...loci in living human cells by an optimized CRISPR/Cas system. Chen B, Gilbert LA, Cimini BA, Schnitzbauer... a colorful Cas9 toolbox, Ma et al. turned to SpCas9 and its orthologs NmCas9 and St1Cas9. Each ortholog...states. dCas9 gets a colorful makeover Even without its nuclease activity, catalytically dead Cas9 (dCas9... colorful system that uses fluorescently labeled Cas9 to label multiple genomic loci in live cells. While...hybridization (FISH) or fluorescently labeled TALEs, CRISPR/Cas9’s ease of use and ability to label live cells make...well-known being transcriptional activation/repression. dCas9 has also previously been adapted to fluorescently...required by each ortholog, a gRNA designed for one dCas9 should be specific to that ortholog and not cross-talk...

...puromycin resistance cassette, such as PX459). After you co-transfect the CRISPR/Cas system with your homologous...article list, the Genome Engineering Using CRISPR/Cas Systems has expanded out of control. To further consolidate...focus on genome editing using the Zhang lab's CRISPR/Cas plasmids. Designing your CRISPR genome editing experiment...that can be inserted into the genome using CRISPR/Cas forHomologous Recombination (HR)? How long should... crispr cas...the Cas9 nickase doesn't have the ability to induce double-stranded breaks like the wildtype Cas9 does...questions and answers. Here are just 4 of the CRISPR-Cas9 FAQs. If you still have more questions after reading...

...CRISPR-Cas system for mammalian use Stanley Qi’s lab has developed a miniature Cas system (CasMINI) engineered...Miniature CRISPR-Cas systems SEND plasmids Prime editing improvements Miniature CRISPR-Cas systems By Brook... yet active Cas system starting with the same Un1Cas12f1 (Cas14). They noted that Cas12f1 had an extra-long...  They did not modify the Cas12f1 enzyme. The result is a highly efficient Cas system with very compact...miniature CRISPR-Cas12f nuclease Quanjiang Ji’s lab describes an effective miniature Cas system that utilizes...the uncultivated archaea Cas12f (Un1Cas12f1, or Cas14a1) system. The CasMINI system was developed by engineering... enzyme with a fully active Cas9 enzyme. PE-Cas9 comprises an active Cas9 fused to the reverse transcriptase...

...Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes." Mol Cell. 66(3) (2017):373-83. PubMed PMID...with the Cas13 scaffold, testing a whopping 21 Cas13a orthologs, 15 Cas13b orthologs, and 7 Cas13c orthologs...identified Cas13a (C2c2) (Abudayyeh et al., 2016) as a RNA-targeting CRISPR enzyme, they adapted Cas13b for ...target locus, making them more flexible than Cas9/Cpf1. Some Cas13 enzymes prefer targets with a given single...sequence, but orthologs like LwaCas13a do not require a specific PFS. Cas13 enzymes do not contain the ...knockdown. In initial tests, Cas13b from Prevotella sep. P5-125 (PspCas13b) yielded 62.9% average knockdown...to deaminate cytidine and used dRanCas13b (catalytically inactive Cas13) to target the cytosine deaminase...

...comparison of GeoCas9 vs. SpCas9, see the table below and read on to learn more about GeoCas9.   SpCas9 GeoCas9... 1. Comparing SpCas9 and GeoCas9. *For in vitro Cas9 RNP activity or longevity. GeoCas9 is active at high... 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 plasma...used to make eSpCas9 and SpCas9-HF1. By mining the treasure trove of naturally occurring Cas9 diversity ...at elevated temperatures. In both cases, GeoCas9 outperformed SpCas9 at high temperatures. See Figure ...et al tested GeoCas9’s editing skills by transfecting HEK293 cells with GeoCas9 or SpCas9 RNP’s and a ...

... the Addgene Podcast.     Additional Resources on the Addgene Blog Listen to Our Podcast Interview with...plasmid info with a new segment on the Addgene podcast - The Hot Plasmids Segment. Click on the player...below to listen to a quick (~5 min) Hot Plasmids podcast that introduces you to 4 new plasmid technologies... the latest and greatest plasmids through this podcast segment. Below, we break down each of the Hot Plasmids...If you have any comments about the Hot Plasmids Podcast Segment, or have recommendations for new Hot Plasmids... podcast...

...variety of related bacteria with similar Cas systems. Some of these Cas systems have enzymes small enough to...knockdown efficiencies. The ability to identify related Cas systems opens up the potential of further development...CRISPR effector protein they named Cas7-11. Testing showed that Cas7-11 cut RNA with unprecedented specificity...luciferase RNA knockdown assays using Cas7-11   Use The Cas7-11 targeting system contains two pieces...computationally described as a fusion of four Cas7 proteins and a putative Cas11-like protein, and a guide RNA. The...efficacy rates of Cas7-11 were reported as up to 85%, broadly similar to shRNA and Cas13 systems in a range... both of which we're featuring in today's blog. Cas7-11 Development and Discovery As part of their quest...

...single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227. ... P., et al. "Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells." Nature Biotechnology...Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell. 2015 Sep 23. pii: S0092-8674(15)01200...An updated evolutionary classification of CRISPR-Cas systems. 5. Nat Rev Microbiol. 2015 Sep 28. doi: ...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...overcome some of Cas9’s shortcomings. Cpf1’s preferred PAM is 5’-TTN, differing from that of Cas9 (3’-NGG) in...

...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...

...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...