...the Cas9 nuclease with engineered variants: eSpCas9, SpCas9-HF1, & HypaCas9. The off-target problem It...Further testing of two of the mutants, SpCas9(K855A) and eSpCas9(1.1), followed by a technique for detecting...different than those found in eSpCas9, the effect on specificity was similar; SpCas9-HF1 generated fewer off-target... nuclease specificity. They found that eSpCas9(1.1) and SpCas9-HF1 each stall in an inactive form after...across the sites tested when compared to eSpCas9(1.1) and SpCas9-HF1. The HypaCas9 plasmids are available... proteins With their enhanced specificity, eSpCas9, SpCas9-HF1, and HypaCas9 should enable researchers...the Cas9 nuclease itself. Design and testing of eSpCas9 Looking at the structure of the Cas9 nuclease (...

...pTargetF into the BL21(DE3)/pCas strain. In response, we modified the pCas/pTargetF system and updated...these CRISPR systems. The original pCas/pTargetF system Our original pCas/pTargetF system consists of one.... In pCas9, we 1) replaced the promoter of gRNA-pMB1 with PrhaB, 2) changed the replicon of pCas into ...the use of CRISPR editing systems in bacteria. Our pCas/pTargetF system has been one of the most popular...pEcCas/pEcgRNA system We speculated that the gRNA on pCas that is specific to the pMB1 replicon of pTargetF...transformants. Our speculation motivated us to update the pCas/pTargetF system.   Figure 1: The editing ...targeting the genomic site of interest. We compared the pCas/pTargetF system with the updated pEcCas/pEcgRNA ...

...identified four novel SpCas9 variants with altered PAM binding specificity: SpCas9 variant Mutations...wild-type SpCas9, indicating that the variants are likely just as selective as wild-type SpCas9. These VQR... Non-SpCas9's bind a variety of PAM sequences, which makes them useful when no suitable SpCas9 PAM sequence...Furthermore, non-SpCas9's may have other characteristics that make them more useful than SpCas9. For example...-type SpCas9, even when targeting sites that are known to produce off-target editing with SpCas9 (Amrani...Mutations (relative to SpCas9) PAM sequence D1135E variant D1135E NGG VQR variant D1135V, R1335Q...pyogenes PAM sequence NGG compared to wild-type SpCas9, which also displays some cleavage with an NGA ...

...iterative genome engineering (Jiang et al., 2015). pCas contains Cas9 and the phage recombination gene lambda... of editing takes two days, and the pTargetF and pCas plasmids can be subsequently cured from the bacteria...pEcCas/pEcgRNA system works similar to the original pCas/pTargetF but has many other advantages including...Chen J, Zhang Y, Jiang Y, Yang S (2021) A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome ...available from Addgene consists of two plasmids: pCas9: carries Cas9 and chloramphenicol resistance pCRISPR...recombineering machinery are first electroporated with pCas9. Then, pCRISPR is introduced along with an oligonucleotide...tuberculosis CRISPRi plasmids Noting that the more common spCas9 system has generally low efficiencies or can be...

...Split-BioID combines a protein fragment complementation (PCA) technique with proximity labelling to validate binary...additional interacting proteins in one simple experiment. PCA is an in vivo technique where two proteins are fused...

...called a protein fragmentation complementation assay (PCA). In the Split-BioID method, each BirA* fragment ...

...bp) DNA Concentration (μg/μL) Volume of DNA (μL) RepCap 7,265 1.00 727.6 pHelper 11,854 1.00 1,185 Transfer..., for each plasmid we need: Sample Calculations RepCap: 0.08 μg/bp × 7,265 bp = 582.1 μg Volume Needed...

...lentiGuide-Puro. pSpCas9n(BB)-2A-GFP (PX461) - Similar to pSpCas9(BB)-2A-Puro (PX458), pSpCas9n(BB)-2A-GFP ... sequence (pSpCas9(BB)). The 2A-GFP allows for screening of transfected cells. Find pSpCas9(BB)-2A-GFP...Addgene!) pX330-U6-Chimeric_BB-CBh-hSpCas9 - The S. pyogenes Cas9 (SpCas9) has been widely popular in genome...chimeric guide RNA. Find pX330-U6-Chimeric_BB-CBh-hSpCas9. pSpCas9(BB)-2A-Puro (PX459) V2.0 - This plasmid from...nick, rather than cleave DNA. Find pSpCas9n(BB)-2A-GFP (PX461). pSpCas9(BB)-2A-Puro (PX459) - This mammalian...encodes both SpCas9 and your gRNA. It’s the former version of our sixth most popular plasmid pSpCas9(BB)-2A-...so it may be best to go with pSpCas9(BB)-2A-Puro (PX459) V2.0. Find pSpCas9(BB)-2A-Puro (PX459). Thank...

... un-tagged versions of SpCas9 and further variants replace SpCas9 with SpCas9n (nickase), which nicks ... express both SpCas9 and your gRNA in a variety of mammalian cell types. Purpose  SpCas9 This plasmid ... as is obvious from our most requested plasmid, SpCas9 is still going strong as the basis for many genome...further ado, the most request plasmid in 2016 was... pSpCas9(BB)-2A-Puro (PX459) V2.0       Similar to last...2016 is an expression vector for CRISPR nuclease, SpCas9 from the Zhang Lab. It's name, though a little ...as directed by a gRNA. 2A-Puro In this plasmid, SpCas9 is fused to the puromycin resistance gene through... information). V2.0 This designation separates pSpCas9(BB)-2A-Puro (PX459) V2.0 from it's predecessor,...

...than SpCas9, in this case, NNGRRT and NNNNGATT, respectively. Researchers also engineered SpCas9 variants...with wt SpCas9 in mammalian cells, finding xCas9 3.7 displayed about the same editing rate as SpCas9 at an...the targeted sequence at the locus of interest. SpCas9’s 3’ NGG PAM occurs frequently in GC-rich genomes... an NGG PAM every 42 bases in the human genome. SpCas9 targeting should be easy, right? Yes and no. If...selection phage (SP) to carry catalytically dead SpCas9 (dCas9) fused to the bacterial polymerase subunit...cleaves multiple PAMs at a higher efficiency than SpCas9: NGT (4.5-fold efficiency), NGC (2.1-fold), NGA...previous observation that NGA is a secondary PAM for SpCas9. Hu et al. performed GUIDE-seq analysis to characterize...

...than the engineered approach used to make eSpCas9 and SpCas9-HF1. By mining the treasure trove of naturally...GeoCas9 vs. SpCas9, see the table below and read on to learn more about GeoCas9.   SpCas9 GeoCas9 ...loving) bacteria, such as Streptococcus pyogene’s SpCas9. These Cas9 proteins don’t work well at high temperatures...Longevity in Serum?* No Yes Table 1. Comparing SpCas9 and GeoCas9. *For in vitro Cas9 RNP activity or...that it functions at temperatures much warmer than SpCas9 can tolerate. Harrington et al tested GeoCas9’s...temperatures. In both cases, GeoCas9 outperformed SpCas9 at high temperatures. See Figure 1 for a summary...skills by transfecting HEK293 cells with GeoCas9 or SpCas9 RNP’s and a guide targeting a site with an overlapping...

...requirements Reference CBE SpCas9 rAPOBEC1 NGG 6 SpCas9 Target-AID (PmCDA1) NGG 5 SpCas9-VQR rAPOBEC1 NGA 8...plant base editors replacing the SpCas9 with the mutant versions of SpCas9 and SaCas9, which recognize PAM...SaCas9-KKH rAPOBEC1 NNNRRT 8 ABE SpCas9 TadA-TadA* NGG 9-12 SpCas9-VQR TadA-TadA* NGA 8 SaCas9 TadA-TadA...compatibility All of the base editors mentioned above are SpCas9 nickase-based and depend on the presence of an ...TadA-TadA* NNGRRT 9 SaCas9-KKH TadA-TadA* NNNRRT 8 SpCas9-VRER TadA-TadA* NGCG 8  Tips for designing base...

...compared to the full-length SpCas9. In 2016, Chew et al. developed a split intein spCas9-AAV toolbox that retains...tissues. However, packaging Streptococcus pyogenes (SpCas9) and a gRNA together (~4.2 kb) into an AAV vector...approximately 1350 amino acids in size, which includes SpCas9, and shortern orthologs approximately 1000 amino...SaCas9 produced indels at a similar efficiency to SpCas9, leading the group to focus their efforts on SaCas9...off-target effects. To compare the off-target effects of SpCas9 and SaCas9, Zhang’s group used an approach called...display higher levels of off-target activity than SpCas9, confirming its suitability for in vivo studies...et al developed a proof-of-concept split intein SpCas9 which had modest editing rates in HEK-293T cells...

...Cas9 (SpCas9) requires an NGG PAM to recognize the target. Osamu Nureki’s lab engineered SpCas9 (SpCas9...NGG. In addition to the engineered SpCas9, they also fused the SpCas9-NG with the activation-induced cytidine...the targeting range of CRISPRCas9 by engineering SpCas9 Article contributed by Shreya Vedantam CRISPR/Cas9...cells. The increased versatility of the engineered SpCas9-NG and its variants can be used to engineer specific...specific sites that were previously difficult. Find SpCas9 and Target-AID-NG plasmids at Addgene! Nishimasu...

...pyogenes (SP); SpCas9 3' NGG SpCas9 D1135E variant 3' NGG (reduced NAG binding) SpCas9 VRER variant...a variant with increased PAM flexibiliy and eSpCas9/SpCas9-HF1 have increased targeting specificity). ...variant 3' NGCG SpCas9 EQR variant 3' NGAG SpCas9 VQR variant  3' NGAN or NGNG  xCas9 3' ...' NG, GAA, or GAT SpCas9-NG 3' NG  Staphylococcus aureus (SA); SaCas9 3' NNGRRT or NNGRR(N)  Acidaminococcus...

...is similar to that of high fidelity Cas9s eSpCas9 and SpCas9-HF1. Both AsCpf1 and LbCpf1 ribonucleoproteins... Cpf1 and its guide RNAs are smaller than their SpCas9 counterparts, so they will be easier to deliver...have a low genetic capacity of ~4.5 kb. At 4.1 kb, SpCas9 barely fits into AAV when you add in a gRNA and...respectively, larger than SaCas9 but smaller than SpCas9. These nucleases should fit well into AAV vectors...only slightly lower than that of the widely used SpCas9 and comparable to SaCas9. As seen with Cas9 orthologs...cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave at ~90 sites, according to Kim et al...off-target activity than that previously observed with SpCas9. Kleinstiver et al. suggest that AsCpf1’s off-target...

... to your experimental target, SpCas9 and its enhanced variants, eSpCas9 and HypaCas9, are great choices...choices. Check out our SpCas9 containing plasmids, optimized for a variety of species.   Specificity concerns...target locus, enhancing specificity over a single SpCas9. The drawback of this approach is that two sgRNAs... using a Cas9 variant with higher specificity (eSpCas9 and HypaCas9) instead.  Choosing a Cas9 for site-specific...anywhere to be found. If you’re lucky enough to find a SpCas9 compatible PAM at your site of interest, that is... Cas9s with looser PAM requirements compared to SpCas9. xCas9 has a PAM requirement of NG, GAA, or GAT...The PAM Requirement and Expanding CRISPR Beyond SpCas9.” Single base pair edits Is the location and type...

...Zhang Lab had described eSpCas9, while the Keith Joung Lab had developed SpCas9-HF1. Both of the enzymes... similar off-target activity, relative to eSpCas9 and SpCas9-HF across a number of different sites. Using...the nuclease. For the most frequently used Cas9, spCas9 from Streptococcus pyogenes, this sequence is 3...The PAM Requirement and Expanding CRISPR Beyond SpCas9 described the PAM specificity of Cas9s isolated...Nureki Lab described a rationally engineered Cas9, SpCas9-NG, which required only the di-nucleotide PAM NG...

...injury, the most utilized PAM sequence, that of SpCas9, is NGG. Assuming all nucleotides are represented...that recognize NG PAMs. The Nureki lab generated SpCas9-NG, a rationally engineered Cas9 with NG PAM compatibility...capable of editing at frequencies comparable to SpCas9. SpRY can also edit NYN PAMs, but with less efficiency...variant didn’t display higher off-target effects than SpCas9, a pleasant surprise since the PAM flexibility ...flexibility of gRNA placement significantly over SpCas9 and more so than Cas9s with an NG PAM.   Cas... Cas PAM sequence  Additional notes SpCas9 NGG - Cas12a TTTV Great for AT-rich regions Cas9...editable with CRISPR technology.     Fig. 1: SpCas9 has sparse PAM coverage in the human genome. SpRY...

...is similar to that of high fidelity Cas9s eSpCas9 and SpCas9-HF1. Both AsCpf1 and LbCpf1 ribonucleoproteins...genomes that lack the 3’-NGG PAM sites favored by SpCas9. Since Cpf1 doesn’t require a tracrRNA, crRNA guides... Cpf1 and its guide RNAs are smaller than their SpCas9 counterparts, they will also be easier to deliver...only slightly lower than that of the widely used SpCas9 and comparable to SaCas9. As seen with Cas9 orthologs...cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave at ~90 sites, according to Kim et al...off-target activity than that previously observed with SpCas9. Kleinstiver et al. suggest that AsCpf1’s off-target...