...-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...

...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...

...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...

... 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. ...