...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....

...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...

... 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...

...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...

...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.,...

...V, Najgebauer H, Allen F, Gonçalves E, Shepherd R, Doench JG, Yusa K, Vazquez F, Parts L, Boehm JS, Golub..., Tsherniak A, Iorio F (2019) Agreement between two large pan-cancer CRISPR-Cas9 gene dependency data ... capacity of a locus. When using S. pyogenes Cas9 (SpCas9), potential target sites are both [5’-20nt-NGG... (inhibition) technologies – a nuclease-dead Cas9 (dCas9) is directed near the promoter of a target gene...with CRISPR technology is usually accomplished by Cas9-mediated dsDNA breaks: following a cut, the error-prone...even more exquisite for the so-called base editor Cas9, which makes DNA changes in the absence of dsDNA...Incidence of Off-Target Mutations in Individual CRISPR-Cas9 and TALEN Targeted Human Stem Cell Clones Detected...

..., Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Cell. 2014 Feb 27;156(5):935-49. doi...Silvana Konermann, the first generation of CRISPR-Cas9 plasmids were not designed with gene activation ...question for her then was this: Can you engineer CRISPR-Cas9 activators that work well enough on any gene that...describe just such a system, which they call CRISPR/Cas9 Synergistic Activation Mediator or SAM. SAM is an... three components: 1. A nucleolytically inactive Cas9-VP64 fusion; 2. A sgRNA incorporating two MS2 RNA...system, Konermann explained, was new knowledge about Cas9’s crystal structure. “I thought maybe we could use...levels of control in the system. First of all, the Cas9s can be turned on and off, to reversibly activate...

...CRISPR-Cas9. Cell 167:1829-1838.e9 . https://doi.org/10.1016/j.cell.2016.11.017 Shin J, Jiang F, Liu J-J...et al., 2016). While Streptococcus pyogenes Cas9 (SpyCas9) is the most used and well studied CRISPR system... study, the labs found that anti-CRISPR prevents Cas9 activity in HEK293 by reducing editing from ~30%...interact with CRISPR-Cas systems such as Cascade-Cas3, Cas9, Cas12, and Cas13 (Marino et al., 2020). Some Acr...ways: Inhibit DNA binding Example:  AcrIIA4 blocks Cas9’s interaction with the PAM site (Dong et al., 2017... crRNA loadingExample: AcrIIC2 interactions with Cas9 interferes and prevents the correct assembly of ... AcrIIC1 binds to the HNH endonuclease domain of Cas9 and prevents target DNA cleavage (Harrington et ...

...0022-1317-73-3-653. PMID 1372038. Randall, R. E., Young, D. F., Goswami, K. K., & Russell, W. C. (1987).... M., Wang, P., Li, H., Hall, R. N., Wang, B., Prakash, M., & Dunn, A. R. (2024). Split Luciferase Molecular...Overview of the dual-plasmid CRISPR/Cas9 workflow in M. abscessus. A) Sth1Cas9 mediates double strand break ...mScarlet-based tags for live imaging in C. elegans CRISPR/Cas9 for gene disruption in M. abscessus  Bioluminescent...Mycobacterium abscessus using a dual-plasmid CRISPR/Cas9 system By Aryana Khosravani Deborah Hung’s lab has... has developed a novel dual-plasmid CRISPR/Cas9 system for gene disruption in Mycobacterium abscessus,...resistance interfering with selection. Using an inducible Cas9 expression and removing the sgRNA plasmid after ...

...doi.org/10.3389/fgeed.2020.617910 Zou, R., Liu, Y., Wu, B., et al. Cas9 deactivation with photocleavable gRNAs...surprise then that stability of the RNA used in CRISPR-Cas9 experiments – the “gRNA” – suffers from the same...still effectively serving their function of guiding Cas9 to generate a targeted break. These advances in ...modifications The recognition sequence for CRISPR-Cas9 targeting is led by a ~20 bp gRNA which is part ...trans-activating crRNA (tracrRNA) which interacts with the Cas9 protein. These RNA molecules are vulnerable to attack...partially DNA gRNA is surprisingly well-tolerated by the Cas9 protein and can increase on-target efficiency. In...modifications listed above were developed before CRISPR-Cas9, for use in applications like siRNA and antisense...