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  • Biasing genome-editing events toward precise lengthdeletions with an RNA-guided TevCas9 dual nucleaseJason M. Wolfsa, Thomas A. Hamiltona, Jeremy T. Lanta, Marcon Laforeta, Jenny Zhanga, Louisa M. Salemia,b,Gregory B. Gloora, Caroline Schild-Poultera,b, and David R. Edgella,1

    aDepartment of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada; and bRobarts ResearchInstitute, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5B7, Canada

    Edited by Marlene Belfort, University at Albany, Albany, NY, and approved November 15, 2016 (received for review October 3, 2016)

    The CRISPR/Cas9 nuclease is commonly used to make gene knock-outs. The blunt DNA ends generated by cleavage can be efficientlyligated by the classical nonhomologous end-joining repair pathway(c-NHEJ), regenerating the target site. This repair creates a cycle ofcleavage, ligation, and target site regeneration that persists untilsufficient modification of the DNA break by alternative NHEJprevents further Cas9 cutting, generating a heterogeneous popula-tion of insertions and deletions typical of gene knockouts. Here, wedevelop a strategy to escape this cycle and bias events towarddefined length deletions by creating an RNA-guided dual active sitenuclease that generates two noncompatible DNA breaks at a targetsite, effectively deleting the majority of the target site such that itcannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevInuclease domain to Cas9, functions robustly in HEK293 cells and gen-erates 33- to 36-bp deletions at frequencies up to 40%. Deepsequencing revealed minimal processing of TevCas9 products,consistent with protection of the DNA ends from exonucleolyticdegradation and repair by the c-NHEJ pathway. Directed evolutionexperiments identified I-TevI variants with broadened targetingrange, making TevCas9 an easy-to-use reagent. Our results highlighthow the sequence-tolerant cleavage properties of the I-TevI homingendonuclease can be harnessed to enhance Cas9 applications, circum-venting the cleavage and ligation cycle and biasing genome-editingevents toward defined length deletions.

    CRISPR/Cas9 | genome editing | NHEJ | I-TevI homing endonuclease

    Genome editing with engineered nucleases has revolutionizedthe targeted manipulation of the genomes of organisms rang-ing from bacteria to mammals (1). Zinc finger nucleases (ZFNs)(2), transcription-like effector nucleases (TALENs) (3), MegaTALs(fusion of a LAGLIDADG homing endonuclease and TALEdomain) (46), and nucleases based on the CRISPR-associatedprotein 9 (Cas9) all represent programmable genome-editing nu-cleases that have successfully been used to introduce targetedchanges in genomes (711). One of the most common applicationsof genome-editing nucleases is gene knockouts that are performedin the absence of an exogenously added repair template (12). In thecase of Cas9, the blunt DNA ends introduced at DNA cleavage aresubstrates for error-free repair by the classical nonhomologous end-joining repair (c-NHEJ) pathway (13), regenerating the target sitefor recleavage by the nuclease. This cycle of cleavage, ligation, andtarget site regeneration is perturbed when the double-strand break(DSB) is sufficiently modified by exonucleolytic processing byc-NHEJ, or by the alternative NHEJ pathway (alt-NHEJ), to pre-vent cleavage by the nuclease (1418). Imprecise repair by either ofthe NHEJ pathways generates the characteristic spectrum of het-erogeneous length insertions or deletions (indels) centered aroundthe break site (19, 20). The heterogeneous distribution of indels,and the fact that not all indels generate gene knockouts, means thatdownstream screening and confirmation of knockout genotypes isrequired. In addition, the chromatin context of the target site (21,22), the cell cycle stage and cell type (23), and the nature of theDNA overhangs generated by genome-editing nucleases influencethe types of indels and efficiency of gene knockouts (24). Coupling

    the expression of DNA end processing enzymes and genome-editing nucleases can bias gene knockouts by enhancing exonu-cleolytic end processing before ligation by NHEJ, with variable ratesof success depending on the end-processing enzyme used (24, 25).Although more commonly used for homology-directed repair ap-plications (26), paired Cas9 nickase variants can be used to generategene knockouts but also generate heterogeneous length deletions.Here, we provide a simple and robust solution to bypass the

    persistent cycle of cleavage and religation and shift genome-editingevents toward deletions of defined length. We created a dual nu-clease that introduces two noncompatible DNA breaks at a targetsite such that the majority of the target site is deleted, preventingregeneration of the target site and continued cleavage. We fusedthe monomeric nuclease and linker domains from the I-TevIhoming endonuclease to Cas9 (27, 28), creating an RNA-guidedTevCas9 dual nuclease that functions robustly in HEK293 cells atendogenous target sites. TevCas9 generates defined length dele-tions of 3336 bp at high frequencies with minimal DNA endprocessing compared with Cas9. We envision that the non-compatible and directional nature of the TevCas9 overhangs willenhance applications such as site-directed mutagenesis by oligo-nucleotide ligation, deletion of binding sites, or epitope tagging.

    ResultsFusion of I-TevI to Cas9 Creates an RNA-Guided Dual Nuclease.To create aTevCas9 RNA-guided dual nuclease, we fused the I-TevI nucleasedomain (residues 192) and a portion of the linker domain (residues92169) to the N terminus of SpCas9 nuclease derived from theStreptococcus pyogenes type II CRISPR system (27, 28) (Fig. 1A). ATevCas9 target site includes the I-TevI 5-CNNNG cleavage motif,

    Significance

    Most genome-editing nucleases introduce a double-strand breakat a target site, leaving compatible DNA ends that are repaired bythe nonhomologous end joining repair pathway to regenerate thetarget site. This leads to a cycle of cleavage, target site re-generation by ligation, and subsequent recleavage by the nucle-ase. Here, we bypass this cycle by developing and testing anRNA-guided dual active site nuclease that introduces two non-compatible DNA breaks at a target site, deleting the majority ofthe target site so that it cannot be regenerated. The dual nuclease(TevCas9) robustly functions in HEK293 and biases genome-editingoutcomes toward defined length deletions at high frequencies.Potential applications include directional oligonucleotide ligation,deletion of DNA-binding sites, and in-frame protein deletions.

    Author contributions: J.M.W. and D.R.E. designed research; J.M.W., T.A.H., J.T.L., M.L., J.Z., andL.M.S. performed research; J.M.W. contributed new reagents/analytic tools; J.M.W., T.A.H., J.T.L.,G.B.G., C.S.-P., and D.R.E. analyzed data; and G.B.G., C.S.-P., and D.R.E. wrote the paper.

    The authors declare no conflict of interest.

    This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: dedgell@uwo.ca.

    This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1616343114/-/DCSupplemental.

    1498814993 | PNAS | December 27, 2016 | vol. 113 | no. 52 www.pnas.org/cgi/doi/10.1073/pnas.1616343114

    http://crossmark.crossref.org/dialog/?doi=10.1073/pnas.1616343114&domain=pdfmailto:dedgell@uwo.cahttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1616343114/-/DCSupplementalhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1616343114/-/DCSupplementalwww.pnas.org/cgi/doi/10.1073/pnas.1616343114

  • a DNA spacer that interacts with the I-TevI linker and that sepa-rates the cleavage motif from the binding site, the DNA-bindingsite complementary to the gRNA, and the downstream NGGprotospacer-associated motif (PAM) sequence. Previous studiesrevealed that the length of the DNA spacer is crucial for I-TevIcleavage function, with lengths of 1419 bp supporting activity invarious chimeric contexts (27, 29, 30). We coexpressed TevCas9with a C-terminal histidine tag and a gRNA targeting a site in theretinoic acid receptor alpha gene (RARA.233, numbered accord-ing to start of target site in the RARA cDNA) from the sameplasmid in Escherichia coli. The RARA.233 target site (Dataset S1)was predicted according to a binding model that used data fromin vitro profiling of I-TevI linkerDNA spacer nucleotide prefer-ence (SI Appendix, Figs. S1S3) and the activity of the I-TevI nu-clease domain on all possible 64 CNNNG variants (30). TheTevCas9 variant (TC-V) used in this experiment contains an ac-tivity-enhancing V117F substitution in the I-TevI linker.We purified TC-V by metal-affinity chromatography, and

    showed that TC-V was complexed with an RNA species of the sizepredicted for the transcribed gRNA (Fig. 1B, *). To determinewhether the TC-V/gRNA ribonucleoprotein (RNP) complex in-troduced two DSBs, we examined the cleavage profile on a PCRsubstrate that contained the RARA.233 target site. Cleavage wasconsistent with two sequential DSBs, first by the Cas9 nuclease andsecond by the I-TevI nuclease (Fig. 1 C and D). The predicted33-bp product corresponding to the fragment excised between theI-TevI and Cas9 cleavage sites was difficult to reproducibly visu-alize and not used to quantitate reaction progress. Interestingly,cleavage by Cas9 was 30-fold faster than cleavage by I-TevI(Fig. 1E, as judged by time required for 50% product formation).

    TevCas9 Functions Robustly in HEK293 Cells. TevCas9 is targeted bythe Cas9-associated gRNA, a property that allowed us to directlycompare TevCas9 and Cas9 activity at the same sites in HEK293cells. To do so, we used T7 endonuclease 1 (T7E1) mismatchcleavage assays to measure on-target modification at the RARA.232site and at two other sites, one in the tuberous sclerosis 1 gene(TSC1.21