...conducted in Professor Cepko's lab and to get some insight into her management and mentoring styles. If you'd...Cepko's recent work on developing GFP-activated proteins, read our recent blog post. Check out the break...al. “A nanobody-based system using fluorescent proteins as scaffolds for cell-specific gene manipulation...

...lacked an “off-switch” for Cas proteins. Discovery of anti-CRISPR proteins That is, until the discovery...of anti-CRISPR proteins in 2012 (Bondy-Denomy et al., 2012). Anti-CRISPR (Acr) proteins are phage-derived... found here. How do anti-CRISPR proteins work? Anti-CRISPRs proteins are highly diverse but most block... be as effective. This is where Acr proteins come in. Acr proteins are small enough that they could be...technology is constantly evolving. Variants of Cas proteins can be used for genome editing, activating gene...s activity. The concern is that the longer Cas remains active in a cell, the greater chances there are...CRISPR-Cas immune system of bacteria. These Acr proteins were originally found in type I CRISPR-Cas systems...

...current vaccines against the variants? Alejandro Balazs, a Group Leader at the Ragon Institute of MGH, MIT...vaccines against SARS-CoV-2 variants, this study was the first to compare so many different strains. It was...study. Figure 1: Neutralization levels against spike pseudovirus decreases depending on the variant..., Tegally H, Lustig G, Giandhari J, Pillay S, Wilkinson E, Naidoo Y, Karim F, Ganga Y, Khan K, Bernstein...

... plant hormones called auxins to conditionally and reversibly degrade proteins of interest fused to a ...   New toolkit for N or C terminal tagging of proteins for degradation using AID Article contributed ...ubiquitin ligase pathway needed for AID. By tagging proteins with a 7kD degron called mAID and expressing the...developed a CRISPR/Cas9 based method to tag endogenous proteins in HCT116 cells with mAID. Using this method, ...generate conditional alleles of a few essential proteins (Natsume et al., 2016). Now, the Kanemaki lab ... plasmids for the N- or C- terminal tagging of proteins with mAID and other tags (Yesbolatova et al., ...sufficiently suppresses basal degradation of tagged proteins, a common problem of the AID technology (Yesbolatova...

...into the 2in1 vector instead of the GatewayTM cloning BP reaction, where insertion of the GOI occurs through...technique using small scaffold proteins to identify novel binding proteins that bind to either the light... anti-CRISPR podcast segment Anti-CRISPR (Acr) proteins potently inhibit type II CRISPR systems including...temporally ubiquitous, making them a rather blunt instrument for controlling CRISPR. To bring greater control...optogenetics. Through the use of light-sensitive protein domains, they have developed a system to control CRISPR-Cas9...Bubeck et al., 2018). They accomplished this by inserting a photo-sensitive LOV2 domain from A. sativa into...study the electrical activity in the brain by inserting electrodes into the brain. But this process is...

...majority of knock-ins were occurring 24 hours post-injection, making screening for knock-ins much more efficient...unc-119) (Dickinson et al., 2013). These approaches allowed effective recovery of knock-ins, but did result...visible phenotype enriches for knock-outs and knock-ins at other genomic loci. The first approach – co-CRISPR...representative co-selection experiment, five knock-ins were recovered from seven rescued F1 animals (gel...from 11-100% of F1 animals carrying precise knock-ins, and homozygous knock-in animals can be obtained ...repair templates are critical for efficient knock-ins (Ghanta et al., 2020). Together, these improvements..., obtaining as many as 100 independent GFP knock-ins from a single injected animal.  PAM sequence considerations...

...donor vector to create knock-ins of fluorescent proteins, fusion proteins, or transcriptional regulators...research! This time: GeneWeld vectors to create knock-ins CasX Drug inducible CRISPR/Cas9 activation E. coli...knockout libraries for cancer research Creating knock-ins with GeneWeld vectors GeneWeld from Jeffrey Essner...library from David Bikard’s lab. The Bikard library contains ~93,000 unique gRNAs that target random positions...

...sequencing confirmation. All knock-ins A method applicable to knock-ins both large and small is a software...desired edit is. Large knock-ins Are you introducing a large insertion, something 20 base pairs or bigger... to knock-in size ratio below 10:1. Small knock-ins Is your knock-in edit small, with only a few base...appropriately edited for your desired mutation - insertion, deletion, or site-directed knock-in. We’ll also...edit – is it any frameshift mutation? A specific insertion or deletion? A knock-in? A base edit? Once you...interest will get the job done. In other words, any insertion or deletion that isn’t a multiple of three will...simple algorithm to quantify editing efficiency by insertion and deletion frequency. Fig. 1: Overview...

...get done. However, this is not the case for knock-ins. Knock-in mutations typically require an exact DNA...for HR-related mutations before attempting knock-ins by HDR.   Fig. 1: Early steps in repair of...Banan. Recent advances in CRISPR/Cas9-mediated knock-ins in mammalian cells. J Biotechnol., 16(4): 387-390...less traveled and generating a knock-in cell line instead of a knock-out? We’ve got you covered in this blog...template. The highest HDR efficiency is observed when inserts are within 10 bps of the break, making the cut ...further, you can inhibit cell cycle regulatory proteins responsible for the transition out of S phase,...events compete with NHEJ-mediated edits of small insertions and deletions.   Start your knock-in experiment...

...selection-based CRISPR/Cas9 method to generate knock-ins on any genetic background. Their new method uses ...added to the plates to select for candidate knock-ins. These candidate animals can then be further screened...it possible fluorescently label proteins in C. elegans, they inserted the SEC into a synthetic intron ...sequences are inserted into the genome. Figure 1: Self-excising cassette schematic from Dickinson et al...Self-Excising Drug Selection Cassette. Dickinson DJ, Pani AM, Heppert JK, Higgins CD, Goldstein B. Genetics. 2015...Homology directed repair (HDR), which enables insertion of custom genomic modifications, is very robust...making the detection of rare events difficult. Dickinson et al. sought to create a new and efficient selection-based...

... with reduced error rate for long sequence knock-ins Visual barcodes for live cell clonal multiplexing... with reduced error rate for long sequence knock-ins by: Lucie Wilson Le Cong’s lab has developed a method... large inserts. However, this system has a much lower error rate. For a one kilobase insertion, the researchers...limitations of number of flourescent proteins that can be inserted into a cell without disrupting its functions...peptides with four different fluorescent proteins, and inserting each visual barcode into its own subclone...TurboID-nanobody protein and allows TurboID to start labeling proteins in close proximity to the GFP-tagged protein (...need to generate “single-use” transgenic lines. Instead, researchers can generate and maintain transgenic...

...are appropriate for generating knockouts or knock-ins, or introducing mutations and tags, while a catalytically...homology directed repair (HDR) template to create knock-ins, point mutations, or to add a tag, you will also ...used in conjunction with activator or repressor domains to control gene expression. Base editors can help...will evaluate the outcome: Will you be detecting insertions/deletions using a mismatch repair assay? Or is... to identify and enrich positive cells. Likely contains a selection marker to generate stable cell lines... (packaging limit ~4.5kb). CRISPR elements are inserted into an AAV transfer vector and used to generate...Topic Page Learn about CRISPR Delivery Using Ribonucleoproteins (RNPs) Read Tips for a 1st Time CRISPR User...

...amplification. Given the inefficiency of HDR for knock-ins, some labs have used NHEJ for whole plasmid integration...is thought to be ideal for HDR-independent knock-ins, but this possibility is still being explored. More... DSB repair. NHEJ joins the ends of a DSB in an error-prone fashion, with insertions and deletions common...construct is inserted into the PITCh vector via In-Fusion or SLIC cloning. Step 2: Design an insertion locus-... of PITCh-based genomic insertion would compare to CRISPR sticky-end insertion using the nuclease Cpf1...introduce additional nucleotides flanking the insertion. If the DNA ends anneal incorrectly, MMEJ may ...constructed with 5’ and 3’ microhomology to the insertion locus flanking the GFP-Puro cassette. Three double...

...concept and go beyond the basics - diving into the ins and outs, tackling complex questions, and giving ...around the corner. Here at Addgene, we’re always inspired by the new seasons, so we've made some big summer...Social Media Are you following us on Twitter or Instagram (or Facebook or LinkedIn) but want to hear and...love sunshine and warm weather - check out our Instagram all summer long to see what kind of shenanigans...

...CRISPR can also be used to generate cell line knock-ins of patient mutations which are variants of unknown...drugging some targets – the dreaded ‘undruggable’ proteins – having multiple hits or an entire pathway of...

...be used in a lab? Here we’ll be talking about the ins and outs of this classification system, including...minute. Rinse the slide briefly with tap water. Flood the slide with Gram's iodine solution. Rinse briefly...immediately rinse. Flood the smear with the safranin counterstain for 30 - 60 seconds. Rinse the slide ...specific procedural instructions, including timing, preparing the smear, and mixing the stains and solutions...biologist named Christian Gram realized that certain stains were retained only in some species of bacteria....negative (red/pink) staining. Image credit: Mark Perkins https://www.flickr.com/photos/occbio/7092385559...laboratory setting. When designing a plasmid to be inserted into a specific bacterium, it can be important...

...needs. For instance, I had one colleague who preferred detailed instructions and regular check-ins, which ... made similar transitions can provide you with insights, lessons from their experiences, and practical...

...screen vs. a selection and we are here to discuss the ins and outs of both approaches. Screens and selections...players in a biological process. Players can be proteins, genes, enzymes, drugs, or anything else that ...specifically looking at cells that can survive. Instead, all manipulations are assessed, regardless of ...considering. RNAi screens are similar to CRISPR, but instead of knocking out the genes of interest (or the whole...

...One-on-Ones: Managers should set up regular check-ins with their employees. 15-30 minutes weekly or monthly...who could potentially be impacted by a project. Instead, invite the experts on the topic at hand. Invite... people of an updated protocol), send an email instead. The 3 A's - Agendas, Action items, and Accountability...parts where people had questions or suggestions, instead of reading the whole thing together. How long...FInd Tips on Picking a Research Project Read Other Inside Addgene Posts Resources on the Addgene Website...

...developed new CRISPR-based methods for various knock-ins, discussed in our CRISPR 101 eBook. It’s hard to...targeting vector designed to insert a resistance gene. The vector contains a neomycin resistance gene (...sequence using a specially designed vector that contains sequence homology to the locus of interest. To...negative selection marker HSV-tk is used to select against random recombinants.   Design your targeting...recombination, the negative selection marker selects against improper, random recombination into a different...After “floxing” the resistance gene through the insertion of flanking LoxP sequences, you can remove the...Figure 2: A knockout targeting vector designed to insert and later remove a resistance gene. In this construct...