...technology. Nano-lanterns contain a Renilla luciferase variant fused to an FP; when supplied with a luciferase...BRET). Photons emitted by a Renilla luciferase variant are used to excite the fused fluorescent protein... endogenous protein fusions created using CRISPR/Cas9. eNLs offer high luminescent signal for imaging ...Addgene. Suzuki, Kazushi, et al. “Five colour variants of bright luminescent protein for real-time multicolour...

... Check out this post to see if an engineered Cas9 variant might work for your experiment. PITChing MMEJ...Corn’s lab describe how a better understanding of Cas9 biophysics can help enhance HDR. CRISPR 101: Ribonucleoprotein...tool to help you design, pick, and close guides for Cas9 and Cpf1. This tool supports over 250 genomes, and...The PAM Requirement and Expanding CRISPR Beyond SpCas9 The closer your edit is to the PAM site, the higher...

... a colorful Cas9 toolbox, Ma et al. turned to SpCas9 and its orthologs NmCas9 and St1Cas9. Each ortholog...locus, and to do so in vivo. Catalytically dead Cas9 (dCas9) cannot induce gene editing, but it retains its...the other orthologs. Ma et al. tested their dCas9 variants using gRNAs specific for telomeric sequences...success of Ma et al.’s approach, fluorescently labeled Cas9’s come with a number of limitations. Since color...by PAM sequence, each color requires a different Cas9 ortholog, and the target sequence must be located...genome editing. If used with catalytically active Cas9, the short, CRISPRainbow gRNA will permit labeling...publication at Addgene. 4. Kiani, Samira, et al. “Cas9 gRNA engineering for genome editing, activation ...

...protein variant (CyPet) optimized for FRET measurements with a yellow fluorescent protein variant (YPet)...protein variant (YPet) optimized for FRET measurements with a cyan fluorescent protein variant (CyPet)...Technique PI Purpose pCas9 42876 CRISPR Luciano Marraffini Bacterial expression of Cas9 nuclease gRNA. For...Anhydrotetracycline inducible expression of wild-type Cas9 from S. pyogenes for inducing double stranded breaks. For... periplasm. Series contains vectors with a YFP variant that functions in the periplasm and can be co-localized... pCri System Various Plasmids T7/lacO and lacO variants that can be expressed in B. subtilis Various combinations...Anhydrotetracycline inducible expression of catalytically inactive Cas9 ( S. pyogenes ) which can be combined with expression...

...pDGE Dicot Genome Editing Kit contains Cas9, Cas9 nickase, and Cas9 activator expressing plasmids that can...CRISPR-Cas9-based tool for targeting CpG methylation by fusing catalytically inactive Cas9 (dCas9) with...high-throughput CRISPR/Cas9 gene modification in Caenorhabditis elegans CRISPR/Cas9 technology has revolutionized...reported on a novel Cas9-based activator developed by the Church lab, termed “dCas9-VPR”, which facilitates...gRNA expressing cells were then transfected with a Cas9-expressing plasmid to generate a population of mutant...post entitled “Genome-wide Screening Using CRISPR/Cas9”. Detailed information regarding the Wu Lab library...gene was chosen to represent the primary splice variant in 38 types of human cancer. Many of the transcripts...

...Screening immune cell lineages in vivo using the CRISPR-Cas9 system CHIME Article contributed by Aliyah Weinstein...custom nucleases for each gene of interest. CRISPR-Cas9 tools have been used in immunology for in vitro ...Medical School developed a solution: CHIME, a CRISPR-Cas9 system that can edit genes in any immune lineage...in vivo, the non-immunogenic violet-excited GFP variant Vex. To understand the impact of this gene editing...expressed this construct in bone marrow stem cells from Cas9-expressing mice, and used the cells to reconstitute...

...organism without a specific preferred mutation Cas9 (or Cas9 nickase) Single (or dual) gRNA targeting 5′ ...generating a point mutation or inserting a tag Cas9 (or Cas9 nickase); Base editor Single (or dual) gRNA ...concern, Cas9-gRNA ribonucleoprotein (RNP) complexes are advantageous due to the transient Cas9 expression... RNA delivery of Cas9 and gRNA In vitro transcription reactions generate mature Cas9 mRNA and gRNA, which...generate transgenic embryos Cas9-gRNA ribonucleoprotein (RNP) complexes Purified Cas9 protein and in vitro transcribed...want to consider alternative Cas enzymes ( see Cas9 variants and PAM sequences ). Once possible PAM sequences...and/or Cas9 expression or (2) a lack of efficient target cleavage in cells expressing both Cas9 and gRNA...

...CRISPR Antimicrobials Day 7: Multiplexing and Cas9 variants: CRISPR 101: Multiplex Expression of gRNAs and...Pooled libraries: Genome-Wide Screening Using CRISPR/Cas9 and Plasmids 101: NGS Quality Control for Pooled... CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9 Day 10: Transcriptional regulation:...The PAM Requirement and Expanding CRISPR Beyond SpCas9 Day 8: Repair mechanisms: CRISPR 101: Non-homologous...

...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...

...of plasmids expressing different functional variants of Cas9 and empty gRNA backbones. A few examples of... an intronized Cas9 series of vectors and DNA modules for assembly of CRISPR/Cas9 constructs - Sylvestre...CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants - Niko Geldner. CRISPR-Cas9 vector...truncatula , wheat, and barley - Daniel Voytas. A CRISPR/Cas9 Toolkit for use in a variety of plant species (e.g...or more gRNA expression cassettes - Qi-Jun Chen. Cas9 and gRNA backbone for use in rice. Includes a protocol...species and genotypes - Zhenghe Li. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex...MoClo-compatible zCas9i Cloning Kit includes a high efficiency intron-optimized SpCas9-coding gene, zCas9i, and an...

... 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...

...Uses Viral Integration Serotypes Pseudotyping AAV Variants References AAV Components The small (4.8 kb) ssDNA...plasmids available from Addgene. The popular CRISPR/Cas9 genome editing system has been modified for use ...of AAV-DJ, but with enhanced brain uptake. AAV Variants As mentioned above, AAV improvements have included...hybrid viruses with new properties. Other system variants include: Self-complementary AAV (scAAV): One downside...Mol Ther. 2009 Aug;11(4):442-447. PubMed . AAV variants: Self-complementary recombinant adeno-associated...use in AAV, or containing Staphylococcus aureus (SaCas9) . AAV is also a promising method for gene therapy...

...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 )....