...S., Mojica, F. J. M., Terns, R. M., Terns, M. P., White, M. F., Yakunin, A. F., Garrett, R. A., Van Der... available with multiple Cas9 variants, including high fidelity Cas9s, Cas9s with various PAM requirements...H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., & Nureki...e1007749. PMID: 30403660 Sanson, K. R., Hanna, R. E., Hegde, M., Donovan, K. F., Strand, C., Sullender, M. E...strand SpCas9-HF1 - disrupt Cas9’s interactions with DNA phosphate backbone HypaCas9 - increase Cas9 proofreading...new tools. Figure 3: Overview of Cas9n Nickase and dead Cas9 The two Cas9 nuclease domains, RuvC and HNH...enhanced specificity by engineering Cas9 into high fidelity Cas9s (hfCas9) . There is currently no defined...

...Behan FM, Iorio F, Picco G, Gonçalves E, Beaver CM, Migliardi G, Santos R, Rao Y, Sassi F, Pinnelli M, Ansari... Ansari R, Harper S, Jackson DA, McRae R, Pooley R, Wilkinson P, van der Meer D, Dow D, Buser-Doepner ...CRISPR/Cas9 is an enormously plastic tool and has taken the scientific world by storm. While Cas9 has been...required for Cas9-based activation. CRISPRa for genome-wide studies One of the best uses for Cas9 activators...context of the gene, Cas9 activators efficiently solve these problems.   Which Cas9 activator should I ...Best Cas9 Variant for Your Next Experiment Find out More about Genome-wide Screens with CRISPR/Cas9 Learn...with AAV Resources on Addgene.org Find Cas9 Activators Find Cas9 Repressors Catch Up on Your CRISPR Background...

... SI, Dohmae N, Ishitani R, Zhang F, Nureki O (2014) Crystal Structure of Cas9 in Complex with Guide RNA...one AAV, Cas9 must be made smaller. Previous attempts to “shrink” Cas9 include the use of St1Cas9 (~3.3 ...amino acids in length, Cas9 from Campylobacter jejuni (CjCas9) is the smallest Cas9 ortholog characterized...vehicle for Cas9. However, to allow for more room for regulatory sequences while still fitting Cas9 and its...equally potent Cas9 protein, the Zhang lab took a different approach. They analyzed over 600 Cas9 orthologs...EV, Sharp PA, Zhang F (2015) In vivo genome editing using Staphylococcus aureus Cas9. Nature 520:186–191... Zhang F (2014) In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol...

...S., Mojica, F. J. M., Terns, R. M., Terns, M. P., White, M. F., Yakunin, A. F., Garrett, R. A., Van Der..., Demircioglu, F. E., Moeller, L., Kocalar, S., Oshiro, R., Makarova, K. S., Macrae, R. K., Koonin, E....Regev, A., Lander, E. S., Voytas, D. F., Ting, A. Y., & Zhang, F. (2017). RNA targeting with CRISPR–Cas13...10.1093/nar/gku241 Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X.,..., Alkhnbashi, O. S., Costa, F., Shah, S. A., Saunders, S. J., Barrangou, R., Brouns, S. J. J., Charpentier...Mojica, F. J. M., Scott, D., Shah, S. A., Siksnys, V., Terns, M. P., Venclovas, Č., White, M. F., Yakunin...-021-03886-5 Pinilla-Redondo, R., Mayo-Muñoz, D., Russel, J., Garrett, R. A., Randau, L., Sørensen, S....

...Jump to: SpCas9 Plasmids GeCKO SAM AAV - In vivo Delivery AAV - Cas9 Mouse CRISPR ( C lustered R egularly...Return to top Cas9 SAM transcriptional activation plasmids and screening library CRISPR/Cas9 Synergistic...of cells and nuclei. Return to top AAV - Cas9 mouse CRISPR-Cas9 is a versatile genome editing technology...constitutively expressing Cas9 knockin mice (Platt et al ., Cell 2014). In these mice the CRISPR-Cas9 system can be...constitutively Cas9-expressing mouse. Described here are AAV vectors that can be combined with Cas9 in a wide...are below. The Cas9 knockin mouse can be purchased from Jackson Labs: Cre-dependent Cas9 mouse Constitutively...cloning information: CRISPR-Cas9 mouse toolbox protocol 637.8 KB CRISPR-Cas9 Cre expression vectors for...

...S., Mojica, F. J. M., Terns, R. M., Terns, M. P., White, M. F., Yakunin, A. F., Garrett, R. A., Van Der...deletions. Many of these methods rely on Cas9 or a derivative of Cas9, but the ever-expanding repertoire of...bound to the DNA. Cas3 or Cas9? Large genomic deletions can be achieved using Cas9, but Cas3 may be a better..., Alkhnbashi, O. S., Costa, F., Shah, S. A., Saunders, S. J., Barrangou, R., Brouns, S. J. J., Charpentier...considered weak, especially compared to the power of Cas9. However, by continually passing the ssDNA past ...better option. To get large deletions using Cas9, two or more gRNAs must be used along the same chromosome... with expressing all Cascade components can make Cas9 easier to work with than Cas3. Future targets Cas3...

...High fidelity Cas9s: Three high fidelity Cas9 variants , SpCas9-HF, eSpCas9, and HypaCas9 display very ...III and Cas9 enzymes, which together separate the individual crRNAs. (3) The tracrRNA and Cas9 nuclease...EV, Sharp PA, Zhang F. 2015. In vivo genome editing using Staphylococcus aureus Cas9. Nature . 520(7546...Ebert BL, Root DE, Doench JG, Zhang F. 2014. Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells....Zetsche B, Scott DA, Yan WX, Zhang F. 2016. Rationally engineered Cas9 nucleases with improved specificity...Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. 2013. Multiplex genome...KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJ, Charpentier E, Haft ...

... Zhang Y, Yin C, Zhang T, Li F, Yang W, Kaminski R, Fagan PR, Putatunda R, Young WB, Khalili K, Hu W. ...or apoptosis with Cas9/gRNA expression. A pooled analysis found no evidence of Cas9 cleavage at predicted...were located near the Cas9 cut site, leading Wang et al. to conclude that some Cas9-derived indels may not...Pan Q, Gendron P, Zhu W, Guo F, Cen S, Wainberg MA, Liang C. CRISPR/Cas9-Derived Mutations Both Inhibit...Michailidis E, Bhatta A, Scott DA, Zhang F, Rice CM, Bhatia SN. CRISPR/Cas9 cleavage of viral DNA efficiently...CRISPR/Cas9 escape occurs in an animal model. If these escape mutations are frequent, the dCas9-mediated...destroy infected cells, the other using wild-type Cas9 to remove the HIV-1 genome from infected cells. Read ...

...prime editor consists of a Cas9 nickase fused to a reverse transcriptase. The Cas9 nickase is guided to the... that most Cas9-induced DSBs are repaired by non-homologous end joining. As a result, Cas9 treatment causes... ~ ~ ✓ ✓ ✓ R221K N394K Cas9 mutations Improve Cas9 nuclease activity ~ ~ ~ ~ ...Hussmann, J. A., Yan, J., Knipping, F., Ravisankar, P., Chen, P.-F., Chen, C., Nelson, J. W., Newby, G...Suiter, C. C., Lee, C., Chardon, F. M., Yang, W., Leith, A., Daza, R. M., Martin, B., & Shendure, J. (... M., Davis, J. R., Randolph, P. B., McElroy, A., Gao, X. D., Raguram, A., Richter, M. F., Everette, K....Schmidt, R., Solley, S. C., Gilbert, L. A., Chan, M. M., Bauer, D. E., Marson, A., Parsons, L. R., & Adamson...

...H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., & Nureki...Hussmann, J. A., Yan, J., Knipping, F., Ravisankar, P., Chen, P.-F., Chen, C., Nelson, J. W., Newby, G...Schmidt, R., Solley, S. C., Gilbert, L. A., Chan, M. M., Bauer, D. E., Marson, A., Parsons, L. R., & Adamson...editing technique that relies on a fusion enzyme of Cas9 nickase + reverse transcriptase and a prime editing...., 2019), disrupting its canonical structure and Cas9 binding (Nishimasu et al., 2014). Figure...pegRNAs that use other bases in this location bind to Cas9 with canonical sgRNA structure (right). Created ...along with your intended edit. This prevents the Cas9 nickase from re-binding and nicking the newly synthesized...

...codon-optimized Cas9 constructs 44249 pdCas9-bacteria A catalytically inactive bacterial Cas9 expression plasmid...system, CRISPR ( C lustered R egularly I nterspaced S hort P alindromic R epeats) pathway, as an RNA-guided...machineries, requiring only a nuclease-deficient Cas9 (dCas9) protein and a customized s ingle g uide RNA ...Description 44246 pdCas9-humanized A catalytically inactive, human codon-optimized Cas9 expression plasmid...plasmid 44247 pdCas9::BFP-humanized A catalytically inactive, human codon-optimized Cas9-BFP fusion expression... plasmid 44250 pwtCas9-bacteria A wild-type Cas9 expression plasmid for use editing bacterial genomes ...plasmid for use with the bacterial Cas9 constructs 46910 pHR-SFFV-dCas9-BFP Human expression vector containing...

...Photoswitchable Cas9 Richter et al., 2016 tsRC9 RsLOV from R. sphaeroides Cas9, dCas9 Gene editing ...proteins from N. crassa Split Cas9, Split Cas9 nickase, Split dCas9 Genome editing by NHEJ and HDR, repression...heterodimerizing domain to each of the Cas9 fragments, the authors created a photoactive Cas9 tool, as shown in the figure...modular nature of Cas9, Nihongaki et al. showed that it was possible to swap out the Cas9 domains in their...light-activated split-Cas9 (FAST) system constitutively expresses the C-terminal fragment of Cas9 while transcription...p65 and VP64 which in turn transcribes N-Cas9. The N- and C-Cas9 fragments then dimerize to form a functional...Off Genome Editing Single-chain Cas9 photoswitches Photoactivation of Cas9 can also be achieved using single-chain...

..., Perrimon N, Weiss R, Aach J, Collins JJ, Church GM (2015) Highly efficient Cas9-mediated transcriptional...Nureki O, Zhang F (2014) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature... Guilak F, Crawford GE, Reddy TE, Gersbach CA (2013) RNA-guided gene activation by CRISPR-Cas9–based transcription...Perrimon N, Collins JJ, Church G (2016) Comparison of Cas9 activators in multiple species. Nat Methods 13:563...References Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA (2013) Programmable repression and ...Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, P R Iyer E, Lin S, Kiani S, Guzman CD, Wiegand DJ, Ter-Ovanesyan...Chavez A, Tuttle M, Pruitt BW, Ewen-Campen B, Chari R, Ter-Ovanesyan D, Haque SJ, Cecchi RJ, Kowal EJK, ...

...alternative Cas9, from Streptococcus aureus, is as effective at editing mouse embryos as Cas9 from S. pyogenes...A, Suchy F, Yamaguchi T, Nakauchi H (2018) Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated ...Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex Genome..., Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-Step Generation of Mice Carrying ...guest bloggers, Wenning Qin and Haoyi Wang. CRISPR/Cas9 is revolutionizing the mouse gene-targeting field...ever to manipulate the mouse genome using CRISPR/Cas9. Similar to the human genome, the mouse genome ...robust in performance. In its natural setting, CRISPR-Cas9 is an acquired immune system in bacteria and archaea...

...consisting of a catalytically inactive form of Cas9 (dCas9) plus guide RNA (gRNA) for biochemical purification...CRISPR Cas9 gRNA plasmids from Dr. Hodaka Fujii's lab. Genome...(gRNA) to target human IRF-1 promoter for the Sa-Cas9 system. 105283 hIRF-1 gRNA #351 (Sa) Expresses a...(gRNA) to target human IRF-1 promoter for the Sa-Cas9 system. References An enChIP system for the analysis...lines expressing the 3xFLAG-dCas9 protein for enChIP analysis. Fujita T, Kitaura F, Oji A, Tanigawa N, Yuno...Persson CU, Eriksson P, Veerla S, De Preter K, Speleman F, Fujii H, Påhlman S, Mohlin S. Biochem Biophys Res...promoter in a chicken B cell line. Fujita T, Kitaura F, Yuno M, Suzuki Y, Sugano S, Fujii H. DNA Res. 2017...

...H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., & Nureki...the basic parts of the Cas9 enzyme.   Figure 1: A cartoon depiction of Cas9’s two major lobes, REC...   Unbound Cas9 Now that we know what we’re looking at, let’s peek at unbound—“apo”—Cas9 from Streptococcus...Figure 8: Comparison of Cas9:RNA crystal structure (left, PDB ID 4ZT0) with Cas9:RNA:DNA electron microscopy...10.1038/s41594-022-00756-0 Jiang, F., & Doudna, J. A. (2017). CRISPR-Cas9 Structures and Mechanisms. Annual..., D. R. (2013). High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease...Components of CRISPR  xCas9: Engineering a CRISPR Variant with PAM Flexibility Cas9 vs the Other Cases Further...

...overcome some of Cas9’s shortcomings. Cpf1’s preferred PAM is 5’-TTN, differing from that of Cas9 (3’-NGG) in...KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJ, Charpentier E, Haft ...than that of the widely used SpCas9 and comparable to SaCas9. As seen with Cas9 orthologs, Cpf1 efficiency...rate is similar to that of high fidelity Cas9s eSpCas9 and SpCas9-HF1. Both AsCpf1 and LbCpf1 ribonucleoproteins...two Cpf1 nucleases, distant cousins of well-known Cas9. Cpf1 cleaves DNA in a staggered pattern and requires...rather than the two (tracrRNA and crRNA) needed by Cas9 for cleavage. Now, two new studies show that Cpf1...Cpf1 displays lower off-target editing than Cas9, confirming that this protein is well suited for genome...

...as a binding signal for Cas9 and this sequence is a strict requirement for Cas9-mediated DNA cleavage. ...of novel S. pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9 homologs derived from...2020). Characterization of Cas9 from additional bacterial species Many more Cas9 orthologs have been isolated...EV, Sharp PA, Zhang F (2015) In vivo genome editing using Staphylococcus aureus Cas9. Nature 520:186–191...them more useful than SpCas9. For example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase... in length, Cas9 from Campylobacter jejuni (CjCas9) is even smaller than SaCas9 and is also compatible...VQR, EQR and VRER SpCas9 variants effectively double the targeting range of CRISPR/Cas9 within the human...

...Beijersbergen, R. L., Medema, R. H., & van Steensel, B. (2021). Impact of chromatin context on Cas9-induced ...J., Chardon, F. M., McDiarmid, T. A., Daza, R. M., Kim, H., Lalanne, J.-B., Nathans, J. F., Lee, D. S....024-02268-2 Schep, R., Brinkman, E. K., Leemans, C., Vergara, X., van der Weide, R. H., Morris, B., van...2023). This method uses a prime editor (typically a Cas9 H840A nickase fused to a reverse transcriptase) ...Meuleman, W., De Ridder, J., Berns, A., Wessels, L. F. A., Van Lohuizen, M., & Van Steensel, B. (2013). ....2013.07.018 Anzalone, A. V., Randolph, P. B., Davis, J. R., Sousa, A. A., Koblan, L. W., Levy, J. M., Chen, ... Wilson, C., Newby, G. A., Raguram, A., & Liu, D. R. (2019). Search-and-replace genome editing without...

...Wang Q, Xu J, Dong F, Yang S, Yang J, Zhang Z, Qian Y, Chen J, Zhang J, Liu Y, Tao R, Jiang Y, Yang J, ...effectors employed by Type II systems (such as CRISPR-Cas9). Unlike in regular Type I CRISPR-Cas systems, however...bacteria, alongside more established tools such as Cas9-recombineering, CRISPRa/i, and base editing. Leveraging...INTEGRATE - hereafter VchINT) and encodes a Type I-F CRISPR-Cas system. The four major components required...interest flanked by ~50-150 bp transposon end sequences (R and L ends).  Figure 1: (A) Components ...) Mechanism of RNA-guided transposition in type I-F INTEGRATE. The requirement for multiple Cas proteins...orientations, but is strongly biased towards T-RL, where the R end is proximal to the target site (Klompe et al.,...