...in bioRxiv Find the GeneWeld vector kit CasX, a small Cas protein, is active in E. coli and human cells...ago, Jennifer Doudna’s lab discovered a new Cas protein, CasX, from metagenomic analysis of microbial DNA...GeneWeld vectors to create knock-ins CasX Drug inducible CRISPR/Cas9 activation E. coli genome-wide inhibition...demonstrating that CasX is active in both E. coli and human cells. Using the structure of CasX at eight different...and inactive CasX for E. coli and human expression. Read the paper in Nature Find the CasX plasmids ...inducible CRISPR/Cas9 systems for transcriptional activation and genome editing by fusing Cas9 to a mutated...vectors GeneWeld from Jeffrey Essner’s lab uses CRISPR/Cas9 and a donor vector to create knock-ins of fluorescent...

...other CRISPR/Cas systems used for endogenous protein tagging, please visit our website CRISPR/Cas Plasmids...example, Masato Kanemaki's lab has developed a CRISPR/Cas-based system for tagging endogenous proteins with...this system lies in the ability of the endonuclease Cas9 to create a DNA double stranded break (DSB) at a..., and co-injection of the targeting vector and a Cas9 expression plasmid will insert the desired genetic...reactions are then mixed and transfected along with a Cas9 expression vector. They can also be introduced directly...into a Drosophila S2 cell line stably expressing Cas9. This PCR toolkit offers C- and N-terminal tagging... promote homology directed repair after a CRISPR/Cas9 induced break. Learn more about these constructs...

...to target new genomic sites. The discovery of the Cas proteins in 2005 was a major breakthrough in the ...the world of genetic engineering as CRISPR-Cas editing soon replaced the protein-based DNA-binding domain...domain with a single-guide RNA sequence.  The Cas proteins, just like its predecessor induce a double-strand...editors (meganucleases, ZFNs, TALENs, and CRISPR-Cas) is that they induce double-strand breaks (DSBs) ... the genome. They fused a catalytically impaired Cas9 protein to a DNA-editing enzyme to generate the ...based on a prime editor which is a fusion between Cas9 and reverse transcriptase. Using an elongated template...The new frontier of genome engineering with CRISPR-Cas9. Science 346:1258096 . https://doi.org/10.1126/science...

...for drug discovery, there is more on the horizon. Cas enzymes have been engineered to have many unique ...References Fellmann, C., et al., Cornerstones of CRISPR-Cas in drug development and therapy. Nat Rev Drug Discov...characterized or have a simple pathology. In these cases, preliminary identification of potential targets...odds of finding a quality drug downstream. In some cases, there may already exist a drug which targets some... of genome edits accessible, fast, and easy. In cases where the mutational basis of a disease is known...CRISPR 101: Making a Knock-In Cell Line Using CRISPR-Cas9 to Edit Disease Out of the genome Tips for a first...

... engineering from ZFNs through TALENs and CRISPR-Cas nucleases, provided important context for the rest...to plan your CRISPR experiment for our new CRISPR/Cas users) and updated Synbio page to people who were...the CRISPR-Cas9 system. Mehmet Fatih Bolukbasi from Scot Wolfe's lab described a chimeric Cas9 that is improving...followed by three of the biggest names in the CRISPR-Cas9 field – Jennifer Doudna, Feng Zhang, and Keith Joung...

...et al. "Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases." Genome...large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes." Development, growth & differentiation...mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering." Cell 153.4 (2013): ...plasmid contains humanized Cas9 expression cassette and sgRNA expression cassette. The target sgRNA can be...expression cassette reconstitution. When the target sequence was digested by sgRNA guided Cas9 endonuclease... encoding Cas9 into the oocyte. This original method was developed as the use of CRISPR/Cas9 in mammalian... guest blogger Samantha Young. The use of CRISPR/Cas9 for gene editing has expanded since its adaptation...

...as a template. To do so, they used a Cas nickase, instead of dCas9. The resulting editor, BE3, nicks the...editing requires three elements. They are, broadly: A Cas protein (catalytically dead or nickase) fused to ...a deaminase that makes the edit A gRNA targeting Cas to a specific locus A target base for editing within...within the editing window specified by the Cas protein These elements were the starting point towards ...evolution of an adenine base editor with improved Cas domain compatibility and activity. Nature Biotechnology...individual Cas9. They also mutagenized the cytidine deaminase portion of the base editor to create SpCas9 base..., a base editor containing high fidelity Cas9 variant HF-Cas9 (Rees et al., 2017). HF-BE3 showed 37-fold...

... Addgene blog PRIME Editing CRISPR 101: Cas9 vs the Other Cas(s) Progress Towards a PAM-free CRISPR... transposases or the Cascade-Cas3 system. We also describe newly engineered Cas9s with more flexible requirements...and enhanced editing, as well as alternatives to Cas9. All of this is accompanied by new or refreshed ...

...these kits, as there are many options! CRISPR CRISPR-Cas can harness the power of MoClo to expand both targetability...sgRNAs, MoClo can also be used to test a variety of Cas enzymes with different PAM requirements and editing...optimization, and more. While there are many CRISPR-Cas libraries and tools available for certain model organisms...elements impact viral titer and to compare different Cas enzymes in all-in-one AAV delivery. While Fragmid... chromosomal integration of larger expression cassettes (Shaw et al., 2023). Those looking to express ...unique sgRNAs into a multi-TU targeting construct (Cas9, sgRNA, reporter, etc.). This allows for the multiplexing...repurposed for CRISPR applications (gene insert = Cas9 + gRNA), Addgene distributes specialized CRISPR ...

...    References and resources References Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The...https://www.ncbi.nlm.nih.gov/books/NBK27106/  Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The...complement system proteins, which can initiate a cascade that helps attack extracellular pathogens.  Transportation...encode those proteins are designated by the lower-case Greek letters μ, δ, γ, ε, and α. Some isotypes have...