...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...