...possibilities for Cas proteins With their enhanced specificity, eSpCas9, SpCas9-HF1, and HypaCas9 should enable...specificity of the Cas9 nuclease with engineered variants: eSpCas9, SpCas9-HF1, & HypaCas9. The off-target...portions of Cas9 might be important for nuclease specificity. They found that eSpCas9(1.1) and SpCas9-HF1 each...sites tested when compared to eSpCas9(1.1) and SpCas9-HF1. The HypaCas9 plasmids are available from Addgene...off-target effects” have included using a Cas9 nickase variant, lowering Cas9 expression, and truncating gRNA sequences... target effects of CRISPR/Cas9 by altering the nuclease activity of the Cas9 nuclease itself. Design and... and testing of eSpCas9 Looking at the structure of the Cas9 nuclease (PDB ID: 4OO8 and 4UN3), Slaymaker...

... A multitasking Cas Most Cas effectors have one job — Cas see DNA, Cas chop DNA. Cas3, however, has two...CRISPR 101: Cas9 vs. The Other Cas(s) CRISPR 101: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems INTEGRATE...CRISPR has brought many other Cas effectors to light. One lesser-known Cas is waiting for its chance in...and Cas11. Type I-E Cascades contains all five of these Cas proteins, while type I-C Cascades do not contain... the spotlight — Cas3. Cas3 can induce genomic alterations on a scale that other Cas effectors can’t, ...a multi-Cas complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade is brought...complex” system Cas3 is part of the Class 1 CRISPR family, which means it requires multiple Cas effectors to...

... for a candidate Cas for your experiment. Target requirements As we all know, a Cas guided by a gRNA ...properties for a Cas entry (SpyCas9a) from CasPEDIA.   How can I use it? CasPEDIA operates like many...Addgene and CasPEDIA have got you covered!     Fig. 1 – CasPEDIA Cas ID display information for...for a given Cas (SpyCas9a) from CasPEDIA.   Helpful resources and information For some researchers,...CRISPR 101: Which Cas9 do I choose for my CRISPR experiment? CRISPR 101: Cas9 The Other Cas(s) The PAM Requirement...Once upon a time, not so long ago, spCas9 was the only Cas enzyme widely available and applied by researchers...What are the features? CasPEDIA has collected relevant information on all the Cas enzymes you know and ...

...the nuclease Cas9, but how does it stack up to the new Cas enzymes on the block? The Cas family of proteins...targeting. RNA targeting Cas enzymes Move over Cas9, CRISPR isn’t just for DNA anymore! Cas enzymes have been... facts on Cas family proteins and genome editing abilities   Choosing a Cas While Cas9 may be the...the most adaptable Cas currently available, the other Cas proteins should not be overlooked. The unique...DNA edits.   Cas3 – The shredder What sets Cas3 apart from Cas9 and almost all other Cas enzymes is its...delivery.   Cas14 – Mini, but single strand mighty Unlike many other Cas enzymes, Cas14 targets ssDNA...lesser-known Cas enzymes while also highlighting their similarities and differences to Cas9. DNA targeting...

...Separate Cas proteins then express and process the CRISPR loci to generate RNA, which guide a Cas nuclease.... Many CRISPR/Cas moieties carry out these functions using slightly different Cas proteins and PAM ...CRISPR/Cas defense by generating a single point mutation in the PAM sequence, preventing the Cas nucleases... information on CRISPR/Cas, check out Addgene’s CRISPR Guide or find CRISPR/Cas plasmids for your research... bacterial genome. Cas genes, which code for polymerases, nucleases, and helicases, were also disrupted...clustered regularly interspaced palindromic repeats) Cas (CRISPR associated genes) system. This popular genome...look at how prokaryotes originally utilized CRISPR/Cas as a powerful and adaptive defense strategy against...

... Jennifer Tsang. CRISPR-Cas technology is constantly evolving. Variants of Cas proteins can be used for...missing: a way to shut off Cas’s activity. The concern is that the longer Cas remains active in a cell,...protein inhibitors of CRISPR-Cas systems that help phages evade the CRISPR-Cas immune system of bacteria.... II-A CRISPR-Cas systems. A full list of Acr proteins organized by the type of CRISPR-Cas system they ...that interact with CRISPR-Cas systems such as Cascade-Cas3, Cas9, Cas12, and Cas13 (Marino et al., 2020)....AcrIIA4 blocks Cas9’s interaction with the PAM site (Dong et al., 2017), and AcrIIC3 causes Cas proteins to...interactions with Cas9 interferes and prevents the correct assembly of the crRNA-Cas complex (Zhu et al...

...mutations arise from base/prime editing or Cas cleavage. In the case of Cas cleavage, off-target mutations can...confer specificity of the nuclease; however, many Cas and Cas-like nucleases are promiscuous. In other words...consider your Cas enzyme. Even if there are similar sequences present in the genome, certain Cas enzymes are...approaches have been developed. Cas ChIP-Seq, where only DNA sites bound by Cas are sequenced, has been tested...appropriate method for an activator or repressor Cas. Similarly, Cas ChIP-Seq would also identify all DNA binding...CRISPR 101: Which Cas9 do I choose for my CRISPR experiment? CRISPR 101: Cas9 The Other Cas(s) CRISPR 101... of Maya Kostman. The most commonly used Cas, spCas9, has gone through multiple rounds of optimization...

...type VI, each relying on a unique Cas effector — Cas9, Cas12, or Cas13. If you’d like to explore Class ...while Class 2 use single protein Cas effectors, like Cas9. The Cascade complex doesn’t usually carry out...attributed to the multi-Cas effector, Cascade. Developing tools using a Cascade complex is tricky, as you...the blog CRISPR 101: Cas9 vs. The Other Cas(s) CRISPR 101: RNA Editing with Cas13 Build your CRISPR vocabulary...Class 1 CRISPR systems use multi-Cas effectors, referred to as Cascade (CRISPR-associated complex for antiviral... subtypes use Cas10 as the primary effector complex but are defined by the accessory Cas proteins in the...which are among the smallest Cas enzymes (400 to 700 amino acids), use a Cas14 enzyme, and uniquely target...

...gene cas14. cas14 encodes for a small Cas protein (40-70 kDa), which is half the size of other Cas proteins...characteristic of CRISPR-Cas enzymes. In contrast to other Cas enzymes, Cas14 has not been found in bacterial...integrase, cas1. They discovered a family of CRISPR-Cas systems containing cas1, cas2, cas4, and a new...CRISPR-Cas systems. There are 24 variants of the cas14 gene that cluster into 3 subgroups (cas14a-c). All.... Identifying Cas14 variants In this quest to discover new and maybe more efficient Cas systems, Jennifer...of virtually any organisms on this planet, CRISPR-Cas systems were merely adaptive immune systems that ...try to determine whether simpler and maybe smaller Cas systems could exist in nature. To do this, they ...

...Protein Cas: Different CRISPR systems involve different proteins, but many of them are called Cas, short...by the Cas9 protein from the appropriate species. Some Cas proteins, like LbCas12a or AsCas12a, do not...encodes a tracrRNA (in some systems including Cas9), Cas proteins, and a CRISPR array. The CRISPR array...spacer. In bacteria, these crRNAs are bound by Cas proteins (Cas9 shown here). The repeat sequence base pairs... famous of these proteins is Cas9 nuclease, but there are many other Cas proteins as well, both natural...organized. Target strand: The strand of foreign DNA that Cas proteins recognize using an RNA guide. Non-target... to be cut, which varies between CRISPR systems. Cas proteins won’t cut DNA that doesn’t have their specific...

...-flexible Cas enzymes   The solution to the PAM problem seems simple, in theory: find a Cas enzyme or ...placement significantly over SpCas9 and more so than Cas9s with an NG PAM.   Cas PAM sequence  Additional...Expanding Beyond Cas9 xCas9: Engineering a CRISPR Variant with PAM Flexibility History of CRISPR Cas – A Tale ...are some available.   Cas proteins that don’t utilize NGG   There are plenty of Cas9 variants which utilize...etc.). Additionally, alternative Cas enzymes exist, such as Cpf1/Cas12a, which recognizes the PAM sequences... of generating Cas enzymes with alternative PAM requirements (Zhao et. al, 2023). xCas9 has been shown...Walton et. al, 2020). These Cas proteins have everything we know and love about Cas9, but with broader PAM ...

...catalytically impaired Cas protein (dCas or Cas nickase) fused to a DNA-modifying enzyme, in this case a deaminase...change before the Cas domain lets go of the DNA. ABEs are somewhat slow and need a strong Cas9 domain to “hold... are only weakly compatible with many Cas domains beyond SpCas9. In order to expand the gene-editing applications...specifies the editing target site on the DNA, the Cas domain directs the modifying enzyme to the target...substitutions.  The need for speed: base editing before Cas lets go Figure 1: David Liu gives a talk at...to evolve ABEs with an increased compatibility to Cas homologs. Evolving better base editors with the help...deamination rate so that deamination can happen before Cas homologs let go. The Liu lab developed the Phage-...

...engineered variants of Cas nucleases. In doing so, they also created a modular toolkit for Cas-mediated genome...editing toolkit New Cas protein identified Jennifer Doudna’s lab recently identified Cas14, a family of small...small DNA-targeting Cas proteins. Cas14 targets and cleaves ssDNA which they have adapted for detecting...genome editing in plants While there have been many Cas nucleases developed for plants, it has been difficult...dual-nuclease Cas9-Cas9 chimeras Scot Wolfe’s lab has developed dual-nuclease Cas9-Cas9 chimeras with ...compared to monomeric Cas9’s. Read the paper in Nature Communications Find the Cas9-Cas9 chimera plasmids...Science Find the Cas14 plasmids Anti-CRISPR proteins for optogenetic control of CRISPR-Cas9 Anti-CRISPR ...

... targeting specificity). Cas9 endonuclease functions While native CRISPR/Cas systems have a variety of...sometimes sgRNA) In the native Type II CRISPR/Cas system, Cas9 is guided to its target sites with the aid...vary (for example, xCas9 is a variant with increased PAM flexibiliy and eSpCas9/SpCas9-HF1 have increased...sequence, Cas9 will neither bind nor cut the target. Cas9 homologs from different organisms or Cas9 mutants...pyogenes (SP); SpCas9 3' NGG SpCas9 D1135E variant 3' NGG (reduced NAG binding) SpCas9 VRER variant...3' NGCG SpCas9 EQR variant 3' NGAG SpCas9 VQR variant  3' NGAN or NGNG  xCas9 3' NG, GAA,...basic level, CRISPR/Cas9 genome editing systems use a non-specific endonuclease (Cas9 or closely related...

...Bacterial Strains by Use of Genome-Targeting CRISPR-Cas Systems. Ahmed A. Gomaa, et al. mBio. 2014 January...CRISPR/Cas9 system to precisely edit specific genes in monkeys. "Our study shows that the CRISPR/Cas9 system...for CRISPR Plasmids? CRISPR-Cas9 Plasmids available at Addgene CRISPR-Cas9 References, Protocols, and ...advances in the understanding and application of CRISPR/Cas9 technology are coming along at a fast and furious...Beisel and Rodolphe Barrangou, had applied the CRISPR/Cas9 system to developing an antibiotic that selectively...important details in their understanding of how CRISPR/Cas9s actually work. Last month, the Lawrence Berkeley... Nogales presented the first detailed picture of Cas9's three-dimensional shape via x-ray crystallography...

...multiple Cas proteins to form the Cascade targeting complex is a hallmark of Type I CRISPR-Cas systems,... (such as CRISPR-Cas9). Unlike in regular Type I CRISPR-Cas systems, however, Cascade from VchINT does...to Type I-F, but instead employ a Cas12k protein instead of three Cas proteins (Fig. 1A, bottom), and require...transposases and the target programmability of CRISPR-Cas, these systems represent the next generation of tools... hereafter VchINT) and encodes a Type I-F CRISPR-Cas system. The four major components required for RNA-guided... of DNA targeting by a transposon-encoded CRISPR–Cas system. Nature 577:271–274 . https://doi.org/10.1038...TS, Sternberg SH (2019) Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration. Nature...

...Cambridge, MA and Sequana Therapeutics in San Diego, CA. She earned a B.S in Biology from MIT.   Kenneth...at Credit Suisse’s Technology Group in Palo Alto, CA.  Benjie Chen is a co-founder of Addgene and received... In this episode of the Addgene podcast, we sit down with Addgene’s Founders - Melina Fan, Ken Fan, and...Listen to the interview with Addgene's founders Podcast breakdown Intro Chapter 1 - Who are Addgene's Founders...the audio engineer and producer for the Addgene Podcast.      Additional Resources from the Addgene Blog... podcast...

...shifts. This backbone model of Cas9 is based on Fuguo Jiang’s structure of Cas9 – primed for cleavage. This...researchers for the past fifteen years. On those occasions when we are fortunate to hand-deliver the model...contacted us in 2014 to ask if we could make a model of Cas9 based on the structure published by Martin Jinek...other aspects of the structure. While this first Cas9 model is certainly useful, it is also wrong in at... at least one serious way. We represented the Cas9 protein in a surface format made of rock-hard, inflexible... the amazing conformational flexibility that the Cas9 protein must have in order to scan along the double-stranded...model of a protein. We now have another model of Cas9 – a flexible alpha-carbon backbone model made of...

... sequencing data.”  Recently, Seqera published a case study on how Addgene has used Nextflow to improve...our existing resources. You can read it, and other case studies, on the Seqera website. ...