...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...

...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 ...

... 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...

...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 ...

...fusing catalytically inactivated RNA-targeting Cas13 (dCas13) with the adenine deaminase domain of ADAR2.... as these recent blog posts on prime editing, IgnaviCas9, and Nanoblades), but we hope that this blog ...toolkit for epitope tagging Lentiviral capsid-based Cas9/gRNA ribonucleoprotein delivery Cytosine deaminase... a catalytically impaired Streptococcus pyogenes Cas9. They called this editor Target-ACE (adenine and...Read the paper in bioRxiv Find Target-ACE   CRISPR/Cas9-based toolkit for epitope tagging in neurons Harold... the paper in bioRxiv Find the ORANGE toolkit   Cas9/gRNA ribonucleoprotein delivery by lentiviral capsid-based...capsid-based bionanoparticle Transient expression of CRISPR/Cas9 in cells has advantages like reduced off-target ...

...Pint series. Chapter 4: The SITN Podcast, Sit'N Listen - In their podcast, the SITN team brings in scientists...The Goal of the SITN Podcast - Vini and Amy would like to develop the SITN podcast as a means for listeners...senior.  To learn more about the STIN's podcast, email sitnpodcast@gmail.com    For more info about starting...have to be crazy complicated! Listen to the full podcast for more great science communication tips or listen...of Science in the News, and member of the SITN podcast team. You can follow Vini on twitter @vinidhra....Science in the News, and is a member of the SITN podcast team. You can follow Amy on twitter @aigilson. ...the audio engineer and producer for the Addgene Podcast.     Additional Resources on the Addgene Blog ...

...the usefulness of CPPs must be evaluated on a case-by-case basis. While setting up their CPP system to ...would be compatible with CRISPR/Cas9 genome editing. Although CRISPR/Cas9 is all the rage, they had another... method and its potential applications to CRISPR/Cas9 genome editing.   Protein-only transduction? Niels...expression. Image courtesy of Diego D'Astolfo. CRISPR/Cas9: a potential application of iTOP Having tested iTOP...expression has a long-lasting effect, such as Cre- or Cas9-mediated recombination. Both primary hESCs and immortalized...cells treated with iTOP efficiently took up both Cas9 protein and a gRNA (yes, iTOP can also be used for... indicating that iTOP may be valuable for CRISPR/Cas9 stem cell editing. The small molecule NSDB...

...To overcome this obstacle, a positive selection cassette can be incorporated, and used during the differentiation...the development of site-specific nucleases (CRISPR/Cas9, TALENs, and ZFNs). However, several challenges ...piggyBac™ transposon system can be combined with CRISPR/Cas9 to efficiently perform genetic manipulations in ...along with a site-specific nuclease (such as CRISPR/Cas9) to create a double-stranded DNA break. After DNA..., but defective for integration [4], the donor cassette is removed leaving behind no accessory transposon...recombination and Footprint-Free™ removal of the selection cassette by excision-only piggyBac™. Image courtesy of ...recombinase technology (Cre/loxP) to remove the donor cassette. While this method can be effective, a loxP site...

...fluorescent protein to various Cas9 variants, SpCas9, NmCas9, and St1Cas9. These fusions localized to specific...The catalytically dead Cas9 protein (dCas9) is well known for its ability to bind DNA targets without...inhibition, among others. Over the past few years, dCas9 has also become a robust visualization tool to study...University of Massachusetts Medical School developed a dCas9-based multicolor labeling system to visualize chromosomal... al. “Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow.” Nature...