..., 2021) NG Cas9 NG - SpG Cas9  NG  - SpRY nuclease  C to T Base Editor SpRY A to G Base Editor...PAM-interacting domain of Cas9. After multiple rounds of engineering, they developed the SpRY Cas9 variant (Walton...several Cas9s that recognize NG PAMs. The Nureki lab generated SpCas9-NG, a rationally engineered Cas9 with... 2020). SpRY uses NRN PAMs and is capable of editing at frequencies comparable to SpCas9. SpRY can also...    Fig. 1: SpCas9 has sparse PAM coverage in the human genome. SpRY Cas9 has PAM sites covering... loci will have a PAM located next to it. The SpRY Cas9 can also work with NYN PAMs; it just isn’t always...notes SpCas9 NGG - Cas12a TTTV Great for AT-rich regions Cas9 EQR NGAG - xCas9 NG Less...

...using a Cas9 variant with higher specificity (eSpCas9 and HypaCas9) instead.  Choosing a Cas9 for site-specific... CRISPR/Cas9 has made it easier than ever to make precise, targeted genome modifications. Cas9 has been...introducing Cas9-mediated frameshift mutations in the gene of interest. The genomic target for Cas9 and the... system. This system uses a “nicking” Cas9, in which one of Cas9’s two critical enzymatic residues has...Further, this group also characterized a second Cas9 variant, SpRY, which cuts at NRN PAMs with high efficiency...can be used with a variety of impaired Cas9 proteins (such as SpRY) with altered PAM requirements, giving...targeted to the promoter region. Cas9 activators include Cas9 fused to VP64, VP64 along with other SAM factors...

...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...development of near-PAMless Cas9 variants. These variants, named SpG and SpRY, can target NGN PAMs and NRN...2020). Characterization of Cas9 from additional bacterial species Many more Cas9 orthologs have been isolated...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...

...Walton et al., 2020 SpRY Cas9, available fused to ABEmax(7.10) or to BE4max   Evolved SpCas9 with NRN or NYN..., a base editor containing high fidelity Cas9 variant HF-Cas9 (Rees et al., 2017). HF-BE3 showed 37-fold...other methods. In a head-to-head comparison with Cas9, Cas9 modified 9/12 known off-targets with a 14% indel...individual Cas9. They also mutagenized the cytidine deaminase portion of the base editor to create SpCas9 base... 1.1%. These numbers are a vast improvement over Cas9-mediated HDR for the loci tested, where average ...system is also subject to off-target editing based on Cas9 off-target activity.  Find CRISPR base editing plasmids...using a cytidine deaminase from sea lamprey fused to Cas9 nickase (Nishida et al., 2016). Target-AID acts ...

... C or T SpRYc - SpRY PAMs plus NNG; a chimeric version of SpRY Though these PAM-flexible Cas9s are useful... available with multiple Cas9 variants, including high fidelity Cas9s, Cas9s with various PAM requirements...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...application. Cas9 Orthologs While SpCas9 is the most commonly used Cas enzyme, additional Cas9 orthologs ...constructs with Cas9 fused to proteins that modulate HDR or NHEJ components, like miCas9 , Cas9-DN1S , or others...

... C or T SpRYc - SpRY PAMs plus NNG; a chimeric version of SpRY Though these PAM-flexible Cas9s are useful... available with multiple Cas9 variants, including high fidelity Cas9s, Cas9s with various PAM requirements...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...application. Cas9 Orthologs While SpCas9 is the most commonly used Cas enzyme, additional Cas9 orthologs ...constructs with Cas9 fused to proteins that modulate HDR or NHEJ components, like miCas9 , Cas9-DN1S , or others...

... plasmid contains Cas9 and if so, which function of Cas9. We have plasmids with Cas9 that can cut, activate...Expression System Cloning Enzyme Co-expressed Cas9 Cas9 System Selection PI pCRISPR 42875 Bacteria BsaI...BbsI)_CBh-Cas9-T2A-BFP 64323 Mammalian BbsI yes, cut S. pyogenes BFP Kuhn pU6-(BbsI)_CBh-Cas9-T2A-mCherry...meningitidis Joung Cas9 sgRNA vector 68463 Mammalian U6 none S. pyogenes Zhang Cas9/pTREX-n 68708 Other...independent U6 promoters and Cas9 from CBh promoter. Ventura A CRISPR/Cas9 toolkit for multiplex genome...cassettes are assembled into a Cas9 expressing vector. Yang A Robust CRISPR/Cas9 System for Convenient, High-Efficiency...accessory pack is available for dCas9 and FokI-dCas9. Yamamoto Multiplex CRISPR/Cas9-based genome engineering...