We narrowed to 614 results for: CaS

... 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...

...-for-one solution: TurboCas!TurboCas provides the genomic targeting power of dCas9 with the dynamic labeling... While dCas9 has been used in combination with proximity labeling enzymes before, such as CasID, TurboCas...background labeling. By fusing miniTurboID with dCas9 to create TurboCas, the Shilatifard Lab created a very quick...editing with STITCHR Dynamic protein labeling with TurboCas Enhancer-AAV viral preps targeting interneurons...Bentley For RNA-guided genome editing, think beyond Cas9 with STITCHR: a combined CRISPR / retrotransposon... Figure 4: STITCHR consists of a nicking Cas9 fused to a shortened R2Tocc transposon protein, ...for scarless insertion of the payload (teal). The Cas9 gRNA targeting guide and the transposon RNA homology...

...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...

...) 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...

...consultants as short-term experts to work on “cases”. Cases present specific operational or commercial challenges...performance, and occasionally being faced with “shifting goal posts” as a part of a case, consulting can...include efforts like flexible staffing on case teams, where case managers are encouraged to use the wider...clients face imminently. Consulting firms deploy case teams, usually with 3-6 consultants, who work on...facing a crunch, and using lessons from previous cases to better staff future teams. As with any highly...become cauldrons on the verge of boiling. On other occasions, clients can be excessively demanding. For instance... critical analyses skills were applicable to my cases. I can directly use my understanding of the molecular...

... found led them back towards Cas9 fused base editors (nCas9-BE), as Cas9 is mechanistically more favored...first thought to enable MBE by using Cas12a fused base editors (dCas12a-BE). However, the poor editing efficiencies...also exhibited reduced Cas9-dependent off-target editing and do not cause higher Cas9-independent off-target...only minimal payloads. DAP array  Although CRISPR-Cas12a, with abilities to process and release its own ...leading the edge of the precision gene editing. CRISPR-Cas9, the key component to enable efficient base editing...would independently generate multiple gRNAs for nCas9-BE, or any other kinds of CRISPR tools, without ...organisms and has previously been used for multiplex Cas9-nuclease editing. Typically, a single array encoding...

...lenti-Cas9-VQR plasmid page. Despite their differences, both of these PID formats for lenti-Cas9-VQR contain...resolvable compact identifier for every plasmid. In case these PID formats are new to you, we’ll go through...available through Addgene. Let’s take a look at lenti-Cas9-VQR-Blast, which has the Addgene ID #87155. The ...The resolvable compact identifier format for lenti-Cas9-VQR is the URL “http://n2t.net/addgene:87155” where...n2t.net web address. For the RRID format, lenti-Cas9-VQR is represented as “RRID:Addgene_87155” and the...formats. Let’s go back to our example plasmid, lenti-Cas9-VQR-Blast. If you scroll down to the bottom of this...semi-colons. For our example plasmid it will read “lenti-Cas9-VQR-Blast was a gift from Daniel Bauer (Addgene ...

...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... https://doi.org/10.1038/s41593-021-00969-4.   dCas9-SSAP with reduced error rate for long sequence knock-ins...Wilson Le Cong’s lab has developed a method using dCas9-SSAP to knock-in long sequences with reduced risks...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...

...interest. To get the molecular components of CRISPR/Cas9 (Cas9 protein and the crRNA-tracrRNA complex) and the...accurately as possible. This is especially true in the case of protein tagging to elucidate cellular structures...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 ...