... CHIME works through transduction of Cas9-expressing (Rosa26-Cas9) hematopoietic stem cells with a lentiviral...genes pXPR_219 Violet-excited GFP (vex) Rosa26-Cas9 I-CHIME Inducible knockout of a single gene...Violet-excited GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 L-CHIME Knocks out a single gene in defined... Violet-excited GFP (vex) Lineage-Cre; Rosa26-Cas9 S-CHIME Knocks out pairs of genes sequentially...Violet-excited GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 Table 1: X-CHIME systems Proof of Concept These...Haining, W. N., & Sharpe, A. H. (2019). A CRISPR-Cas9 delivery system for in vivo screening of genes in...

...paper in Communications Biology Find the Cas9-PCV and PCV-Cas9 plasmids Selective curbing of unwanted ...Covalent tethering of DNA template to Cas9 SECURE base editors Nme2Cas9 CRISPR interference in Candida albicans... AAV delivery of Nme2Cas9 with gRNA cassette Erik Sontheimer’s lab identified a Cas9 protein from Neisseria...library Covalent tethering of DNA repair template to Cas9 Homology-directed repair (HDR) can be used to make...oligodeoxynucleotide HDR template covalently attached to the Cas9-gRNA RNP complex via the fused HUH endonuclease ...After testing dCas9 fusions with transcriptional repressors, they found that fusing dCas9 to an Mxi1 repressor...gene. Read the paper in mSphere Find the dCas9 and dCas9-Mxi1 plasmids If you have a new CRISPR tool...

...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...distance"  Even though we had controlled over-active Cas9 and repeated our experiments with blocking mutations...

...effects of gRNA and Cas9 expression, they fail to account for the effects of Cas9-induced dsDNA breaks... either stably expressed Cas9 or that had been lentiviral infected with Cas9. Validating the Library The...Genome-wide CRISPR/Cas9 screens are a high-throughput systematic approach for identifying genes involved.... Unanswered questions about genome-wide CRISPR/Cas9 screen design Are non-targeting guides an appropriate...single-guide studies? Many strategies exist for reducing Cas9 off-target effects, but most studies have focused...previously identified essential genes while previous Cas9 and shRNA library screens only identified  60% of...screen. This toxicity was used a proxy for detecting Cas9 cutting. Safe targeting guides better control for...

...indicates that like GeoCas9 and ThermoCas9, IgnaviCas9 is a type II-C Cas9. IgnaviCas9 is most similar to...thermostable Cas9 enzymes have previously been characterized: GeoCas9 from the Doudna lab and ThermoCas9 from ...Find IgnaviCas9 here! What can you do with IgnaviCas9? Why would a thermostable Cas9 be useful? It can enable...launched in the Quake Lab. Identifying IgnaviCas9, a hyperthermostable Cas9 This year it was wonderful to learn... Ignavibacteriae phylum genome. IgnaviCas9 is a hyperthermostable Cas9 CRISPR nuclease that is active ... our CRISPR blog posts Read about GeoCas9, another thermostable Cas9 Resources on Addgene.org Read our... to a type II-C Cas9 from Parvibaculum lavamentivorans, whose optimal growth temperature is 30°C. Find...

...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...when using a nickase version of Cas9, but were also successful using a dCas9 variant as well. Furthermore...unsuccessful and the wild-type sequence persists, Cas9 will cleave the gene of interest, inducing a lethal...

...prime editor consists of a Cas9 nickase fused to a reverse transcriptase. The Cas9 nickase is guided to the... 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 ~ ~ ~ ~ ...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...ratios of desired edits to indel byproducts than Cas9-initiated HDR. Prime editing tools Prime editing...

...Silvana Konermann, the first generation of CRISPR-Cas9 plasmids were not designed with gene activation ...question for her then was this: Can you engineer CRISPR-Cas9 activators that work well enough on any gene that...describe just such a system, which they call CRISPR/Cas9 Synergistic Activation Mediator or SAM. SAM is an... three components: 1. A nucleolytically inactive Cas9-VP64 fusion; 2. A sgRNA incorporating two MS2 RNA...system, Konermann explained, was new knowledge about Cas9’s crystal structure. “I thought maybe we could use...levels of control in the system. First of all, the Cas9s can be turned on and off, to reversibly activate...sam.genome-engineering.org/. Crystal structure of Cas9 in complex with guide RNA and target DNA. Nishimasu...

...report. A scientist working in China was using CRISPR/Cas9 in an attempt to create gene-edited babies. Several...valid critiques being offered of He’s work: CRISPR/Cas9 genome editing is not far enough along, scientifically...research published on December 13 shows us, CRISPR/Cas9 techniques are continually being refined; it is ...an embryo—even if it’s not necessarily via CRISPR/Cas9. This leaves us with the second critique. Absent...agreed-upon international moratorium on utilizing CRISPR/Cas9 in humans. This topic was to be revisited at meetings...emerging technology. We could do this for CRISPR/Cas9 – or any other germline editing technique—too. As...not “what is the translational pathway for CRISPR/Cas9 research” but whether or not we will stop shuffling...

...enrich for Cas9 variants with activity on non-NGG PAMs, generating xCas9 3.0-3.13. To test xCas9 3.0-3.13...In order to bind DNA, Cas9 and other CRISPR enzymes require a short PAM sequence adjacent to the targeted...problem, researchers have engineered alternative Cas9s binding distinct PAM sequences. Now, Hu et al., ...low predicted on-target efficiency. In cases where Cas9 won’t work, there are other options. Commonly used..., improve targeting in AT-rich loci or genomes. Cas9s from other species, like S. aureus and N. meningitidis...phage-assisted evolution to generate and select for Cas9 mutants with PAM flexibility. In this system, the...the surface of modifications that can be made to Cas9. It is a pleasant surprise that selecting for one...

...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 ...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...other ABEs, the editor they were using consisted of Cas9 fused to an engineered adenosine deaminase capable...

...Overview of the dual-plasmid CRISPR/Cas9 workflow in M. abscessus. A) Sth1Cas9 mediates double strand break ...mScarlet-based tags for live imaging in C. elegans CRISPR/Cas9 for gene disruption in M. abscessus  Bioluminescent...Mycobacterium abscessus using a dual-plasmid CRISPR/Cas9 system By Aryana Khosravani Deborah Hung’s lab has... has developed a novel dual-plasmid CRISPR/Cas9 system for gene disruption in Mycobacterium abscessus,...resistance interfering with selection. Using an inducible Cas9 expression and removing the sgRNA plasmid after ...Figure 4). This work marks the first use of CRISPR/Cas9 in M. abscessus. The simplicity and efficiency of...repair. B) M. abscessus is first transformed with Cas9 on an integrative plasmid containing tetracycline...

... 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... 2014). There are several variants of the CRISPR/Cas9 system floating around, and many pre-designed plasmids...as soon as possible! Selecting your target CRISPR/Cas9 is often used to study the biology surrounding a...the target sequence was digested by sgRNA guided Cas9 endonuclease, homology dependent repair (HR, homologous... fragments. The pX330 plasmid contains humanized Cas9 expression cassette and sgRNA expression cassette...for Testing Your gRNAs in vitro at Addgene CRISPR/Cas9 gene editing in mice Now that you’ve validated your...

...Listen to the Fire-Cas9 Podcast Segment Jerry Crabtree’s lab has developed a new CRISPR/Cas9-based system to...Inducible Recruitment for Epigenome editing by Cas9 (FIRE–Cas9), combines these approaches to enable rapid...epigenetic modulation. While catalytically inactive cas9 (e.g. dCas9) has been fused to a variety of protein modules...Communications. 2017. PubMed PMID: 28924139. FIRE-Cas9 for inducible interrogation of the epigenome Article...type. By varying which sgRNAs are used with FIRE-Cas9, experiments can be designed to target multiple ... epigenetic memory at nearly any locus. The FIRE-Cas9 plasmids can be found here. Braun et al. Nat Communications...

...interest. To get the molecular components of CRISPR/Cas9 (Cas9 protein and the crRNA-tracrRNA complex) and the...regulatory control. But even with the advent of CRISPR/Cas9 technology, inserting large tags into a precise ...We’ve created and tested plasmids that use CRISPR/Cas9 to endogenously tag a wide variety of genes with...process following the double strand break CRISPR/Cas9 makes at the target site. The whole segment—a GFP...the plasmid from being cut and destroyed by CRISPR/Cas9 during transfection. Making the double strand break...the fluorescent tag to the cells, we used CRISPR/Cas9 to make a precise cut in the genome near either ...Brock, et al. "Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization...

...sophisticated”.     New to CRISPR/Cas9 Technology? Learn More: Addgene's CRISPR/Cas9 Guide History of CRISPR Cas...awards that will be bestowed upon discovers of CRISPR/Cas9 genome engineering, Dr. Jennifer Doudna and Dr. ...They are finding they have been able to use CRISPR/Cas9 to generate mice with genes knocked out in the insulin...offered the great analogy of comparing the CRISPR/Cas9 system to Norton Antivirus. Antivirus programs work... crispr/cas9...

...Nat Chem Biol). Mutate the PAM or sgRNA sequences Cas9 likely goes through multiple rounds of cleavage ...guarantee there won’t be re-targeting after HDR. If your Cas9 cuts in the coding region of a gene, make sure that...inhibitors to your culture media prior to and after Cas9 introduction increases HDR, just make sure to remove... promoting HR was as simple as swapping out your Cas9? It can be! Cas12a is a Cas family member which ...protein to the nuclease. Early HR factors fused to Cas9 have been shown to increase HDR frequency by committing...019-0432-1 Mehdi Banan. Recent advances in CRISPR/Cas9-mediated knock-ins in mammalian cells. J Biotechnol...homology-directed repair choice in response to CRISPR/Cas9 Induced-double strand breaks. Int J Mol Sci., 21...

..., 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)   ...The popular CRISPR base editing technique uses dCas9/Cas9 nickase fused to a cytidine deaminase for targeted...Plasmid ID Plasmid Name Cas9 Variant Speacial Features 85169  pJL-SaBE3  SaCas9 (NNGRRT)    85170  pJL-SaKKH-BE3... pJL-SaKKH-BE3  SaCas9 (NNGRRT)    85171  pBK-VQR-BE3  VQR-Cas9 (NGA)  Lower off-target activity than...the effects of dCas9 binding to a genomic locus, or limiting the amount of time that Cas9 is active in ...other cytosines near the target and in predicted Cas9 off-target sites. Two recent papers from blue flame...

..., a base editor containing high fidelity Cas9 variant HF-Cas9 (Rees et al., 2017). HF-BE3 showed 37-fold...other methods. In a head-to-head comparison with Cas9, Cas9 modified 9/12 known off-targets with a 14% indel...individual Cas9. They also mutagenized the cytidine deaminase portion of the base editor to create SpCas9 base...et al., 2020 SpRY Cas9, available fused to ABEmax(7.10) or to BE4max   Evolved SpCas9 with NRN or NYN PAM... 1.1%. These numbers are a vast improvement over Cas9-mediated HDR for the loci tested, where average ...system is also subject to off-target editing based on Cas9 off-target activity.  Find CRISPR base editing plasmids...using a cytidine deaminase from sea lamprey fused to Cas9 nickase (Nishida et al., 2016). Target-AID acts ...

...Addgene!) pX330-U6-Chimeric_BB-CBh-hSpCas9 - The S. pyogenes Cas9 (SpCas9) has been widely popular in genome... with lentiCas9-Blast or with cell lines already expressing Cas9. Find lentiGuide-Puro. pSpCas9n(BB)-2A-GFP... Similar to pSpCas9(BB)-2A-Puro (PX458), pSpCas9n(BB)-2A-GFP (PX461) expresses both a Cas9 protein and...Find pMD2.G. pSpCas9(BB)-2A-GFP (PX458) - This plasmid from the Zhang lab expresses Cas9 from Streptococcus...and a gRNA. But instead of Cas9, this plasmid expresses Cas9n (a D10A nickase mutant) to nick, rather than...vector also from the Zhang lab is used to deliver Cas9 and your gRNA of interest into mammalian cells. ...fact, the Zhang lab has shared over 40,000 CRISPR-Cas9 components over the years on their own or through...