic stem and progenitor cells (HSPCs) so that they become resistant to precise CART CD33-targeted therapy and circumvent any toxicity.[149] The CD33 knockout HSPCs displayed normal multilinear engraftment and differen-tiation in a monkey model. When CART CD33-targeted therapy was introduced, the knockout cells were unaffected, and there was no apparent myeloid toxicity. Overall, this study highlights the use of Cas9 to precisely tailor and generate CART to spe-cific cancerous cells. This paradigm can broaden the scope of potential antigens for CART therapy for all cancers, including breast cancer, and thus increase its therapeutic utility.

Additionally, Cas9 can be used to disrupt genes that inhibit T-cell signaling molecules such as the programmed cell death protein (PD1) checkpoint receptor to intensify treatment.[150] Because transfection efficiency is problematic with T-cells, the researchers opted to use a nucleofection approach rather than viral transduction. This electroporation gene disruption approach was proven to be efficient, nondetrimental to cell viability, and enduring, underscoring the potential for non-viral Cas9-mediated editing. Figure 9A shows the cleavage of PD-1 from three different T-cell donors (H2, G2, and Z2) after PCR products were amplified, and a T7 endonuclease assay

was conducted. Sequencing confirmed that PD1 mutation was achieved in all three donors. However, in the healthy donor (H2), there was a large deletion relative to the gene disruption of the other two donors (G2 and Z2). The researchers explored whether gene disruption would inhibit T-cell proliferation and determined that there was no statistically significant effect. To mimic more of an in vivo situation, PD1 genetically engi-neered knockout T-cells were cocultured with PD-L1 tumor cells to assess the sustainment of PD1 disruption. As shown in Figure 9B, flow cytometry revealed that ≈3% of the modified T-cells expressed PD1, while nearly 18% of the control T-cells expressed PD1 with stimulation of the tumor antigen. Overall, this result illustrates that PD1 disruption is not transient, but sustained in the presence of tumor antigens. The immune response associated with the Cas9 modified primary T-cells from two late-stage melanoma patients was assessed by intro-ducing HLA-epitope matched peptides (Figure 9C). There was an increase in Interferon–γ production in the Cas9 PD1 knockout group, indicating that the PD1 disruption enhanced the overall immune response induced by the T-cells. Given the evident immune response elevation, researchers cocul-tured melanoma patient-derived transduced T-cells with PD-L1 expressing M14 melanoma cells to determine if the tumor

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Figure 9. Cas9 enhances immunotherapy. A) Detection of gRNA1:Cas9-mediated cleavage of hPD-1 by T7EN1 cleavage assay. B) Expression of hPD-1 on T cells was determined by flow cytometry 7 and 21 d post-transfection stimulated by irradiated tumor cells. C) T cells from a melanoma patient was stimulated by melanoma associated peptides pulsed autologous DCs indicating enhanced cytokine production. D) The hPD-1 KO T cells or control T cells from a melanoma patient were co-cultured with CFSE labeled M14 cells. After 6 h, PI was added and the cells were analyzed by flow cytometry. Reproduced under the terms and conditions of the Creative Commons Attribution license 4.0.[150] Copyright 2015, Macmillan Publishers Limited.



cells would be also subjected to greater cytotoxicity. The PD1 knockout cells showed greater dose-dependent lytic abilities in M14 cells (Figure 9D). As such, the disruption of immune inhib-itory checkpoints may be clinically relevant for adoptive T-cell therapy of breast cancer. A phase I clinical trial (NCT02793856) for PD-1 knockout T-cells engineered with Cas9 in patients with small cell lung cancer concluded in 2018, demonstrating prom-ising results for both the safety and efficacy of this therapeutic approach.[151] A phase II clinical trial (NCT03081715) for PD-1 knockout T-cells for esophageal cancer is underway.

Cas9 could have applications in the field of cancer vaccines as well. For instance, dendritic cell (DC) cancer vaccines may be administered in conjunction with Cas9 to improve the therapeutic efficacy and duration of the protection for breast cancer. Immature DCs arise from bone marrow precursors

that demonstrate minimal T-Cell activation. Once immature DCs recognize antigens, such as tumors, they become acti-vated, mature DCs and express high levels of MHC molecules that migrate to the lymph nodes where they activate killers T-cells (CD8+) to induce an antitumor immune response.[152] Patients with breast cancer have DCs that are phenotypically similar to immature DCs in that they have low expression of MHC molecules, do not stimulate leukocytes to the same extent as mature DCs, and paradoxically confer immune toler-ance instead of immunity.[153] Consequently, it may be better to use DCs taken directly from patients and manipulate them ex vivo.[143] Cas9 can be used to precisely guide DC differentia-tion from bone marrow precursors to respond to specific breast cancer antigens. Furthermore, although an in vitro DC vaccine methodology against breast cancer was developed in preclinical

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Table 3. Molecular targets for breast cancer therapies based on CRISPR technologies.

Target Function Reason CRISPR system Reference(s)

ETV6-NTRK3

fusion gene

The ETV6 gene (ETS variant gene 6) encodes a transcriptional repressor

which plays role in hematological malignancies

NTRK3 is a membrane anchored tyrosine kinase and functions as a

receptor for the neurotrophin NT-3, regulating development and mainte-

nance of the vertebrate nervous system

Secretary breast carcinoma

diagnostic biomarker

Cas9 [63]

MAG13-AKT3

fusion gene

MAG13 regulates cell signaling processes and interacts with PTEN to reg-

ulate AKT activity. AKT3 regulates metabolism, proliferation, cell survival,

growth, and angiogenesis through serine and threonine phosphorylation

Enriched in TNBC Cas9 [64]

PTEN Phosphate and tensin homolog tumor suppressor gene that modulates

cell cycle progression

Mutated in breast cancer Cas9 [66]

BRCA1/2 Tumor suppressor genes that maintain genomic stability Mutated in breast cancer Cas9 [100]

Cripto-1 Encodes an epidermal growth factor protein that serves as a receptor

for the TGF signaling pathway and facilitates the maturation of notch

receptors

Mutated notch proteins are often

found in TNBC

Cas9 [71,72]

UBR5 Encodes an E3 ubiquitin-protein ligase and is involved in regulatory

proliferation

UBR5 gene amplification is

common in TNBC and is associated

with poor survival rates

Cas9 [75,76]

KDM(5A,5B,5C,6B) Histone demethylases that regulate transcription of proteins involved in

cell differentiation and favors cell proliferation

Association between expression of

histone demethylases and breast

cancer

Cas9 [77]

BRD4 Recognizes and binds acetylated histones to maintains epigenetic

memory throughout transcription

Sustains TNBC migration and

invasion

dCas9 [95]

ATK1, GATA3,

PIK3CA, and MAP3K1

ATK1/PIK3CA: regulates cell growth, survival

GATA3: regulates epithelial cell differentiation in the mammary gland

MAP3K1: regulates apoptosis

Breast cancer oncogenes Cas9 [99]

SHCBP1 Involved in signaling pathways for cell proliferation Overexpressed in breast cancer and

associated with the proliferation of

malignant MCF-7/ MDA-MB-231 cells

Cas9 [101]

BRM/BRG1 BRM is the ATPase subunit and BRG1 is the catalytic subunit of enzyme

complexes that uses ATP to disrupt chromatin target promoters

Overexpressed in breast cancer Cas9 multiplex [102]

MDR1 Encodes a drug efflux pump involved with drug resistance Sensitize breast cancer cells to

chemotherapy

Cas9 [105]

miR-21 Regulates cellular proliferation, invasion, migration, and apoptosis

through interactions with PTEN

Confers resistance to chemotherapy

in HER2+ breast cancer patients and

predicts TNBC survival

Cas9 [106,107]

LSD1 Transcriptional corepressor through demethylation of histone 3

lysine 4 (H3K4)

Modulates the growth of breast

cancer cells

dCas9 [114,124]

KRAS GTPase that signals messages between the extracellular space

and the nucleus

KRAS mutations found in breast

cancer

Cas9 [66]



studies, specific monotherapy with DCs for breast cancer did not produce a consistent and significant clinical benefit post metastasis.[154] Therefore, Cas9 may be helpful in targeting and prolonging breast cancer-specific immune activation.

To enhance the efficacy of DC vaccines, there are multiple ongoing clinical trials (NCT00088985, NCT00266110, and NCT00978913) exploring the combination of DC immuniza-tion and chemotherapy on breast cancer outcomes; however, gene editing with Cas9 may offer additional benefit. Instead of combining multiple therapies that have limited success independently in the attempt to compound positive outcomes, it may be advantageous to include combinations that enhance the immune response, while limiting immune intolerance. Additionally, the specific reasons for limited DC success in breast cancer should be analyzed. Once triggered by immu-notherapy, CD8+ cells must overcome numerous barriers including, intrinsic regulators such as PD1, inflammation, antigen loss, immune invasion, and obstacles specific to the cancer type such as fatty cells in breast cancer.[155] Cas9 can help overcome several of these barriers by identifying the key molecular antigens specific to each breast cancer subtype and subsequently inactivate T-cell molecules involved in the damp-ening of immune responses or the induction of immune toler-ances, including PD1, which is also involved in the induction of immune tolerance.[156] Regardless, more research will be neces-sary to determine how individual breast cancer patients develop immunity in response to DC vaccines, before the design of gene-editing approaches based on CRISPR/Cas9 technologies.

5. Perspectives and Future Directions

CRISPR technology has the power to advance the precision med-icine approach to breast cancer at all stages of disease, including risk reduction, detection, diagnosis, and therapy. CRISPR offers advantages to other genetic engineering techniques RNAi, Zinc finger nucleases (ZNFs), and transcription activator-like effector nucleases, as it is permanent, relatively simple to engineer, and highly programmable to different functions. The different CRISPR variants can also be combined in a seemingly infinite number of ways to achieve a therapeutic goal. Table 3 shows the genetic targets outlined with its corresponding CRISPR variant that can be used for breast cancer treatment.

While CRISPR technology is advancing, it must be noted that the delivery, efficiency, and off-target mutations still pose potential problems to the clinical applications thereof. CRISPR variants can be delivered virally or nonvirally with varying effi-ciencies and clinical safety. Viral transduction is more potent than its nonviral counterpart, but is also accompanied by immunological concerns and concerns of seamless genetic integration.[157] Regarding gene disruption efficiency, it is still unclear what efficiency is necessary to have a positive thera-peutic effect in vivo, as it is possible that the current efficacy is inadequate to make a significant impact. Numerous studies have been published describing computational algorithms that can predict likely Cas9 off-target effects and counter unin-tended DSBs through careful gRNA design.[158,159] Another CRISPR variant spCas9-HF1 has more than 85% improved on-target efficiency compared with spCas9 and could offer another

solution to off-target effects.[160] Nevertheless, more research is needed in the preclinical stage to ensure that off-target effects are not a point of concern.

In the future, genome-wide screening for breast cancer subtypes should be conducted to identify specific genetic and epigenetic targets for CRISPR technology to be most effective. The functionality of the identified mutations and their related signaling pathways need to be thoroughly analyzed before they are manipulated for therapy with CRISPR. More in vivo studies regarding Cas9 epigenetic modulation should be conducted, as there is only one thus far in Drosophila, and none that spe-cifically investigate Cas9 epigenetics for breast cancer.[161] The use of synthetic biology for Cas9 modulation can be further extended to create real time predictive algorithms for specific metastatic pathways that update as epigenetic regulation pro-gresses and the cancer advances, so that treatment can always be precisely one step ahead of cancer. Ongoing research has the potential to optimize and advance CRISPR technology, culmi-nating in the clinical realization of its full potential for breast cancer diagnosis, modeling, and treatment.

AcknowledgementsR.L.M. and M.A.G. contributed equally to this work. The authors would like to thank Prof. Piero Dalerba, Sara Viragova, and Seetha V. Srinivasan for their technical and language editing. R.L.M. acknowledges the fellowship support from Sigma XI Grant-in-Aid of Research Program (Award 62017031593274167). This work was supported by National Institutes of Health (Grant Nos. AI096305, UG3TR002142, UG3TR002151, HL140275, and GM110494), Guangdong Innovative and Entrepreneurial Research Team Program, China (Grant No. 2013S086), and Global Research Laboratory Program, Korea (Grant No. 2015032163).

Conflict of InterestThe authors declare no conflict of interest.

Keywordsbreast cancer, CRISPR, precision medicine, targeted therapeutics

Received: May 18, 2018Revised: June 30, 2018

Published online: August 17, 2018

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