CRISPR Genome Editing Systems in the Genus Clostridium: aTimely Advancement

Kathleen N. McAllister,a Joseph A. Sorga

aDepartment of Biology, Texas A&M University, College Station, Texas, USA

ABSTRACT The genus Clostridium is composed of bioproducers, which are impor-tant for the industrial production of chemicals, as well as pathogens, which are asignificant burden to the patients and on the health care industry. Historically, eventhough these bacteria are well known and are commonly studied, the genetic tech-nologies to advance our understanding of these microbes have lagged behind othersystems. New tools would continue the advancement of our understanding of clos-tridial physiology. The genetic modification systems available in several clostridia arenot as refined as in other organisms and each exhibit their own drawbacks. With theadvent of the repurposing of the CRISPR-Cas systems for genetic modification, thetools available for clostridia have improved significantly over the past four years.Several CRISPR-Cas systems such as using wild-type Cas9, Cas9n, dCas9/CRISPR inter-ference (CRISPRi) and a newly studied Cpf1/Cas12a, are reported. These have the po-tential to greatly advance the study of clostridial species leading to future therapiesor the enhanced production of industrially relevant compounds. Here we discuss thedetails of the CRISPR-Cas systems as well as the advances and current issues in thedeveloped clostridial systems.

KEYWORDS CRISPR, CRISPRi, Clostridium, genome editing

Clostridia are comprised of obligate anaerobic endospore-forming firmicutes, andthe best studied act as either important producers of industrially relevant compo-

nents (e.g., Clostridium acetobutylicum or C. cellulolyticum) or pathogens (e.g., C. botu-linum or Clostridioides difficile) (1, 2). Genetic tool development in clostridia increasedover the last few years and continues to expand (3). Counterselection tools forhomologous recombination or allelic exchange are common across many bacterialorganisms, including clostridia (4). Such counterselection markers include I-SceI (5), upp(6), mazF (7), codA (8), galK (9), pyrE (10), and pyrF (11). Though common, each tool haslimitations, which have been discussed in length (12–15). More recently, single-stranded DNA annealing protein was used to engineer a recombineering system for C.acetobutylicum (16). This protein, RecT, is an ortholog from C. perfringens and is used byphage (i.e., Rac or lambda prophages) to help increase the efficiency of homologousrecombination of the single-stranded DNA into bacterial genomes. Through the help ofRecT, a short oligonucleotide is introduced into the target site (16, 17). This genetic toolshows the potential for exploitation of bacteriophage-derived single-stranded DNAbinding proteins for homologous recombination (16).

In the mid-2000s, TargeTron technology (ClosTron) was repurposed for use in clostridia(18, 19). This development led to a surge in our understanding of the physiology andmolecular processes that occur in clostridia, especially in C. difficile. The TargeTronsystem relies on the retargeting of mobilizable group II introns to create insertionmutations in the desired target site. The inclusion of retrotransposable activated markers(RAMs) permits the activation of an antibiotic resistance gene upon splicing from theintron RNA, thereby providing antibiotic resistance upon insertion into the genome (18,

Citation McAllister KN, Sorg JA. 2019. CRISPRgenome editing systems in the genusClostridium: a timely advancement. J Bacteriol201:e00219-19. https://doi.org/10.1128/JB.00219-19.

Editor William Margolin, McGovern MedicalSchool

Copyright © 2019 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Joseph A. Sorg,jsorg@bio.tamu.edu.

Accepted manuscript posted online 13 May2019Published

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19). Despite the popularity of this tool, it has major drawbacks compared to cleandeletions of target genes. The TargeTron target sites can be difficult to identify incoding regions which are relatively small, �400 bp. Because of this, not every gene canbe targeted for mutation (7, 12). Moreover, due to the nature of the mutation, theinsertion results in polarity for downstream genes (12). Also, due to target sites beingchosen by an algorithm, targeting at some sites results in aberrant insertions at othersites in the genome. Lastly, the antibiotic resistance marker has led to perceivedhypervirulence of mutant strains in the hamster model of C. difficile infection (20, 21).

With the advent of the CRISPR-Cas systems for genome editing, there has been asurge in the application of this tool in many organisms. The ease of use and pliabilityof these systems make them an attractive target to be developed for use in clostridia.Here we discuss CRISPR-Cas9/Cpf1 genetic engineering and the CRISPR-cas tools developedso far in Clostridium species.

CRISPR

The clustered regularly interspaced short palindromic repeats (CRISPR), along withtheir CRISPR-associated (Cas) proteins, have been developed into one of the mostpromising and successful genome editing tools to date (22). Although the significancewas unknown at the time, the CRISPR system was first discovered in 1987 (23). CRISPRloci have been found in �47% of bacteria and �87% of archaea and have beensuggested to have spread through horizontal transfer among prokaryotes as a basis ofadaptive immunity. CRISPR arrays within bacteria and archaea are divided into threemajor types and sixteen different subtypes based on the cas genes present within theorganisms (24).

CRISPR systems function as defense mechanisms to evade attacks from phage andother mobile genetic elements by incorporating a small fragment of the invader’s DNAinto the host’s genome. When an invading organism inserts its DNA into the bacterialcell, the native Cas proteins adapt to the infection by facilitating the incorporation of�30-bp fragments of this invading DNA (spacers) into the CRISPR locus on the host’schromosome. Within the CRISPR locus is a series of spacers, which have identity to prioradaptation events, flanked by repeat sequences of �30 bp in length, forming theCRISPR array. The acquisition of spacers occurs in an ordered fashion, where eachspacer is incorporated at the beginning of the array behind the leader repeat sequence,resulting in a timeline of the previous infections (25).

The CRISPR array is constitutively transcribed into pre-CRISPR RNAs (pre-crRNAs).These pre-crRNAs are processed and cleaved into individual crRNAs which are loadedonto a Cas endonuclease (26). In type II CRISPR systems, a trans-activating crRNA(tracrRNA) is upstream of the CRISPR locus and is essential for crRNA maturation byRNase III and Cas9 (27). The tracrRNA and crRNA form a duplex that is loaded onto theCas9 endonuclease (26). The most well-known and characterized Cas endonuclease isCas9 from Streptococcus pyogenes (22). Once the Cas proteins are loaded with duplexedtracrRNA:crRNAs, the Cas-RNA complex will find complementary sequences withininvading nucleic acids (28). The ability of the Cas protein to discriminate this foreignDNA from the complementary sequence encoded by the CRISPR array of the host isthrough the use of a protospacer adjacent motif (PAM). The PAM sequence is two tofive nucleotides in length and found at either the 5= end or 3= end of the protospacersequence in the invader’s genome; the location and sequence of the PAM sequencerecognized on the invading DNA is unique for each type of Cas endonuclease (29, 30).Upon recognition of the complementary sequence by the CRISPR RNA and the PAM bythe Cas endonuclease, the Cas endonuclease introduces a double-stranded DNA breakinto the invading DNA or a single-stranded RNA break into the invading RNA (29–31).

Soon after the discovery of the function of the Cas9 endonuclease, the first CRISPRgenome editing systems were developed in human cells, zebrafish embryos, andbacteria (Streptococcus pneumoniae and Escherichia coli) (32–39). In bacteria, a Casendonuclease, a single guide RNA (sgRNA), and a donor region for homologousrecombination are required for in vivo editing (Fig. 1A) (39). The Cas endonuclease

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cleaves double-stranded DNA. In order for the endonuclease to cleave at a specificlocation, a guide RNA is used to direct the endonuclease to the intended target site(40). To simplify the system, the gRNA has been engineered to be produced as a singleRNA molecule by fusing the crRNA and the tracrRNA. The result is an sgRNA which loadsonto the endonuclease and directs it to the target site by binding to the DNA (30). Insilico identification of sgRNAs is chosen where a PAM site is directly up- or downstreamfrom the target sequence (29). The sgRNA brings the Cas endonuclease to the intendedtarget, and endonuclease recognizes the PAM sequence and cleaves the DNA (29–31).To date, the most commonly used Cas endonuclease for genome editing in bacteria isCas9 from S. pyogenes; the Cpf1 endonuclease from Acidaminococcus sp. is beingdeveloped as an alternative to Cas9 (22, 41, 42). Because many bacteria either do nothave or have inefficient nonhomologous end joining (NHEJ) repair systems, repair ofdouble-stranded DNA breaks through homologous recombination is preferred (43). Tofacilitate this repair, a donor region that encodes the desired change in the genome isincluded in these systems (40). In prior work, Jiang et al. (39) demonstrated thathomologous recombination post-Cas9 cleavage of the DNA resulted in only a smallincrease in editing. Their work suggests that Cas9 cleavage of the bacterial DNAcounterselects the population of cells that has not recombined with the donor region;recombination of the donor region with the chromosome removes the sgRNA site fromthe chromosome. Here, we review the developed CRISPR systems for genetic modifi-cation in clostridia, which greatly expanded in 2015 and continue to expand today.

FIG 1 Comparison of CRISPR system plasmids. Representative sample mutagenesis plasmids for CRISPRediting systems are shown. Each contains the three regions: a homology region (denoted by homologyarms [HA]), an endonuclease (Cas9 or Cpf1), and a gRNA. Plasmids for editing using Cas9 or Cas9n (A),dCas9 with no necessary homology region (B), Cpf1/Cas12a with one target (C), and Cpf1/Cas12a withtwo targets (D) are shown. Examples of CRISPR-Cas mutagenesis plasmids are listed inside each plasmid.

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WILD-TYPE Cas9

In 2012, the function of the type II Cas9 endonuclease was characterized and kickedoff the CRISPR-Cas9 genome editing revolution (30, 32, 33). The Cas9 enzyme relies onthe base pairing between the tracrRNA and the crRNA to cleave the cDNA adjacent tothe PAM sequence (5=-NGG-3= in S. pyogenes) located at the 3= end of the targetsequence (29–31). Cas9 has two nuclease domains (HNH and RuvC-like) that areessential for the nuclease to induce a double-stranded DNA break (Fig. 2A). The HNHdomain, a ���-metal fold which contains the active site, cleaves the DNA strandcomplementary to the targeting sequence located on the crRNA and the RuvC-likedomain cleaves the opposite strand of DNA and shares an RNase H fold similar to the

FIG 2 Review of CRISPR-Cas genetic modification systems. Shown are graphical representations of each CRISPR-Cas system discussed in this review. Eachcontains the different endonucleases: Cas9 (A), Cpf1/Cas12a (B), and dCas9 with an activator/repressor (C), as well as the sgRNA or crRNA, PAM site andsequence, and cleavage locations and their respective cleavage domains for each. A table is included in panel A to describe the different mutant alleles of Cas9.Panel C also shows the promoter region area and the start codon for reference.

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RuvC Holliday junction resolvase (30, 44, 45). Mutation of either domain results in aCas9 nuclease that only cleaves a single strand of DNA (Fig. 2A). For example, intro-ducing an aspartic acid to alanine mutation at the 10th amino acid (D10A) inactivatesthe RuvC-like domain (Cas9n) and results in an enzyme that only makes single-strandedDNA breaks (“nickase”) (30). In addition, mutation of both nuclease domains, D10A andH840A, results in a catalytically dead nuclease, also known as dCas9, which will bediscussed later (34, 46).

In some organisms, including clostridia, the expression of the Cas9 endonucleasehas a high cost on the bacterial cell in terms of toxicity (12, 13, 47–50). Despite this cost,CRISPR-Cas9 systems have been successfully applied in C. acetobutylicum (13, 14, 51),Clostridium autoethanogenum (52), Clostridium beijerinckii (12, 13, 50, 53), C. cellulolyti-cum (48, 54), C. difficile (15, 55), Clostridium ljungdahlii (56), Clostridium pasteurianum(49, 51), and Clostridium saccharoperbutylacetonicum (57) (Table 1) by tightly regulatingthe expression of the cas9 gene. Specifically, the implementation of inducible promot-ers (e.g., tetracycline-, xylose-, or lactose-inducible promoters) has helped to circumventthe issue of Cas9 toxicity (14, 15, 50, 55, 57). Except for C. cellulolyticum, Clostridiumcellulovorans, and Clostridium tyrobutyricum, all of these clostridia use CRISPR-Cas9systems that encode wild-type Cas9 and have mutation efficiencies exceeding �50%(Table 1).

A few clostridial CRISPR systems have used the Cas9n allele (13, 48, 53, 54). Themajor advantage of this allele of Cas9 is for use in bacteria which have poor double-stranded DNA break repair systems or in bacteria where expressing both wild-type Cas9and the gRNA is lethal (48, 58). By using the Cas9n allele, the organism can overcomethe toxic effects induced by wild-type Cas9. In C. cellulolyticum, Cas9n was required tomerely obtain transformants of the mutagenesis plasmid (48). In that study, however,the endonuclease was under the control of a constitutive promoter (48). It is unclearif the use of an inducible system in C. cellulolyticum would allow for tighter regulationof the wild-type Cas9 endonuclease and overcome the limitation of using the wild-typecas9 allele. Interestingly, other clostridia that have developed Cas9n as a tool also havetools that use wild-type Cas9 (i.e., C. acetobutylicum [13, 14, 51] and C. beijerinckii [12,13, 50, 53]). It is unclear if it is necessary to use the Cas9n allele in these systems; theediting efficiencies are high for both Cas9 alleles.

The most recent use of the Cas9n allele in C. beijerinckii is different than what is usedin any other clostridia (53). Here, the nickase is fused to both a cytidine deaminase anda uracil DNA glycosylase inhibitor. In this unique system, the complex is guided to thetarget site by the sgRNA, a nick is made in the DNA, specific base pair substitutions (C/Gto T/A) are introduced, and the site is repaired. These changes result in possiblemissense mutations or null mutations in the targeted gene/coding region. The effi-ciencies reported here for base editing are comparable to those seen for gene editingusing Cas9. While this method avoids the use of a donor region on the plasmid (decreasingthe size of the plasmid as well as obviating the need of the bacteria to repair the lesion),it does not introduce specific mutations within the chromosome (53).

One aspect of the CRISPR-Cas9 system is the composition of the target sequenceand the design of the sgRNA that recognizes this sequence (e.g., G�C content andspecific nucleotide positions within the intrinsic sequence). Oddly, none of the devel-oped clostridial CRISPR-Cas9 systems have discussed this aspect of the system. Theauthors report differences in editing efficiencies when targeting different genes, butthere are no direct discussions about the sgRNAs used or if this impacted the efficien-cies of gene editing. In all of these systems, one sgRNA is used to make one mutation.Whether or not the authors tested other potential target sites within the mutated geneis unclear. The inclusion of multiple sgRNAs within the same plasmid to make multiplemutations at one time has not been accomplished in clostridia using the wild-type Cas9allele. To accomplish this, each sgRNA would require its own promoter to drive itsexpression, or different spacers could be used if the tracrRNA is expressed separately.Unfortunately, this would result in an increase in the size of the mutagenesis plasmid,which may result in issues when working with such a large plasmid. Smaller promoter

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TAB

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regions such as the C. beijerinckii sCbei_5830 small RNA promoter (49–51, 59) or the C.cellulolyticum P4 synthetic promoter (48, 54, 60) used in some clostridia CRISPR systemscould be functional in other Clostridium species. Both promoters, the sCbei_5830 smallRNA promoter and P4 promoter, are small (42 bp and 36 bp, respectively) and arefunctional in several clostridia (Table 1) (48–51, 54, 55, 57, 59–62). This is promising forthose clostridia which do not have many genetic tools and for which strong promotersare unknown.

The use of a donor region for homologous recombination is often necessary inbacteria to achieve high editing efficiencies (39). A double-stranded chromosomalbreak is highly toxic to cells, and recombination of the homology region with thechromosome protects against Cas9 targeting by removing the targeting site (48, 63).Nearly 28% of bacteria have the NHEJ component Ku (64). NHEJ components either arenot present in some bacteria or have low expression. This poses a problem for genomeediting when trying to repair a Cas9-induced double-stranded DNA break. Withoutadded expression of NHEJ components, such as on a multicopy plasmid, repairing thelesion using this method is not practical (65). Therefore, homologous recombination-based protection is much more efficient and sensible. The most commonly usedhomology arm length in clostridia CRISPR-Cas systems is 1 kb each, with 500 bp eachbeing the second most common(12–15, 48–52, 55–57). The size of the homology armsmay differ for each bacteria or CRISPR-Cas9 system, but the lowest reported homologyregion that achieves reasonable editing efficiency in clostridia is 100 bp (48).

dCas9/CRISPRi

CRISPR-Cas9-mediated genome editing creates a markerless edit within the targetDNA (e.g., bacterial chromosome or plasmid) (22). Not all genes can be targeted, assome genes are essential for the survival of the bacteria. For these reasons, a geneticsystem which does not make chromosomal deletions and only regulates the expressionof genes is necessary (66). CRISPR interference (CRISPRi) has been developed, whereboth catalytic sites of Cas9 have been mutated: D10A (located in the RuvC-like domain)and H840A (located in the HNH domain) (Fig. 2A) (30, 46). These mutations render Cas9catalytically dead. Here, Cas9 and the sgRNA function to bind and block a region ofDNA from transcriptional activity. First developed and implemented in E. coli (46), theauthors suggest that the Cas9:sgRNA complex collides with the elongating RNA poly-merase (RNAP). They found that targeting the template strand permits RNAP to readthrough and not come in contact with the Cas9:sgRNA complex. Thus, targeting thenontemplate strand of DNA has been reported to yield higher repression than targetingthe template strand in bacteria (46). A common use of this tool is to target the promoterregion of a gene or operon to downregulate expression upon induction of the CRISPRisystem (46, 67). A positive aspect to this system is that the plasmid size for the CRISPRisystem is smaller than that of the original genome editing CRISPR-Cas systems, in thatno donor region for DNA repair is necessary (Fig. 1B). More importantly the functionalityor characterization of essential genes can be explored using the CRISPRi system (66).

A few Clostridium species have developed CRISPRi systems: C. acetobutylicum (13,51), C. beijerinckii (13, 59), C. cellulovorans (68), C. difficile (69), C. ljungdahlii (60), and C.pasteurianum (51) (Table 1). In all cases, the successful repression of genes in theseorganisms ranges from 20% (e.g., plasmid-carried afp in C. acetobutylicum [51]) to 99%(e.g., pta in C. ljungdahlii [60]). These values depended on the gene being targeted and,more likely, the target sequence used for the sgRNA. The target sequence chosen forthe implementation of CRISPRi is more important here due to the function of repres-sion; an inefficient target sequence may not hold dCas9 on the DNA as tightly orconsistently as a more efficient target sequence. Li et al. targeted the spo0A gene inboth C. acetobutylicum and C. beijerinckii, using the same system, and obtained differentrepression percentages, 45% and 84%, respectively (13). The two sgRNA sequences areonly 35% identical. Because the two sequences are different, and in different organisms,direct comparisons between the two studies cannot be made. Similarly, the location oftargeting has an effect on the efficiency of repression. Target sites that are farther away

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from the transcription start site have lower efficiencies than those closer to thetranscription start site. Moreover, the use of multiple sgRNAs increases the efficiency ofrepression, as long as the target sequences do not overlap. Finally, truncated sgRNAtarget sequences, those with less than 12 bp, do not result in repression of the targetgene; full-length sgRNAs, those of 20 bp in length, are preferred for efficient repression(46). The only obvious issue with the CRISPRi genetic tool is that of polarity fordownstream genes. The effects of knocking down one gene in an operon will likelyhave an effect on the downstream genes.

Cpf1/Cas12a

Cpf1 (Cas12a) is a type V CRISPR system effector protein which has been studied inFrancisella novicida, Acidaminococcus sp. (AsCpf1), Moraxella bovoculi, and Lachno-spiraceae bacterium (47). The Cpf1 endonuclease specifically recognizes T-rich PAMsinstead of G-rich PAMs, as in the case for Cas9 (45). In another divergence from how thetype II CRISPR system works, Cpf1 itself is responsible for the maturation of pre-crRNA;no tracrRNA is needed. AsCpf1 is guided to its target by the mature crRNA to recognizethe PAM sequence 5=-TTTN-N23-3= (i.e., 5=-TTTN-3= followed by a 23-bp protospacer).AsCpf1 then cleaves the double-stranded DNA resulting in a staggered, 5-nucleotide(nt) 5= overhang that is 18 to 23 bp downstream from the PAM site (42). Unlike Cas9,AsCpf1 only uses one domain (RuvC-like) to digest both DNA strands rather than onedomain for each strand of DNA (Fig. 2B) (42, 70). Even though this endonuclease is notas well-studied/characterized as the Cas9 endonuclease, Cpf1 has been used forgenome engineering in Escherichia coli, Yersinia pestis, Mycobacterium smegmatis, andCorynebacterium glutamicum (71, 72) as well as in C. beijerinckii and C. difficile (Table 1)(61, 62).

One advantage of using Cpf1 over Cas9 is that the T-rich PAM sequence used byCpf1 could be more probable in AT-rich organisms, such as clostridia, as well as forpromoter regions, which are commonly AT-rich (45). It has been suggested that Cpf1 isbetter suited for bacteria, such as C. difficile, which have low DNA conjugation efficien-cies. This suggestion is based on the described lower toxicity of the Cpf1 endonuclease(62). These toxicity claims were based upon a study of Corynebacterium glutamicumwhere the authors could not obtain transconjugants when introducing their CRISPR-Cas9 plasmids. However, they were able to obtain several transformants from the samesystem using Cpf1 instead of Cas9 (72). A separate study found that Cas9 is toxiccompared to Cpf1 in Synechococcus sp. strain 2973 (73). However, because this is notan exhaustive list, the lower toxicity of Cpf1 than of Cas9 may be organism dependent.Finally, Cpf1 was suggested to have lower off-target effects than Cas9 (62, 74). However,the study referenced, as well as other studies, found this to be true in human cells andwas not tested in bacteria (74, 75). In C. difficile, Cas9 has no known or detectableoff-target effects as of the date of this review (15).

Another proposed advantage of using Cpf1 is that since a tracrRNA is not needed,the cost for constructing and using plasmids would be less due to the shorter gRNA(Fig. 1C). For Cpf1, a gRNA consisting of only 42 nucleotides would need to besynthesized compared to the �100-nucleotide sgRNA (crRNA and tracrRNA) needed forother type II endonucleases, such as S. pyogenes Cas9 (45, 73). Moreover, the smallersize of the gRNA could lead to the use of multiple gRNAs on a single plasmid tosimultaneously make multiple mutations in a single application of this system (Fig. 1D)(61, 62, 76).

Only two clostridia have developed CRISPR-Cpf1 systems, C. beijerinckii and C.difficile, and the efficiencies of mutagenesis of these systems range from 25% (i.e., C.difficile ermB1-ermB2) to 100% (i.e., C. beijerinckii spo0A, C. beijerinckii pta, and C. difficilefur) (61, 62). These values are similar to those obtained from using wild-type Cas9 (Table1). More genes would have to be targeted to identify significant differences betweenusing these two endonucleases in their respective CRISPR systems. Using C. difficile asan example, the promoters used by Wang et al. (55) (Cas9) and Hong et al. (62) (Cpf1)for the endonuclease and the sgRNA are the same. Even though the efficiencies for the

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CRISPR-Cas9 system are higher than that of Cpf1, few genes were targeted and thesame genes were not targeted between the studies (Table 1) (55, 62). As for C.beijerinckii, the same mutation efficiencies were reported for two genes, spo0A and pta,using either Cas9 or Cpf1 as the endonuclease (61). In that study, the same gRNA wasnot used in each system (61). Based on these few studies, there is low evidence of eitherendonuclease, Cas9 or Cpf1, being superior to the other in clostridia.

ENDOGENOUS CRISPR SYSTEMS

To overcome the toxic effects of S. pyogenes Cas9 or Acidaminococcus sp. Cpf1endonucleases, some genome editing systems were designed that rely on the endog-enous CRISPR array for editing (77–79). Seventy-four percent of clostridial speciesharbor CRISPR-Cas loci, including many of the well-studied clostridia (49, 80–91). Ofthese, C. pasteurianum and C. tyrobutyricum have developed CRISPR-Cas genomeediting systems based on their respective endogenous systems (Fig. 3, Table 1) (49, 91).

As a general starting place to develop endogenous CRISPR systems as genetic tools,Pyne et al. (49) analyzed the Cas proteins within the CRISPR array in C. pasteurianumand determined the PAM sequences by analyzing spacer sequences within the CRISPRarray to find common nucleotide sequences among them. The authors then developeda single-plasmid system which mimics the native CRISPR system by including a syn-thetic CRISPR array and crRNAs with spacers corresponding to the target region whichwas to be mutated. The authors also predicted PAM sequences for three otherclostridia: C. autoethanogenum, C. tetani, and C. thermocellum (49). Zhang et al. alsodeveloped an endogenous CRISPR-Cas system but for C. tyrobutyricum (91). The authorsused similar approaches described above to analyze the CRISPR arrays and determinedthe PAM sequence for C. tyrobutyricum. In addition, the authors multiplexed the gRNAsand made simultaneous genomic deletions using one CRISPR-Cas plasmid construct(91).

Both of the tools using endogenous CRISPR-Cas systems differ from those usingCas9 or Cpf1 in that the endogenous systems rely on the endonucleases encoded in thegenome to form mature crRNAs as well as make the double-stranded DNA breaks

FIG 3 Endogenous CRISPR genome editing in clostridia. Shown is a graphical representation of CRISPR-Cas genomeediting in a clostridial vegetative cell. (1) The endogenous CRISPR region contains a cas operon which encodes aCas endonuclease that is subsequently generated. (2) Meanwhile, a plasmid contains a synthesized CRISPR arrayunder the control of an inducible promoter and a donor region for homologous recombination containing anupstream homology arm (HA) and a downstream HA. When induced, the plasmid transcribes the crRNAs to be usedby the endogenous system to generate individual crRNAs. (3) The endogenous Cas endonuclease complexes witha crRNA to target and cleave the DNA. This is then repaired by the donor region located on the CRISPR plasmid.

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within the genome; no endonucleases are encoded by the plasmid. Genome editingcontrol lies within the design of the CRISPR array containing the pre-crRNAs as well asthe homology arms to be used as donor regions for homologous recombination. TheCRISPR array would contain multiple pre-crRNAs, all under the control of a singlepromoter. Once transcribed, the endogenous system processes the pre-crRNAs intomature crRNAs that are then loaded onto the endogenous Cas endonucleases. Thisunique system potentially allows for multiple targets to be engineered using a singleplasmid system. This avoids the use of multiple different promoters for each gRNA (49,78, 91).

The development of a CRISPR-Cas gene editing tool using an endogenous systemrequires the presence of a known CRISPR system within that organism. Due to a largenumber of bacteria harboring CRISPR-Cas systems, there is great potential for thedevelopment of this tool in nonmodel organisms (24). For those organisms which havepreviously identified or well-studied endogenous CRISPR systems, the development forgenetic modification is streamlined. For those that do not have characterized systems,there is work to be done prior to developing the genetic tool. The type/class of CRISPRarray needs to be determined, and this will help determine how the system willfunction. Another vital part of developing an endogenous CRISPR system is identifyingthe PAM recognition sequence for the encoded endonuclease. Once the CRISPR arrayand PAM are identified, the endogenous CRISPR system could be exploited for genomeediting (49, 91). Unfortunately, different strains of an organism might harbor slightlydifferent components to their CRISPR systems and, therefore, may have differentrequirements for editing (e.g., PAM recognition). This could be seen as a drawback forexploiting the endogenous CRISPR locus, but the benefits could outweigh the longdevelopment time to establish other genetic systems. Because the majority of clostridiahave endogenous CRISPR systems, these could be exploited for use as genetic tools (49,92). In particular, those bacteria which have few to no genetic tools available are goodcandidates for using this type of genetic engineering, since it relies on existingcomponents of the genome, provided they can easily be transformed or have goodconjugation systems.

FUTURE DIRECTIONS AND CONCLUSIONS

In the past few years, the CRISPR-Cas systems have become a fast-growing andbeneficial contribution to the Clostridium field as genetic tools. These tools have thepotential to be applied to all Clostridium species (as well as most other bacteria). Whilesignificant strides have been made toward developing CRISPR-Cas systems, there is alot to be learned about the basic biology of CRISPR-Cas systems as well as the endogenoussystems within clostridia in order to develop genetic tools.

A main consideration in clostridia is ensuring tight regulation of Cas9, or any otherendonuclease, in order to overcome any potential toxic side effects. The use of a strongregulated promoter that drives the expression of each Cas9 and the sgRNA seems to bekey to having a successful CRISPR-Cas genetic tool. In particular, the small RNApromoter from C. beijerinckii has been successfully used in four other clostridia toregulate expression of the sgRNA (49–51, 55, 57, 59, 61, 62). It is possible that thispromoter could be efficient in other clostridia.

Application of the CRISPR-Cas9 system to other organisms may require someoptimization. As observed in C. saccharoperbutylacetonicum, the C. beijerinckii CRISPR-Cas9 system, developed by Wang et al., was directly applied to this organism and wassuccessful (50, 57). Unfortunately, there is probably not a “universal” CRISPR-Cas systemfor use in clostridia, because not all promoters or plasmid replicons will likely work inevery organism. The future of using this system in other clostridia would be to first startwith a preexisting system of a closely related Clostridium species and then modify thissystem as needed. Should this strategy not prove fruitful, the exploitation of theendogenous CRISPR-Cas system from the organism of interest is a viable option. Withthe successful application in C. pasteurianum and C. tyrobutyricum, these systems workwell and with high efficiency (49, 91).

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Another aspect of the system which will need to be worked on is understandingwhat makes a quality sgRNA, i.e., what makes one sgRNA result in a higher mutationefficiency than another sgRNA. The “rules” for designing sgRNAs developed for eu-karyotes, or even other prokaryotes, may not be applicable to clostridia. Currently,many studies have designed multiple sgRNAs with different target sequences in orderto see which will result in a mutant at a high efficiency. Elucidating a set of rules forselecting target sequences will aid in more rapid gene editing.

It was previously demonstrated that a system can be made where dCas9 can becoupled to an activator or repressor to regulate the transcription of a specific gene (Fig.2C) (67, 93). While it has been demonstrated that CRISPRi works effectively in someclostridial species, it would be interesting to apply a system which uses a transcriptionalactivator coupled to dCas9 to enhance the transcription of specific genes, for example,for the fine-tuning of biofuel production and increasing the use of clostridia to generatevaluable end products.

Overall, several labs have laid the groundwork for developing CRISPR-Cas systems asgenetic tools (i.e., Cas9, Cas9n, dCas9, Cpf1/Cas12a, and endogenous systems) inclostridia. The wide range of mutations and regulatory control that can be made withthese systems has already proved beneficial, and the future use of these technologiesis promising.

ACKNOWLEDGMENTSWe thank members of the Sorg lab at Texas A&M University for their helpful

comments and suggestions during the preparation of the manuscript.This project was supported by awards 5R01AI116895 and 1U01AI124290 to J.A.S.

from the National Institute of Allergy and Infectious Disease.The content is solely the responsibility of the authors and does not necessarily

represent the official views of the National Institute of Allergy and Infectious Diseasesor the National Institutes of Health.

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