...sites tested when compared to eSpCas9(1.1) and SpCas9-HF1. The HypaCas9 plasmids are available from Addgene...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... proteins With their enhanced specificity, eSpCas9, SpCas9-HF1, and HypaCas9 should enable researchers...evidenced by all the CRISPR publications, press, and plasmids out there, it’s obvious that CRISPR is a ground-breaking...

...express both SpCas9 and your gRNA in a variety of mammalian cell types. Purpose  SpCas9 This plasmid expresses... un-tagged versions of SpCas9 and further variants replace SpCas9 with SpCas9n (nickase), which nicks ...sure, as is obvious from our most requested plasmid, SpCas9 is still going strong as the basis for many...the top requested plasmid from 2016 is an expression vector for CRISPR nuclease, SpCas9 from the Zhang Lab...sequence as directed by a gRNA. 2A-Puro In this plasmid, SpCas9 is fused to the puromycin resistance gene through...without further ado, the most request plasmid in 2016 was... pSpCas9(BB)-2A-Puro (PX459) V2.0       Similar... V2.0. pSpCas9(BB)-2A-Puro (PX459) V2.0: What's not in the name? Not included in this plasmid's name are...

...GeoCas9 vs. SpCas9, see the table below and read on to learn more about GeoCas9.   SpCas9 GeoCas9 ...temperatures Figure 1: Thermostability of SpCas9 vs GeoCas9. SpCas9 or GeoCas9 RNPs were incubated in reaction... Figure 2: Human plasma longevity of SpCas9 vs GeoCas9. SpCas9 or GeoCas9 RNPs were incubated in human...than the engineered approach used to make eSpCas9 and SpCas9-HF1. By mining the treasure trove of naturally...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...80 °C. Activity is expressed as Kcleave (min-1). SpCas9 has maximum activity from 35-45 °C, while GeoCas9...

...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 ...

...than SpCas9, in this case, NNGRRT and NNNNGATT, respectively. Researchers also engineered SpCas9 variants...transcription from the accessory plasmid (AP) encoding phage gene III. The accessory plasmid library contained all...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...PAM flexibility. In this system, the mutagenesis plasmid (MP6) permits quick, large-scale mutation of the...selection phage (SP) to carry catalytically dead SpCas9 (dCas9) fused to the bacterial polymerase subunit...

...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles ...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...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... were previously difficult. Find SpCas9 and Target-AID-NG plasmids at Addgene! Nishimasu H et al., Science...need. If you'd ever like to write about a recent plasmid deposit please sign up here.        New toolkit... the Kanemaki lab has created a new toolkit of plasmids for the N- or C- terminal tagging of proteins ...

...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...

...compared to the full-length SpCas9. In 2016, Chew et al. developed a split intein spCas9-AAV toolbox that retains...gene-targeting capabilities of full-length SpCas9. This set of plasmids includes AAV-Cas9C-VPR for targeted ...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...

...is similar to that of high fidelity Cas9s eSpCas9 and SpCas9-HF1. Both AsCpf1 and LbCpf1 ribonucleoproteins... is well suited for genome editing.  Find Cpf1 Plasmids at Addgene   Image attribution: Zetsche... 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...

...including SpCas9, SaCas9, LbCas12a, enAsCas12a, SpCas9-NG, SaCas9-KKH, CP1028-SpCas9, and CP1041-SpCas9. The...following components: A plasmid in the host bacterium expressing dCas9 A plasmid in the host bacterium ... weakly compatible with many Cas domains beyond SpCas9. In order to expand the gene-editing applications... of gene III to create infectious particles. A plasmid-based genetic circuit in the bacteria links the...encoding the deaminase TadA-7.10 A mutagenesis plasmid controlling the phage mutation rate Phage infection...Phage-assisted evolution of base-editing activity. A plasmid-based genetic circuit in the bacteria links the...off-target RNA editing rate of ABE7.10. Find the ABE8e plasmids at Addgene! Why engineer and optimize base editors...

...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...all CRISPR plasmids and resources at Addgene Find CRISPR base editors Find CRISPR plasmids for plants ...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...

...is similar to that of high fidelity Cas9s eSpCas9 and SpCas9-HF1. Both AsCpf1 and LbCpf1 ribonucleoproteins...Francisella Cpf1 (FnCpf1), they employed an E. coli plasmid depletion assay to discover FnCpf1’s PAM sequence...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...

...choices. Check out our SpCas9 containing plasmids, optimized for a variety of species.   Specificity concerns... to your experimental target, SpCas9 and its enhanced variants, eSpCas9 and HypaCas9, are great choices...target locus, enhancing specificity over a single SpCas9. The drawback of this approach is that two sgRNAs...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...frequently fused to Cas9 for this purpose. dCas9-KRAB plasmids for repressing target genes in a variety of species...

...applications. This plasmid was the top requested plasmid of 2017. Find pMD2.G. pSpCas9(BB)-2A-GFP (PX458...and an envelope expressing plasmid, such as our second most requested plasmid of all time, pMD2.G. Find... that expresses both SpCas9 and your gRNAs and was also the top requested plasmid from 2016. You may notice...the end of this plasmid which indicates that there was a prior version of this plasmid. Indeed the first...envelope expressing plasmid pMD2.G. Find pMDLg/pRPE. scramble shRNA - This plasmid from David Sabatini....1 protocol. Pro-tip: this plasmid grows more slowly than standard plasmids. Find pLKO.1 - TRC cloning...pX330-U6-Chimeric_BB-CBh-hSpCas9. pSpCas9(BB)-2A-Puro (PX459) V2.0 - This plasmid from the Zhang lab is ...

...Visit our CRISPR plasmids and resources page Read our CRISPR guide Find CRISPR plasmids for mammalian systems...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...

...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief articles...articles provide a synopsis of a plasmid or group of plasmids' functions and applications. We hope that... you to find and use the plasmids you need. You can find all the hot plasmids from 2017 below. With over...over 50,000 plasmids, we can't write posts for every great plasmid that comes into the repository, but...write about your plasmids in a future blog post. No time to read? Listen to our hot plasmids segment on the...editing for precise genome modification. Plasmid ID Plasmid Name Cas9 Variant Speacial Features 85169...85174  pBK-YE1-BE3  SpCas9 (NGG)  Editing window ~2 nt  85175  pBK-EE-BE3  SpCas9 (NGG)  Editing window...

...exact Cas9 plasmid used by Jinek et al. 2012, or browsing for the latest AsCas12a plasmids, Addgene and...Once upon a time, not so long ago, spCas9 was the only Cas enzyme widely available and applied by researchers...find a link to search Addgene's collection for plasmids containing that enzyme, and embedded in the References...lists on every page you can find direct links to plasmids from the article if they're available through ...getting the desired features and cutting (note: spCas9 isn’t always the winner!).   Fig. 3 – Comparison...The PAM Requirement and Expanding CRISPR Beyond SpCas9 More resources from the Innovative Genomics Institute...

...changes we’ve made: New CRISPR plasmid pages We’ve given our CRISPR Plasmids and Resources a fresh new design...design, sorting the plasmids we have available by category. Each plasmid page now has a helpful graphic...introduce you to the plasmid class covered on that page. To help you find popular plasmids, we’ve also added... new plasmid pages that cover some of the hottest CRISPR tools. Base Edit: Base editing plasmids allow...Enhancing CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9 Day 10: Transcriptional regulation...revolution, Addgene has distributed over 100,000 CRISPR plasmids. But that’s not our only job - we strive to also...up with new developments (and, of course, find plasmids that will be useful to you.) With these goals...

...CRISPR, and specifically Cas9 from S. pyogenes (SpCas9), is truly an exceptional genome engineering tool... species, and has many applications. That said, SpCas9 is not the only game in town, and other Cas proteins... can circumvent the limitations associated with SpCas9. Cas13a (previously referred to as C2c2), has several...27256883. PubMed Central PMCID: PMC5127784. Find plasmids from this paper at Addgene. 2. East-Seletsky,...) (2016):270-273. PubMed PMID: 27669025. Find plasmids from this paper at Addgene.  3. Gootenberg, Jonathan...28408723. PubMed Central PMCID: PMC5526198. Find plasmids from this paper at Addgene. 4. East-Seletsky,...) (2017):373-83. PubMed PMID: 28475872.  Find plasmids from this paper at Addgene.  5. Abudayyeh, Omar...

...CRISPR beyond spCas9 Cas proteins More resources on the Addgene Website CRISPR Plasmids and Resources...the fact that it is smaller than the traditional SpCas9 enzyme (about a third of the size) and does not...cleave RNA through the same mechanism as traditional spCas9. If you are looking to modify RNA, Cas9 just won...