...great about CASANOVA? It works with the S. pyogenes Cas9, dCas9-effector fusions, and xCas9, and requires... CRISPR-Cas9 by CASANOVA Article contributed by Cary Valley  Listen to the anti-CRISPR podcast segment...potent Acr-LOV fusion protein inhibitor CASANOVA for “CRISPR-Cas9 activity switching via a novel optogenetic...AcrIIA4.” CASANOVA responds to blue light quickly. Within minutes of inactivating the CASANOVA complex,...CRISPR systems including the popular S. pyogenes Cas9 (Rauch et al., 2017, Hynes et al., 2017). However..., they have developed a system to control CRISPR-Cas9 activity using blue light (Bubeck et al., 2018)...., inhibiting function of Acr. This allows CRISPR-Cas9 to function on its target.  They name their most...

... AAV delivery of Nme2Cas9 with gRNA cassette Erik Sontheimer’s lab identified a Cas9 protein from Neisseria...Covalent tethering of DNA template to Cas9 SECURE base editors Nme2Cas9 CRISPR interference in Candida albicans...paper in Communications Biology Find the Cas9-PCV and PCV-Cas9 plasmids Selective curbing of unwanted ...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...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 ...

...range of CRISPRCas9 by engineering SpCas9 Article contributed by Shreya Vedantam CRISPR/Cas9 is widely...sequence targeted by the Cas9 nuclease. The Streptococcus pyogenes Cas9 (SpCas9) requires an NGG PAM to...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...  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...cells. The increased versatility of the engineered SpCas9-NG and its variants can be used to engineer specific...

...different Cas12a inhibitors. Some of these anti-Cas12a proteins have broad inhibitory effects on Cas12a orthologs... research.     CRISPR-Cas12b for human genome editing The best studied Cas12b nuclease from Alicyclobacillus...The Feng Zhang lab identified a Cas12b from Bacillus hisashii, BhCas12b that is functional at 37 °C but...Communications Find the Cas12b plasmids  Plant base editing with a Cas9-adenosine deaminase fusion... - this time using the Staphylococcus aureus Cas9 (SaCas9). This AAV-based genome editing system is regulated...Alicyclobacillus acidoterrestris (AacCas12b) exhibits optimal DNA cleavage at 48 °C, a temperature incompatible...tRNA adenosine deaminase that is fused to a nicking Cas9. This base editor can be used for A•T to G•C edits...

...combining specific sgRNAs with Cas9 or Cas9 derivatives. Unmodified Cas9 causes double stranded DNA breaks... loss of function while nuclease-deactivated Cas9 (dCas9) can be tethered to a repressor (CRISPRi) or ...an inactive RNA-guided DNA binding protein (dCas9). dCas9 is then tethered to a repressive domain, such... issues. CRISPRi libraries utilize an engineered Cas9, which contains multiple point mutations in the ...library In addition to inhibiting gene expression, dCas9 can also be used for transcriptional activation....developed to recruit transcriptional machinery to dCas9 and its RNA directed binding site. These methods... fusion of an activation domain such as VP16 to dCas9 or the recruitment of multiple copies of VP16 termed...

...nicking Cas9. A guide directs Cas9 to a site of interest, where it nicks its target DNA (left). Cas9 dissociates...enhance nCas9 (enCas9) EvolvR with a targeted mutagenesis rate ~9-fold higher than the original nCas9, while...error prone PolI3M that’s fused to a nicking Cas9 (nCas9). Similar to mutagenesis with error prone PCR...used to direct the PolI3M-nCas9 complex to a DNA site of interest, which nCas9 nicks and then dissociates...amino acid changes were introduced to nCas9. These changes promote nCas9’s dissociation from DNA after nicking... a user-defined genomic region. EvolvR, a CRISPR-Cas9 based targeted mutagenesis method developed by the...diversification by targeting a site of interest with dCas9 fused to the Activation Induced Cytidine Deaminase...

...releasing Cas9. Nanoblades can also deliver Cas9 variants, such as a transcriptional activator dCas9-VPR, ...centrifugation. Gag-Cas9 fusion protein The MLV Gag protein fused to a Flag-tagged SpCas9 is incorporated ...Nanoblades by association with the the Cas9 portion of the Gag-Cas9 fusion protein. A single Nanoblade particle...along with the gRNA and Cas9. An “all-in-one” Nanoblades which carries a gRNA, Cas9, and a HDR donor template... leukemia virus (MLV)-like particles loaded with Cas9-gRNA ribonucleoproteins (RNPs). According to the...generation. Key components of Nanoblades Nanoblades are a Cas9 RNP delivery vehicle that’s based on mouse leukemia...just like the unmodified MLV Gag protein. After Gag-Cas9 is incorporated, the MLV protease, supplied as part...

... CRISPR tools for your application: Wildtype Cas9 or Cas9 nickase are appropriate for generating knockouts...in vivo viral delivery. Cas9 mRNA and gRNA Plasmids containing gRNA and Cas9 are used in in vitro transcription...generating transgenic embryos. Cas9-gRNA ribonucleoprotein complexes Purified Cas9 protein and in vitro transcribed... mutations and tags, while a catalytically dead dCas9 can be used in conjunction with activator or repressor...need to deliver. Minimally, one or more sgRNAs and Cas9 are required for any application. If you want to...Application Mammalian expression vector Promoter driving Cas9 expression can be constitutive or inducible. U6 ...stable cell lines. Transient or stable expression of Cas9 and/or gRNA in a mammalian cell line that can be...

...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...ways. In some cases, however, combining an evolved RT domain from PE6a-d with an evolved Cas9 domain from... ~ ~ ✓ ✓ ✓ 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 ... make a specific edit defined by the pegRNA. In cases when bystander editing is unacceptable, prime editors...

...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...advantage of repair pathways within the cell — in some cases, the same repair pathways that were considered problematic...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...

...CRISPR/Cas9 over previous genome editing techniques is its simplicity and versatility. CRISPR/Cas9 consists...in which Cas9 is included on the gRNA-containing plasmid, or a 2-plasmid system in which Cas9 must be ...of two components: the non-specific endonuclease (Cas9) and a single stranded guide RNA (gRNA). The ~20...the user, and it can be easily modified to target Cas9 to virtually any genomic locus, provided the target...adjacent motif (PAM) sequence. Co-delivery of wild-type Cas9 and a gRNA generates a double-strand break in the...genomic target. The goal of a CRISPR screen is to use Cas9 and a pool of gRNAs to identify genes that are essential...lentivirus to deliver a pooled population of gRNAs to Cas9 expressing cells. Pooled lentiviral CRISPR libraries...

...activate a cascade of caspases, protease enzymes that cleave proteins. Executioner caspases are activated... executioner caspase reporters. In these reporters, the FRET pair is linked by a caspase cleavage site...one of the linkers is a consensus caspase-3 (an executioner caspase) cleavage site (DEVD). Upon cleavage...activated last in the cascade and are responsible for the degradation of over 600 cellular components ultimately...used genetically encoded fluorescent executioner caspase reporters. Several apoptosis reporters have been...site. Cutting the linker by a caspase results in a change in fluorescence emission. FRET and fluorescence...Studying apoptosis in vivo using the FlipGFP-based caspase reporter The Shu Lab successfully used FlipGFP ...

... preassembled Cas9/gRNA complexes or with guide RNA- and Cas9-expression plasmids. Cas9 guide RNA delivery...Targeting Specificity with eSpCas9 and SpCas9-HF1 See How Cpf1 Stacks Up to Cas9 and NgAgo Resources on ...genome, which seem resistant to Cas9 cleavage (1). Both the CRISPR-Cas9 and NgAgo genome engineering methods...engineer sharing the stage with much-lauded CRISPR-Cas9 is DNA-guided endonuclease NgAgo! We'll discuss ...head-to-head.  Exciting features of NgAgo First, unlike Cas9, NgAgo does not require a PAM sequence for target... guide sequence rather than an RNA guide as with Cas9. Third, it can be used to target “difficult” GC-...the other hand, the widespread success of CRISPR-Cas9 across multiple model organisms (3) lies in the ...

...genomic locus: Caspex  Article contributed by Susanna Bachle  Listen to the Caspex podcast segment Steven...lentiviral plasmids, catalytically inactive Cas9 (dSpyCas9) fused to APEX2 is expressed from a Tet/Shield1...please sign up here. Listen to the Hot Plasmids Podcast!  MarathonRT: A highly processive reverse transcriptase...contributed by Will Arnold  Listen to the MarathonRT podcast segment Researchers from Dr. Anna Pyle’s lab at...Marcy Patrick  Listen to the nanobody toolkit podcast segment Nanobodies are single domain antibody fragments...genomic loci. He made use of catalytically inactive dCas9 by fusing it to an engineered peroxidase, (APEX)... proteins with biotin as an affinity tag. This ‘Caspex’ construct is guided by a pre-defined sgRNA sequence...

...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...CAPTURE 2.0:  Replace the biotinalatable tag on dCas9 with a BioTAP-tag. The BioTAP-tag is a 69 aa long...three to two. Lentiviral delivery of BioTAP-tagged dCas9 and gRNAs. Using lentiviral delivery of the two ...

...summarize, CRISPRa uses a nuclease-dead variant of Cas9 (dCas9) and engineered combinations of transcriptional...with neuron-specific expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed by the Church...limited to in vitro settings due to the large size of Cas9 activators, which largely precludes delivery by ...mouse expresses a modified version of the “Suntag” Cas9 activator that recruits up to twenty copies of the... transcriptional activator to each genome-bound dCas9 (see Figure 1a for the transgenic construct). More...packaging limits (i.e., how many sgRNA expression cassettes will fit). Cre-dependent expression of the SPH...here. Neuron-optimized viral vectors Alternatively, Cas9 activators can be delivered to the CNS by lentivirus...

... CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9 Day 10: Transcriptional regulation:...contains Cas13 constructs for mammalian and bacterial systems. Plasmids with wild-type Cas13a (formerly...RNA cleavage in bacteria. In mammalian systems, Cas13a degrades only the specified RNA target. Bonus: ...This characteristic probably explains why pC016 - LwCas13a guide expression backbone with U6 promoter has...CRISPR? I certainly didn’t, but now we can with Cas13b/ADAR constructs from the REPAIR (RNA Editing for...and CRISPR Antimicrobials Day 7: Multiplexing and Cas9 variants: CRISPR 101: Multiplex Expression of gRNAs...The PAM Requirement and Expanding CRISPR Beyond SpCas9 Day 8: Repair mechanisms: CRISPR 101: Non-homologous...

... 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..., with only a few base pairs modified? In these cases, it’s best to do some planning in the design phase...

...containing Cas9 and the PITCh-gRNA. Step 3: Contransfect the PITCh vector with the vector carrying Cas9 and ...protocol for MMEJ-mediated knock-in of a GFP-Puro cassette into a given locus, just upstream of a stop codon...microhomology to the insertion locus flanking the GFP-Puro cassette. Three double stranded breaks are necessary for...for knock-in: one on either side of the GFP-Puro cassette and one in between the 5’ and 3’ microhomologies...microhomologies (5’ and 3’) to anneal, knocking the GFP-Puro cassette into the locus (see figure below). The double-...of PITCh. The PITCh plasmid contains a GFP-Puro cassette flanked by 5' and 3' microhomology and PITCh-gRNA...plasmic is cotransfected with a plasmid carrying Cas9, the PITCh-gRNA, and the locus-specifc gRNA. The...

...Harris lab showed that human APOBEC3A-Cas9n-UGI and APOBEC3B-Cas9n-UGI base editing complexes are more ...contributed by Klaus Wanisch  Listen to the optogenetics podcast segment LOVTRAP is an optogenetic system that ...contributed by Tyler Ford  Listen to the riboglow podcast segment Researchers from the Palmer lab at the ...contributed by Caroline LaManna  Listen to the mCarmine podcast segment Scientists are always on the lookout for...reporter for rapid quantification of base-editing and Cas9 cleavage activity Article contributed by Angela ...Angela Kaczmarczyk  Listen to the base editing podcast segment The increasing popularity of single base-editors...fluorescence-based quantification of base editors that utilize dCas9 fused to cytidine deaminases like APOBEC. To achieve...