...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...

...) model consists of a catalytically dead Cas9 protein (dCas9) that lacks endonuclease activity coupled.... Using dCas9 as an on/off switch The multiple scRNAs all individually utilize the same dCas9 protein....modulate dCas9 expression, multiple scRNA transcriptional programs can be regulated with dCas9 as a master...an orthogonal dCas9 protein that will still recognize guide RNA but with a different dCas9 binding module...specific proteins via aptamer domains. Combined with dCas9, this new scaffold RNA (scRNA) will bind to a PAM-adjacent...layered with distinct programs controlled by each dCas9 regulatory switch. By no means limited to the field...applied to effectively change metabolic flux. The dCas9 protein and orthogonal variants can be used as regulatory...