...applications. That said, SpCas9 is not the only game in town, and other Cas proteins like SaCas9 and Cpf1 can circumvent...respectively. Amplified nucleotides are combined with Cas 13a in complex with crRNA targeting the sequence ...Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes. " Mol Cell. 66(3) (2017):373-83. PubMed ...functional characterization of diverse class 2 CRISPR-Cas systems." Molecular cell 60.3 (2015): 385-397. PubMed...applications of Cas13a. The origins of Cas13a: An RNA cleaving CRISPR nuclease Cas13a was originally ...Perhaps the biggest difference between Cas13a and Cas9 is that Cas13a binds and cleaves RNA rather than DNA...As with Cas9, mutating key residues in the Cas13a molecule results in a “nuclease dead” Cas13a (dCas13a...

...Joung JK (2016) Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol 34:...Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi.org/10.1016...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...blunt ends created by Cas9. Cas12a  displays lower off-target editing than SpCas9 (Kim et al., 2016).  ...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...

...than SpCas9, in this case, NNGRRT and NNNNGATT, respectively. Researchers also engineered SpCas9 variants...enrich for Cas9 variants with activity on non-NGG PAMs, generating xCas9 3.0-3.13. To test xCas9 3.0-3.13...Characterizing xCas9 in mammalian cells Hu et al. compared xCas9 activity with wt SpCas9 in mammalian cells...actually lower for xCas9 3.6/3.7 than for SpCas9. For some non-promiscuous gRNAs, xCas9 3.7 displayed more...using the xCas9 scaffold. With an NGG PAM, xCas9 3.7-VPR constructs modestly outperformed SpCas9-VPR in ...flexibility of xCas9 3.7. Base editor variants with xCas9 are similarly flexible. Like the xCas9 3.7 nuclease...’t know about xCas9. “I’m not 100% sure xCas9 is going to be flat out better than SpCas9. I want everyone...

...et al., 2016). For editing the genome, the CRISPR/Cas system makes use of 3 components, a guide RNA (gRNA...(2013) Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339:819–823 . https://doi.org/10.1126...Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering. Cell 154:1370–1379 ....Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell 153:910–918 . https...alternative Cas9, from Streptococcus aureus, is as effective at editing mouse embryos as Cas9 from S. pyogenes...Nakauchi H (2018) Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-...guest bloggers, Wenning Qin and Haoyi Wang. CRISPR/Cas9 is revolutionizing the mouse gene-targeting field...

...scientific collaboration across the globe. Listen here! Podcast Breakdown (Time: Topic) 0:00-1:00 : Intro 1:01-...the audio engineer and producer for the Addgene Podcast.     Additional Resources Learn about Seeding ...the Addgene Blog Listen to Additional Addgene Podcasts Learn How You Can Contribute to the Addgene Blog...

... CRISPR/Cas systems only require changing the programmable guide RNA. Gene activation by dCas9, also referred...Prior to the discovery of CRISPR/Cas systems, gene activation across multiple loci was an arduous process...seem next to impossible. The development of CRISPR/Cas systems, however, greatly improved the simplicity...al., 2013). To target specific sequences, CRISPR/Cas systems rely on a guide RNA complementary to the ... proteins while others re-engineer components of Cas systems themselves. SunTag, SAM, and VPR have all...bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Research 41:7429–7437 . https...Crawford GE, Reddy TE, Gersbach CA (2013) RNA-guided gene activation by CRISPR-Cas9–based transcription factors...

... Figure 2: comparison of Cas 9 vs. Cas 12a expression cassettes when encoding multiple guides. ... large size of CRISPR-Cas effectors and fusion proteins, such as Cas9 and Cas12a, creates challenges for...and cell engineering applications. CasMINI, a new miniature CRISPR-Cas system for editing and gene activation...State Protein Ladder Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing .../s41598-021-96051-x Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing...of either Cas9 or Cas12a. Despite its small size, CasMINI is comparable in efficacy to dCas12a systems ...expression cassette is advantageous to Cas9’s larger expression requirements (Figure 1), but Cas12a has generally...

...Considerations when choosing your Cas nuclease When choosing the Cas9 nuclease for your experiments, there...PAM sequence that the Cas nuclease recognizes determines site accessibility. Cas9s with a broader PAM specificity...Since the first use of Cas9 to engineer the genome, there are more and more Cas enzymes and variants to...choosing a Cas protein for your experiment. References Hu, Johnny H., et al. "Evolved Cas9 variants with...knowledge of both the advantages and limitations of each Cas enzyme can help determine which enzyme to use. Remember...CRISPR 101 blog posts Find more information about Cas proteins Download the CRISPR 101 eBook Resources...frequently used Cas9, spCas9 from Streptococcus pyogenes, this sequence is 3’-NGG. But Cas9s found in other...

... Is Cas9 not your Cas of choice?  There are plenty of other enzymes to choose from within the Cas family...Koblan, L. W. & Liu, D. R. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors...using a Cas9 variant with higher specificity (eSpCas9 and HypaCas9) instead.  Choosing a Cas9 for site-specific...your experimental target, SpCas9 and its enhanced variants, eSpCas9 and HypaCas9, are great choices. Check...target two Cas9-nickases (Cas9n) to the target locus, enhancing specificity over a single SpCas9. The drawback... expanding number of Cas9s with looser PAM requirements compared to SpCas9. xCas9 has a PAM requirement... making a break.  Base editors are dead Cas9 (dCas9) or nCas9 enzymes fused to either cytosine or adenosine...

...Expanding beyond Cas9 Since the development of CRISPR/Cas9 editing methods, other Cas proteins have been...experimental options. Expanding and improving the CRISPR/Cas editing toolbox for C. elegans Recent work has further...development of toolkits such as the SapTrap CRISPR/Cas Toolkit and SapTrap-SEC kit for C. elegans genome... some cases, injection of Cre is required, however the development of the self-excising cassette (Dickinson...University of California, Santa Cruz. Emerging CRISPR/Cas9 editing technologies have transformed the palette...regulation during developmental processes, CRISPR/Cas9 allows us to delete sequences and introduce mutations...remain in the edited animal. Development of CRISPR/Cas9 editing strategies in C. elegans As brilliantly ...