...the Addgene Blog Learn about High Fidelity Cas9 Variants How to Design Your gRNA Delivering CRISPR Ribonucleoproteins...at the Rockefeller University in NYC. The CRISPR/Cas9 system is a versatile tool for precise gene editing...organisms and model systems. We have used CRISPR/Cas9 extensively for the purpose of making sequence-specific...induced pluripotent stem cells (iPSCs). The CRISPR/Cas9 com­plex is very efficient at introducing double...Cas-blocking mutations After genome editing with CRISPR/Cas9 in human cells was described in 2013 (Cong et al...amounts of single cell clones. Furthermore, CRISPR/Cas9 editing is often bi-allelic, meaning that even if...systematically analyzed: introducing silent CRISPR/Cas9 blocking mutations in either the PAM (changing one...

...such as long or highly structured templates. Cas9 variants PE6e–g often had even higher editing efficiencies...vivo. Incorporating reverse transcriptase (RT) variants, PE6a and PE6b are 500–800 bp smaller than previous...PE6d are optimized for dual-AAV systems. These variants use different RT enzymes and performed large edits...edit.   Figure 1: Choosing the best PE6 variant for your application. Image reused form Doman and...

... activation CRISPR activation uses dCas9, a CRISPR protein variant lacking its endonuclease ability, to...J, Collins JJ, Church GM (2015) Highly efficient Cas9-mediated transcriptional programming. Nat Methods...Perrimon N, Collins JJ, Church G (2016) Comparison of Cas9 activators in multiple species. Nat Methods 13:563...transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517:583–588 . https://doi.org/10.1038...Gersbach CA (2013) RNA-guided gene activation by CRISPR-Cas9–based transcription factors. Nat Methods 10:973–... cell lines.   dCas9-VP64 Description CRISPR activation can occur through fusing dCas9 with VP64, a strong... for CRISPR-based gene activation. dCas9-CBP In this method, dCas9 is fused to CBP, an histone acetyltransferase...

...time CRISPR users Learn how to Choose the Best Cas9 Variant for Your Next Experiment Learn More About Designing...Additional Resources on Addgene.org Find Other Cas9 Variants for Purifying Genomic Regions of Interest Here...co-expressed, the gRNA targets the dCas9 to the loci of interest and dCas9 is biotinylated by BirA. From here...To increase the resolution, the Xu lab created a dCas9-based CAPTURE (CRISPR Affinity Purification in situ... cell line that co-expresses three components: dCas9 with an added biotin acceptor site BirA, a biotin...be pulled down via streptavidin IP. Biotinylated dCas9 goes along for the ride throughout all of these ... CAPTURE cell lines will have varying levels of dCas9 and gRNA expression. Additionally, CAPTURE 3C-based...

...option is to use variants of Cas9, like split Cas9, or a smaller Cas9 ortholog like SaCas9. You could even...at double-stranded break points caused by CRISPR-Cas9 editing. The good news is that this integration ...approximately 4.7 kb — which means that when using Cas9, you’re already almost out of space! Don’t let this... you have one AAV vector dedicated to expressing Cas9, while the other houses the gRNA and scaffold. If...even venture away from the comfort of Cas9, and opt for a completely different Cas effector, like Cas12a...

... eSpCas9(1.1), or HypaCas9. Read more about Cas9 options in our blog post here. Dealing with low editing...version of Cas9 that has been specifically engineered for reduced off target editing, such as SpCas9-HF1, eSpCas9...gRNA and introduced it into your target cells with Cas9. Hooray! Now it’s time to make sure your genome ... product of both an unedited population and your Cas9-targeted cells. Simply upload both trace files to...with the estimated editing frequency of your sgRNA-Cas9 combo. Pro tip: When designing your PCR, ensure ...introduced a donor template along with your gRNA and Cas9 combo and are hoping for homology-directed repair...despair, you have options! If you think delivery of Cas9 or gRNA is the issue, check out or blog on optimizing...

...CRISPR-Cas9 genome editing may be the hot new way to manipulate gene expression, but other gene manipulation...expression; these deletions are defined using 2 loxP variants (only identical loxP sites can mediate recombination...either deletion removes one of the other loxP site variants, preventing further recombination. Brainbow-1.1...with recombination events specified by three loxP variants, but the principles remain the same. The first...Variations Between Brainbow Systems   Brainbow variants and applications beyond the mouse brain In addition...Brainbow to be used in a variety of species. More variants have been created by other labs, including R26R-Confetti...

...Use Cas9 Activators Perform Genome-Wide Screens with CRISPR Generate Mouse Models with CRISPR/Cas9 Additional...techniques, please see “Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity” by Tycko et al. 2016...improving the genome-wide specificities of CRISPR–Cas9 nucleases”
by Tsai and Joung 2016. Many thanks ...high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing...sequencing: a comparison of methodologies for KRAS variant detection." Biotechniques 53.3 (2012): 182-188....of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity." Nature biotechnology 31.9.... "Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells." Nature methods...

...fluorescent protein to various Cas9 variants, SpCas9, NmCas9, and St1Cas9. These fusions localized to specific...The catalytically dead Cas9 protein (dCas9) is well known for its ability to bind DNA targets without...inhibition, among others. Over the past few years, dCas9 has also become a robust visualization tool to study...University of Massachusetts Medical School developed a dCas9-based multicolor labeling system to visualize chromosomal... al. “Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow.” Nature...

... years, scientists have utilized CRISPR-Cas9 protein variants, Cas13, and Cas12a, to develop simple, portable...Cas12a (Cpf1) is a CRISPR Cas variant that can also cleave dsDNA similarly to Cas9 (Zetsche et al., 2015 )....

...for oxidizing environments to photoconvertible variants, it seems like GFP is always learning new things...proteins with a modular structure, constructing split variants should be relatively straightforward, but it is...versions of non-modular proteins like Cre. Could Cas9-DOG be developed soon to allow genome modification...

...plasmids available! Interested in purifying Cas9? Check out pET-28b-Cas9-His from Alex Schier's lab, designed...the lab of Yonglong Chen, pCS2-3xFLAG-NLS-SpCas9-NLS is a Cas9 expression plasmid that was used by Chen...colleagues, FokI-dCas9 expresses Fok1 nuclease domain fused to catalytically inactive Cas9 DNA-binding domain...which is 45 nm more red-shifted compared to other variants. The group further optimized Chrimson through ...off-kinetics (15.8 ± 0.4ms from 21.4 ± 1.1 ms); this new variant was named ChrimsonR. Plasmids containing Chronos...lentiviral and AAV expression vectors for certain variants. Klapoetke et al., Nat Methods 2014 Mar 11;(...gene/region of interest is a key step for CRISPR/Cas9 gene editing. Dr. Masahito Ikawa has created a GFP...

...Gram-positive bacteria (Kellenberger et al., 2013). Other variants of Spinach include Baby Spinach, a miniaturized...cellular RNA to specific intracellular locations using Cas9 and track the localization of the complex. Broccoli...short non-coding RNAs (Autour et al., 2018). Three variants of Mango, coined Mango II, III, and IV, were then...high binding affinity to TO1-biotin. Two of these variants exhibit increased brightness compared to Mango...

...editors use either catalytically dead Cas9 (dCas9) or a Cas9 nickase (nCas9) to avoid introducing double-strand...need from Cas9 are sequence specificity and the R-loop structure. In order to bind DNA, Cas9 requires ...expanded CGBE target scope by creating BE variants with other Cas9s that recognize different PAMs. Their computational...non-target strand of DNA for Cas9 to bind.   R-loop The structure formed by Cas9 binding to DNA and separating...PAM) on the opposite strand. Once it finds a PAM, Cas9 unwinds the adjacent DNA to create a region of separate...single-stranded DNA (ssDNA) region, using enzymes fused to Cas9 to alter the exposed ssDNA bases. Thus, base editors...window restricted to the stretch of ssDNA created by Cas9 binding. Pro tip! In CRISPR, the non-target strand...

... samples. Ma et al. designed plasmids containing Cas9 and two different gRNAs, and electroporated them...genome sequencing in selected control and CRISPR-Cas9 injected embryos. In all cases, they did not observe...CRISPR Topic Page Read about High Fidelity CRISPR Variants Target Multiple Genes at Once with CRISPR Resources...

...2010 Yuhai Cui Red mScarlet Split-wrmScarlet variant of mScarlet Split-wrmScarlet and split-sfGFP: tools...-10) and sfCherry2(11), also photo-activatable variant PAsfCherry2(1-10) Improved split fluorescent proteins...Split-horseradish peroxidase (Alice Ting Lab 2016)  Split-Cas9 for inducible genome editing (Feng Zhang Lab 2015...

...Blainey’s lab has developed a multiplexing approach to Cas9-based pooled CRISPR screens called CROPseq-multi...Anzalone et al., 2019, including modifications to the Cas9 or reverse transcriptase enzymes, inhibition of ...end of the pegRNA, which is not well protected by Cas9. This is similar to the proposed mechanism for engineered...domain conferred several new improvements. The new variant has increased signal-to-noise ratio and better ...of applications, including CRISPR activation by dCas9-VPR, prime editing, and delivery of disease-relevant...

... line knock-ins of patient mutations which are variants of unknown significance – mutations with unclear...CRISPR 101: Making a Knock-In Cell Line Using CRISPR-Cas9 to Edit Disease Out of the genome Tips for a first...

...) model consists of a catalytically dead Cas9 protein (dCas9) that lacks endonuclease activity coupled...effectively change metabolic flux. The dCas9 protein and orthogonal variants can be used as regulatory ON/OFF.... Using dCas9 as an on/off switch The multiple scRNAs all individually utilize the same dCas9 protein....modulate dCas9 expression, multiple scRNA transcriptional programs can be regulated with dCas9 as a master...an orthogonal dCas9 protein that will still recognize guide RNA but with a different dCas9 binding module...specific proteins via aptamer domains. Combined with dCas9, this new scaffold RNA (scRNA) will bind to a PAM-adjacent...layered with distinct programs controlled by each dCas9 regulatory switch. By no means limited to the field...

...blog CRISPR 101: Which Cas9 do I choose for my CRISPR experiment? CRISPR 101: Cas9 The Other Cas(s) CRISPR...more specific variants. HypaCas9, sSpCas9(1.1), SpCas9HF1, and evoCas9 are a few spCas9s which have been...Ma X, Gao F, Guo Y. Off-target effects in CRISPR/Cas9 gene editing. Front Bioeng Biotechnol. 2023 Mar ...of Maya Kostman. The most commonly used Cas, spCas9, has gone through multiple rounds of optimization...