The pLentiGuide-Puro vector (Addgene, no. 52963) was used as a backbone for all sgRNA expression vectors with the exception of the sgRB1 expression vector, which was made with the lentiCRISPRv2-zeo vector (a gift from S. McBrayer, Kaelin Laboratory). The lentiCRISPRv2-zeo vector was created by PCR amplification of a cDNA encoding the zeocin resistance gene from the pLenti4/V5-DEST vector (Invitrogen, no. V49810) using primers that introduced 5′ and 3′ homology arms targeted to regions of the lentiCRISPR v2 vector (Addgene, no. 52961) flanking the puromycin resistance gene cDNA. Primers corresponding to these homology arms were used in an inverse PCR with the lentiCRISPR v2 vector as a template. The zeocin resistance gene cDNA was gel-purified and used in an InFusion exchange reaction with the inverse PCR product.

pLentiGuide-Puro or lentiCRISPRv2-zeo vectors were digested with Bsm BI (New England Biolabs, no. R0580) or FastDigest Esp 3I (Life Technologies, no. FD0454) for 30 min at 37°C, and the resulting linearized vectors were gel-purified. sgRNA oligonucleotide sequences were designed using the Broad Institute Genetic Perturbation Platform Web Portal(https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design) with corresponding Bsm BI/Esp 3I overhangs added to facilitate ligation. Oligonucleotides were synthesized by IDT. Oligonucleotides were annealed using 0.15 nmol of each sense and antisense oligonucleotides. The oligonucleotides were heated at 95°C for 4 min and allowed to slowly cool to room temperature. Annealed oligonucleotides were then diluted 1:100 in nuclease-free water and ligated into the linearized vectors using T4 ligase in a 4-hour incubation at room temperature. A 2-μl aliquot of the ligation mixture was then transformed into 25 μl of HB101 chemically competent E. coli cells (Promega, no. L2011). Plasmids from ampicillin-resistant colonies were isolated by QIAprep Spin Plasmid Miniprep Kit (Qiagen, no. 27106) and validated by DNA sequencing. The sgRNA oligonucleotides used for editing (including Bsm BI/Esp 3I overhangs) are listed in table S1.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.