...journal club discussion on Kevin Esvelt’s CRISPR/Cas9 gene drive paper was very popular. People kept asking...filing cabinets, a rickety old incubator, a few pipettes, and some home-made equipment. It was humble in...year with a computer science degree and had zero pipetting experience. However, after a few months of dedicated...

...classroom Engineering the Plant Genome Using CRISPR/Cas9 pSiM24: Simplifying Plant Genetic Engineering Resources...fluorescent lights. Other experiments require only seeds, petri dishes, and filter paper. Another benefit of using...

...well plate for further analysis. To assess CRISPR/Cas9 knockout efficacy, we have used both Western blotting...200 ul pipettor is set to 200 ul, wiped down with 70% ethanol, and a box of sterile truncated pipet tips...the pipettor, placed into wells, and partially dislodged from the agarose plug by rapid pipetting, as ...rapidly punch out cell clones with sterile truncated pipet tips. Indeed, an experienced operator can pick 48.... We highly recommend the use of an electronic pipettor with variable spacing (such as the Viaflo from...

...   For CRISPR libraries, Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening...plasmids, they are generally electroporated into electrocompetent cells to ensure the highest possible transformation...use for your transformation(s). The amount of competent cells you need total and per transformation may...liquid culture, all of the bacteria can grow and compete in the same container, making it easier for a smaller...be its own colony, and will not be in direct competition with other colonies containing smaller, recombined...recommend ensuring the cells used are meant for repetitive sequences (such as STBL4 or Endura Duo), growing...

...single 72h harvest is best for larger genes such as Cas9. See figure 1 for an example of how changing transfection... formation of replication competent lentivirus (RCL). Replication competence can arise from recombination...

... the guide from binding to the genome or prevent Cas9 from cutting, both of which can introduce significant...mapped to a SNP would be ignored. Working with Lia Petronio and Katherine Huang from Broad’s Pattern team,...between biology and ancestry group as a means of perpetuating racial bias. It is easy, even unintentionally...Noorbakhsh, Isabella Boyle, Joshua Dempster, Lia Petronio, Katherine Huang, Alham Saadat, Thomas Green, ...

...Screening Read about Genome-Wide Screening Using CRISPR/Cas9 Additional Resources on Addgene.org Check out ...system, the possibility of creating a replication-competent virus via recombination between the transfer and...recombination events necessary to create a replication-competent virus. Second, Tat is eliminated through the addition...

...community has created hundreds of useful variants of Cas9. By providing access to the plasmids encoding antibodies...validation experiments performed will be useful in perpetuity.  Second, plasmids are easy to store and share...

...primer for beginners Adenoviral delivery of CRISPR/Cas9 5 tips for troubleshooting viral transductions ...response against AdV. Importantly, recombination-competent adenovirus (RCA) can be generated if the E1 gene...

...vectors fantastic options for delivering shRNA, CRISPR/Cas9 components, and fluorescent sensors. Retroviral...forming a vector that could create replication competent virus. Even though this system was efficient at...potential risk for the generation of replication competent lentiviruses (RCL) especially if you are working...positive cells could make the virus replication competent. The 3rd generation of lentiviral vectors was...8 kb) Potential for generation of replication-competent lentivirus (RCL) Efficient gene transfer Potential...Potential for insertional mutagenesis: Replication-incompetent lentiviruses with human tropism can still infect...

...CRISPR-Cas9 genome editing may be the hot new way to manipulate gene expression, but other gene manipulation...Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells...

...sequential homology directed repairs (HDR) with CRISPR/Cas9. Donor A contains: 400bp homology arms to the site... expression. References: Allocca M, Doria M, Petrillo M, Colella P, Garcia-Hoyos M, Gibbs D, Kim SR,...

...other technologies such as optogenetics and CRISPR/Cas9 will help immunologists create even better tools...Neutrophils travel to the sites of infection or damage (Peters et al. 2008, Chtanova et al. 2008, Kamenyeva et... immune system and its dynamics. References 1. Peters, Nathan C., et al. "In vivo imaging reveals an ...

...cloned into it. gRNAs are used for directing CRISPR/Cas9 to a targeted genomic location, while shRNAs are... propagated in DH5α. For plasmids with highly repetitive sequences (which can be prone to recombination...

...genetic tools. Mobile-CRISPRi from the labs of Jason Peters, Oren Rosenberg, and Carol Gross provides a streamlined...antibiotic resistance selection markers, gRNAs, and dCas9 proteins. Second, it can be introduced into a broader...Mobile-CRISPRi's modular components. Image from Peters et al., 2019 with permission.   Two methods... with an integrated copy of CRISPRi. Image from Peters et al., 2019 with permission.    The team showed...strains and environmental isolates. For example, Peters and colleagues used Mobile-CRISPRi to knockdown...possible with Mobile-CRISPRi. As a proof of principle, Peters and colleagues constructed a 40-member library ...promoters, selection markers, and different versions of dCas9. Following integration, Mobile-CRISPRi is stably...

...an EGFP reporter. Expression of the dCas9-KRAB activator or dCas9-VPR repressor in hPSCs is also driven...piggyBac gRNA delivery and the all-in-one dCas9-KRAB and dCas9-VPR targeting plasmids can be found at Addgene...plasmids were expressed in SHuffle T7 Express competent E.coli (NEB) using IPTG induction. To biotinylate...approaches rely on continual expression of both dCas9 and the gRNA. This expression can vary significantly...expression of single or multiplexed gRNAs and of dCas9 fusions. The dCas-KRAB activator and the dCas-VPR... could quantify and track cells expressing both dCas9 and gRNAs. The multi-gRNA piggyBac vector ...plasmids for piggyBac gRNA delivery and the all-in-one dCas9 targeting plasmids! Hazelbaker et al., Sci Reports...

...permuted green fluorescent protein or cpGFP) to the petunia SL enzymatic receptor DAD2. SLs induce a conformational...editing in moderate thermophiles.  These evolved SpCas9 variants have improved activity on NAG PAMS and...

...been full of dark matter: enhancers, lncRNAs, repetitive elements, repressors, insulators, and more. We...regions and their reverse complements were parsed for spCas9 PAM sites (NGG) and then filtered based on two ...

... series of retroviral vectors expressing tagged dCas9 and guide RNA. The retroviral enChIP system enables...survive as a researcher because of the tough competition for academic positions and poor grant situation...