... 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...ortholog was fused to a different fluorescent protein to create three colors. The specificity of these orthologs...estimate that a minimum of 150-200 fluorescent protein molecules is necessary for a detectable signal,... scaffold that can be used to recruit multiple protein molecules (and also available from Addgene!) Since...

...fluorescent and 3xFLAG-tag fusion proteins. The repair template and Cas9-sgRNA plasmid is then injected ...tagging of every protein in the C. elegans genome. Although they tested fluorescent protein fusions, this...for CRISPR/Cas9 editing in C. elegans: Engineering the Caenorhabditis elegans genome using Cas9-triggered...2013, a remarkable nine papers describing CRISPR/Cas9 genome engineering methods for C. elegans were released...even easier - now, one can generate a fluorescent protein fusion, transcriptional reporter, and loss-of-function...Multiple features of C. elegans have made CRISPR/Cas9 so successful in this species, namely, the robust...create a new and efficient selection-based CRISPR/Cas9 method to generate knock-ins on any genetic background...

...edits) engages the prime editor protein. The prime editor consists of a Cas9 nickase fused to a reverse transcriptase... that most Cas9-induced DSBs are repaired by non-homologous end joining. As a result, Cas9 treatment causes... ~ ~ ✓ ✓ ✓ R221K N394K Cas9 mutations Improve Cas9 nuclease activity ~ ~ ~ ~ ... transcriptase. The Cas9 nickase is guided to the DNA target site by the pegRNA — a guide RNA that also...homology to the genomic DNA locus. After nicking by Cas9, the homologous pegRNA sequence hybridizes to the...-MLV) reverse transcriptase to the C-terminus of Cas9 H840A nickase (PE1). But innovations by the Liu ...generate precise changes. However, the efficiency of Cas9 cleavage is relatively high while the efficiency...

...pCRISPomyces-1 includes Cas9, a tracrRNA, and a CRISPR array, while pCRISPomyces-2 contains Cas9 and a gRNA cassette...functions more similarly to CRISPR/Cas9 systems adapted for eukaryotes in that Cas9-mediated cleavage induces ...targeting either a catalytically dead Cas9 (dCas9) alone or a dCas9 fused to the omega subunit of RNA polymerase... from Addgene consists of two plasmids: pCas9: carries Cas9 and chloramphenicol resistance pCRISPR: carries.... They identified the Cas9 orthologue from Streptococcus thermophilus, Cas9sth1 as a robust and highly...were able to engineer a dCas9 variant fused to bacterial enhancer binding protein required for the activation...when using a nickase version of Cas9, but were also successful using a dCas9 variant as well. Furthermore...

...It’s clear that CRISPR-Cas9 technology has really changed the game for anyone looking to quickly and ...include enrichment against kinases, cell cycle proteins, and more.  “In the past, genome engineering with...Lander library uses sequential infection first of Cas9, then with the sgRNAs, and can result in a higher...leveraged the CRISPR system to target all 19,150 mouse protein coding regions with 87,897 unique gRNAs. Their ...that have previously been stably transfected for Cas9 expression. The Yusa lab has used their library ...aid efforts to understand the function of both protein-coding genes as well as non-coding genetic elements...application offer proof-of-principle that CRISPR-Cas9 can be used to target any sequence in the genome...

...diacetyl coelenterazine-h. Unlike coelenterazine-h, diacetyl coelenterazine-h does not autooxidize and...reporter genes for endogenous protein fusions created using CRISPR/Cas9. eNLs offer high luminescent signal...about Photoactivatable Fluorescent Proteins Check Out Fluorescent Protein Timers Use Rosella to Study Autophagy...Fluorescent Protein Resources Find FRET Plasmids Find Empty Backbones with Fluorescent Protein Tags  ...esterase then converts diacetyl coelenterazine-h to coelenterazine-h, producing a constant supply of substrate...be limited by a few caveats. Since fluorescent proteins (FP) require external light activation, you can...practical due to the low intensity of luminescent proteins. To make luminescent imaging a reality, Addgene...

...available to assess protein-protein interactions. One popular approach is to fuse a protein of interest to ...single-molecule tracking of protein-protein interactions in living cells. Shao S, Zhang H, Zeng Y, Li Y, Sun C...split fluorescent protein (FP) and measure the signal produced when the candidate proteins’ interaction brings... Figure 1: Two proteins are fused to the FP(11) and FP(1-10) fragments. When Protein A and B interact...Multiplexed labeling of cellular proteins with split fluorescent protein tags. Tamura R, Jiang F, Xie J,... and GFP(11) Versatile protein tagging in cells with split fluorescent protein. Kamiyama et al. Nat Commun...

...sequence targeted by the Cas9 nuclease. The Streptococcus pyogenes Cas9 (SpCas9) requires an NGG PAM to...By tagging proteins with a 7kD degron called mAID and expressing the essential plant protein TIR1 in non-plant...previously developed a CRISPR/Cas9 based method to tag endogenous proteins in HCT116 cells with mAID. Using...range of CRISPRCas9 by engineering SpCas9 Article contributed by Shreya Vedantam CRISPR/Cas9 is widely...factors tethered to dCas9, require long time scales with constitutively expressed proteins that are not inducible...light-activated-dynamic-looping (LADL) system. The group fused dCas9 to a truncated CIB1 protein from Arabidopsis (CIBN), which they...   New toolkit for N or C terminal tagging of proteins for degradation using AID Article contributed ...

...lacked an “off-switch” for Cas proteins. Discovery of anti-CRISPR proteins That is, until the discovery...of anti-CRISPR proteins in 2012 (Bondy-Denomy et al., 2012). Anti-CRISPR (Acr) proteins are phage-derived...50 anti-CRISPR proteins that interact with CRISPR-Cas systems such as Cascade-Cas3, Cas9, Cas12, and Cas13...Marino et al., 2020). Some Acr proteins are specific to a particular Cas protein while others can inhibit CRISPR...fourth Acr protein discovered that blocks type II-A CRISPR-Cas systems. A full list of Acr proteins organized... found here. How do anti-CRISPR proteins work? Anti-CRISPRs proteins are highly diverse but most block...blocks Cas9’s interaction with the PAM site (Dong et al., 2017), and AcrIIC3 causes Cas proteins to dimerize...

...choosing a Cas protein for your experiment. References Hu, Johnny H., et al. "Evolved Cas9 variants with...frequently used Cas9, spCas9 from Streptococcus pyogenes, this sequence is 3’-NGG. But Cas9s found in other...assessment of the off-target activity of Cas9. This can occur when Cas9 cleaves a site that is not a perfect...the PAM specificity of Cas9s isolated from different organisms and engineered Cas9 variants that were able...consideration when choosing a Cas9 is balancing specificity and sensitivity. Wild-type Cas9 exhibits high on-target... directed evolution to generate Sniper-Cas9 resulting in a Cas9 with improved specificity. They compared...on-site and off-site activity of Sniper-Cas9 to different Cas9 variants using gRNAs of different lengths...

...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 ...repair proteins fused to CGBEs The David Liu lab also experimented with adding different proteins to the... binding motif protein X-linked (RBMX) (Koblan et al., 2021). Several of these proteins had additive effects...non-target strand of DNA for Cas9 to bind.   R-loop The structure formed by Cas9 binding to DNA and separating...C genome editing with CRISPR-Cas9-directed base excision repair proteins. Nature Communications, 12(1)...PAM) on the opposite strand. Once it finds a PAM, Cas9 unwinds the adjacent DNA to create a region of separate...

...editing technique that relies on a fusion enzyme of Cas9 nickase + reverse transcriptase and a prime editing...., 2019), disrupting its canonical structure and Cas9 binding (Nishimasu et al., 2014). Figure...pegRNAs that use other bases in this location bind to Cas9 with canonical sgRNA structure (right). Created ...along with your intended edit. This prevents the Cas9 nickase from re-binding and nicking the newly synthesized...PE7: This editor includes the fused RNA-binding protein La to protect the end of the pegRNA. Adding 3′ ...Zhang, F., & Nureki, O. (2014). Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell, ...prime editing with an endogenous small RNA-binding protein. Nature, 628(8008), 639–647. https://doi.org/10.1038...

...red fluorescent protein (mCyRFP1). Derived from cyan-excitable organge fluorescent protein (CyOFP1), some...confirmed that the fusion protein retained the PAM properties of the individual Cas9. Kim et al. also mutagenized..., a base editor containing high fidelity Cas9 variant HF-Cas9. They subsequently tested the original BE3...-BE3  EQR-Cas9 (NGAG)  Lower off-target activity than BE3  85173  pBK-VRER-BE3  VRER-Cas9 (NGCG)   ... 87438  pET42b-HF-BE3  HF-Cas9 (NGG)  Very low off-target activity; protein purification vector  87439...self-targeting. Two of these Lmo proteins, AcrIIA2 and AcrIIA4, can work to inhibit Spy Cas9 function in both bacteria...pCMV-HF-BE3  HF-Cas9 (NGG)  Very low off-target activity; mammalian expression vector Rees H, et al. Nature...

...several Cas9s that recognize NG PAMs. The Nureki lab generated SpCas9-NG, a rationally engineered Cas9 with...some available.   Cas proteins that don’t utilize NGG   There are plenty of Cas9 variants which utilize...et. al, 2020). These Cas proteins have everything we know and love about Cas9, but with broader PAM compatibility...PAM-interacting domain of Cas9. After multiple rounds of engineering, they developed the SpRY Cas9 variant (Walton... at NGH PAMs (Zhang et al, 2021) NG Cas9 NG - SpG Cas9  NG  - SpRY nuclease  C to T Base Editor...Plasmids available here)   Nishimasu, H., et al.,Engineered CRISPR-Cas9 nuclease with expanded targeting space...notes SpCas9 NGG - Cas12a TTTV Great for AT-rich regions Cas9 EQR NGAG - xCas9 NG Less...

...CRISPR/Cas9 is an enormously plastic tool and has taken the scientific world by storm. While Cas9 has been...required for Cas9-based activation. CRISPRa for genome-wide studies One of the best uses for Cas9 activators...synthesis. Prior to Cas9 activators, similar tools were made using other DNA binding proteins such as zinc fingers...engineering an entirely new protein. This makes it much cheaper to use Cas9 activators. This larger scale...context of the gene, Cas9 activators efficiently solve these problems.   Which Cas9 activator should I ...Best Cas9 Variant for Your Next Experiment Find out More about Genome-wide Screens with CRISPR/Cas9 Learn...with AAV Resources on Addgene.org Find Cas9 Activators Find Cas9 Repressors Catch Up on Your CRISPR Background...

...STITCHR consists of a nicking Cas9 fused to a shortened R2Tocc transposon protein, and can "retarget" the transposon...the scientists fused the retrotransposon protein R2Tocc to a Cas9 nickase to improve target recruitment ...mini counterpart label nearby proteins with biotin. The biotinylated proteins can then be isolated and identified...for Glypicans DNA editing with STITCHR Dynamic protein labeling with TurboCas Enhancer-AAV viral preps...our bacterial plasmid set for expressing tagged proteins, and stay tuned for mammalian expression and AAV...therapeutic potential. Our partners at the Institute for Protein Innovation (IPI) recently released a set of highly-specific...antibodies against three of the six human glypican proteins (GPC1, GPC3, and GPC4). All IPI antibodies undergo...

...that disrupt the protein-coding capacity of a locus. When using S. pyogenes Cas9 (SpCas9), potential target... (inhibition) technologies – a nuclease-dead Cas9 (dCas9) is directed near the promoter of a target gene...with CRISPR technology is usually accomplished by Cas9-mediated dsDNA breaks: following a cut, the error-prone...code for amino acids near the N’ terminus of the protein, in order to mitigate the ability of the cell to...for amino acids close to the C’ terminus of the protein, to maximize the chances of creating a non-functional...nucleotides, restricting gRNAs to 5 – 65% of the protein coding region will still result in many dozens ...even more exquisite for the so-called base editor Cas9, which makes DNA changes in the absence of dsDNA...

...antibiotic/herbicide resistance and fluorescent proteins. Selection using resistance genes usually involves...transgene free individual. For example, with CRISPR-Cas9 gene editing, transformation may be identified by...CRISPR 101: Engineering the Plant Genome Using CRISPR/Cas9 Tips for Arabidopsis Transformation CRISPR 101: ... a terminator. Image from He, Y., Zhang, T., Sun, H. et al. Hortic Res. (2020).   I can do this by...and Resources References  He, Y., Zhang, T., Sun, H. et al. A reporter for noninvasively monitoring gene...

...evaluate the protein, perform an immunoblot using an antibody against the relevant protein 9 . References...genomic deletions in mammalian cell lines using CRISPR/Cas9 CRISPR...and Daniel Bauer's labs details the use of CRISPR/Cas9 to create genomic deletions in mammalian cell lines...Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9. Bauer DE, Canver MC, Orkin SH. J. Vis. Exp. (95...site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome ...required DNA yield. Perform maxi-prep for each CRISPR/Cas9 construct. Transfecting CRISPRs into Cells of Interest...NOTE: This protocol involves the delivery of CRISPR/Cas9 plasmids by electroporation 4 . This protocol is...

...detect, purify, quantify, deplete, and visualize proteins of interest (Greenfield, 2014). Traditionally,...chain for the antibody and when translated into protein will assemble into a fully functional antibody....encoded in a plasmid. (Right) An assembled antibody protein binds an antigen. Image from Fvasconcellos. ...genes for antibody expression. If starting from a protein sample (for instance, affinity purified antibodies...well in animals because of similarities to host proteins. After the antibody of interest has been cloned...community has created hundreds of useful variants of Cas9. By providing access to the plasmids encoding antibodies... NP, Boeckman JX, Manning CF, Nguyen JT, Bechtold H, Dumitras C, Gong B, Nguyen K, van der List D, Murray...