...fusion for gene activation CRISPR/Cas9 Plasmid Variants Figure 1: Schematic of the CRISPR Collection. Plasmids...in retinal development the lab utilizes a CRISPR/Cas9 based approach to knockout, repress, and activate...taken advantage of the Zhang lab all-in-one (guide/Cas9/reporter) pX458 plasmid family. They have made several... with EF1alpha addition of an mCherry reporter variant nuclear reporters via an H2B fusion shuttle plasmid... plasmids dCas9-KRAB-MeCP2 fusion for gene and non-coding enhancer sequence repression dCas9-VPR fusion...175574 express dCas9-KRAB-MeCP2 for repression. Plasmids 175571 and 175572 express dCas9-VPR for activation...Sources Original pX458 backbone ( Addgene #48138 ) dCas9 + KRAB-MeCP2 ( Addgene #110821 ) VPR ( Addgene #63798...

...expression of S. pyogenes Cas9 or Cas9 variants (hereafter referred to as Cas9) and a single stranded guide...to the nucleus, and several codon optimized Cas9 variants have been created in an effort to increase translation... gRNA backbones also contain Cas9, which enables expression of both Cas9 and the gRNA off of the same ...Nuclease dead Cas9 (dCas9) based activators (such as dCas9-VP64) or repressors (dCas9-KRAB or dCas9-SRDX) can...get more information on multiplexing gRNAs here). Cas9 is commonly tagged with a nuclear localization sequence...repress target genes in plant cells, respectively. Cas9 expression is commonly driven by plant-derived RNA...Examples of commonly used RNA pol II promoters for Cas9 expression include the ubiquitously expressing cauliflower...

... 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...the other orthologs. Ma et al. tested their dCas9 variants using gRNAs specific for telomeric sequences... 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... chromatin organization. This fluorescent CRISPR/Cas9-based method is a great step forward in live cell...As this study joins the pantheon of useful CRISPR/Cas9 techniques, we at Addgene are excited to see what...

...are numerous GFP variants. Here I use the name ‘GFP’, which refers to mEGFP. This variant was made by introducing...nuclease-deficient Cas9 can bind loci in the genome that are defined by a gRNA. Since Cas9 is a protein, a...jellyfish GFP (AvGFP) to create enhanced GFP (EGFP), a variant with improved brightness (Tsien, 1998). Another...necessary to generate the strictly monomeric EGFP variant, mEGFP (Zacharias et al., 2002). GFP needs oxygen...Cranfill et al., 2016 and FPbase.org). A number of variants remain fluorescent under acidic conditions, with...although it still is worthwhile to generate several variants and check the functionality of the fusion (Mastop...

... CRISPR 101: Which Cas9 do I choose for my CRISPR experiment? CRISPR 101: Cas9 The Other Cas(s) The...field has exploded with dozens of Cas enzymes and variants available. Without a comprehensive resource, it...Addgene. So whether you're looking for the exact Cas9 plasmid used by Jinek et al. 2012, or browsing for...CRISPR–Cas systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https...Once upon a time, not so long ago, spCas9 was the only Cas enzyme widely available and applied by researchers...and trans activity of each entry. For example, SpyCas9a (one of the most frequently used Cas enzymes) ...CasPEDIA Cas ID display information for a given Cas (SpyCas9a) from CasPEDIA.   Helpful resources and information...

...single species or PAM binding variant of Cas9. For instance, wild-type SpCas9 must be used with targets that...increases off-target effect. Which species or variant of Cas9 ( S. pyogenes , S. aureus etc.) was this gRNA...Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...

...Components of CRISPR  xCas9: Engineering a CRISPR Variant with PAM Flexibility Cas9 vs the Other Cases Further...the basic parts of the Cas9 enzyme.   Figure 1: A cartoon depiction of Cas9’s two major lobes, REC...   Unbound Cas9 Now that we know what we’re looking at, let’s peek at unbound—“apo”—Cas9 from Streptococcus...residues in this loop to produce SuperFi-Cas9, a high-fidelity variant that still retains high cleavage efficiency...Figure 8: Comparison of Cas9:RNA crystal structure (left, PDB ID 4ZT0) with Cas9:RNA:DNA electron microscopy... with a nearby protospacer-adjacent motif (PAM)? Cas9 is becoming an ever more ubiquitous tool for genome... bridge helix. Created with BioRender.com.   Cas9 has two major lobes, called NUC (for nuclease) and...

...Expanding the base editing scope in rice by using Cas9 variants." Plant biotechnology journal (2018). PubMed...by tethering cytosine deaminase with Cas9 nickase (nCas9). nCas9 creates a nick in the DNA by cutting ...Nickase Cas9 (D10A) Deaminase PAM requirements Reference CBE SpCas9 rAPOBEC1 NGG 6 SpCas9 Target-AID...target genomic locus/loci, choose a base editor variant from Table 1 depending on the PAM sequence. Avoid...Bastian Minkenberg, and Yinong Yang. "Boosting CRISPR/Cas9 multiplex editing capability with the endogenous...Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion." Nature biotechnology...Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion." Nature biotechnology...

.... Repair of Cas9-induced breaks by NHEJ As illustrated below, NHEJ-mediated repair of Cas9-generated breaks... 2020). NHEJ can also be engaged by variants of the canonical Cas9 approach. A pair of CRISPR guides that...Cas system for genome engineering is the fact that Cas9 can be easily programmed to make a DNA double strand...discuss NHEJ, and how it impacts what happens to Cas9-mediated DSBs in the genome. Non-homologous end...consequently the principal means by which CRISPR/Cas9-introduced breaks are repaired. The following factors...introduce indels. Given the end structure of the Cas9 DSB (blunt or near-blunt ends without nucleotide...population of cells constitutively expressing a targeted Cas9 should possess indels in the majority of their chromosomes...

...fully functional Cas9 upon dimerization. Splitting wild-type Cas9 into n-terminal (Cas9(N)-2xNLS) and c-terminal... can write a blog post about it! Split Cas9 system The Cas9 protein is composed of an n-terminal DNA... the bi-lobed architecture of Cas9 to engineer a series of "split" cas9 molecules that cannot function...activation, the c-terminal cas9 fragment was fused with FK 506 binding protein 12 (Cas9(C)-FKBP-2xNLS) and the... the n-terminal cas9 fragment with FKBP rapamycin binding domain of mTor (Cas9(N)-FRB-NES) resulting in...rapamycin-inducible Cas9 for genome editing. Without rapamycin treatment, the Cas9(N)-FRB-NES fragment...sequence. Treatment with rapamycin induces Cas9(N)-FRB-NES and Cas9(C)-FKBP-2xNLS dimerization and net influx...

...enzyme, the development of SaCas9, NmeCas9, Cas12a enzymes, and engineered variants thereof offers additional... capacity of a locus. When using S. pyogenes Cas9 (SpCas9), potential target sites are both [5’-20nt-NGG... (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...to the intended edit rather than some passenger variant that came along with the single cell clone (although...even more exquisite for the so-called base editor Cas9, which makes DNA changes in the absence of dsDNA...2019) Agreement between two large pan-cancer CRISPR-Cas9 gene dependency data sets. Nat Commun 10: . https...

...continuation of that work, they have found at least two variants that display increased neuronal expression in ...side effects, by allowing the user to select for variants that infect the CNS while selecting against those...of up to 20%, comparable to commonly used CRISPR/Cas9 methods for large inserts. However, this system ...in comparison to the 62-90% error rate seen with Cas9 editors. The lab believes that this system could...inactive dCas9 and microbial SSAPs (dCas9-SSAP) which insert genes in a cleavage free manner. The dCas9-SSAP...cleavage-free dCas9-based gene editor using microbial SSAPs. Image from Wang, et al, 2022.  Find dCas9-SSAP...Cre-mediated AAV Targeted Evolution method (M-CREATE) dCas9-SSAP with reduced error rate for long sequence knock-ins...

...using CRISPR and Wednesday will highlight some Cas9 variants. Next, we’ll look at various CRISPR applications...

...sequence targeted by the Cas9 nuclease. The Streptococcus pyogenes Cas9 (SpCas9) requires an NGG PAM to...range of CRISPRCas9 by engineering SpCas9 Article contributed by Shreya Vedantam CRISPR/Cas9 is widely...increased versatility of the engineered SpCas9-NG and its variants can be used to engineer specific sites...  The Kanemaki lab previously developed a CRISPR/Cas9 based method to tag endogenous proteins in HCT116...widely used genome editing tool. However, Cas9’s endonuclease activity can be limited by the need for a specific...recognize the target. Osamu Nureki’s lab engineered SpCas9 (SpCas9-NG) with relaxed preferences for the third ...NGG. In addition to the engineered SpCas9, they also fused the SpCas9-NG with the activation-induced cytidine...

...different strategies with CRISPR-Cas9. There are so many different variants and strategies out there. This...pluripotent stem cell (iPS) technology and CRISPR-Cas9 constructs from Addgene to gene-edit mutations into... of those mutations, they now plan to use CRISPR-Cas9 technology to create cell lines and/or mouse models...ve proposed to use plasmids with modified nickase Cas9 protein in order to target the specific gene region...

...populations that already express Cas9 from an unlinked locus, while a Cas9+sgRNA drive targeting a synthetic...residual sgRNAs and possibly cas9 gene). An immunizing reversal drive is a variant that also spreads through...School. Scientists making transgenic organisms with Cas9 should be aware of the potential hazards of creating...caution. Why does making transgenic organisms with Cas9 risk creating a gene drive? The simplest way to ...last year2, delivering a DNA cassette encoding the cas9 gene and single guide RNAs (sgRNAs) with appropriate...easiest: don't use a DNA vector that encodes both Cas9 and sgRNA. What if I want to work with gene drives...underlying advance enabling RNA-guided gene drives is Cas9 genome editing. Finally, gene drives are an inherently...

...Best Cas9 Variant for Your Next Experiment Find out More about Genome-wide Screens with CRISPR/Cas9 Learn...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...context of the gene, Cas9 activators efficiently solve these problems.   Which Cas9 activator should I ...with AAV Resources on Addgene.org Find Cas9 Activators Find Cas9 Repressors Catch Up on Your CRISPR Background...there has also been significant work on constructing Cas9 transcriptional activators. These constructs allow...upregulation of essentially any gene by fusing mutants of Cas9 deficient in DNA cutting activity to a transcriptional...

...one AAV, Cas9 must be made smaller. Previous attempts to “shrink” Cas9 include the use of St1Cas9 (~3.3 ...amino acids in length, Cas9 from Campylobacter jejuni (CjCas9) is the smallest Cas9 ortholog characterized...vehicle for Cas9. However, to allow for more room for regulatory sequences while still fitting Cas9 and its...equally potent Cas9 protein, the Zhang lab took a different approach. They analyzed over 600 Cas9 orthologs...Resources on the Addgene Blog Learn more about Cas9 Variants and the Expanded CRISPR Toolbox Read about CRISPR... aureus Cas9 (SaCas9, 1053 amino acids) displayed cleavage activity in mammalian cells. SaCas9 produced...truncated Cas9. Unfortunately, certain drawbacks limit the utility of these systems: St1Cas9 requires ...

...efficiently, they’ve recently adapted the CRISPR/Cas9 system for protein tagging. We spoke with Mendenhall...context? Mendenhall: The benefits are two-fold: CRISPR/Cas9 offers high efficiency of inducing double strand...technologies. Generally speaking, anyone using CRISPR/Cas9 in the lab already should be able to run this protocol...be doing something similar. There may be other variants of this that are better for some experiments than... crispr/cas9... cas9...

...CRISPR-based genetic screening methods have primarily used Cas9 to knock out genes, researchers at the Genetic Pertubation... a single expression cassette is advantageous to Cas9’s larger expression requirements (Figure 1), but...optimizing enzyme activity using an enhanced Cas12a variant (enAsCas12a), minimizing off-target effects by ... guides. While two guides require two arrays for Cas9, two guides can be streamlined into a single array...-wide library capable of performing similarly to Cas9 screens in a compacted library. Overall, Cas12a’...CRISPR-Cas effectors and fusion proteins, such as Cas9 and Cas12a, creates challenges for in vivo delivery...529 amino acids, less than half the size of either Cas9 or Cas12a. Despite its small size, CasMINI is comparable...