CRISPR nuclease (CRISPRn) genome editing

Leonardo A Parra-Rivas

Published: 2023-11-12 DOI: 10.17504/protocols.io.dm6gp35qpvzp/v1

Abstract

Steps

LentiCRISPRv2 plasmid (RRID:Addgene_52961) was used for α-syn genome editing. All sgRNAs

were designed using CRISPick https://portals.broadinstitute.org/gppx/crispick/publicc) and examined for genome-wide sequence specificityusing the National Center for Biotechnology Information’s (NCBI) Basic Local Alignment Search Tool NCBI BLAST

(RRID:SCR_004870)( http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sgRNAs (Control 5’

GACGGAGGCTAAGCGTCGCAA3’ and α-syn CRISPRn 5’ GGTTCATGAAAGGACTTTCAA3’) were

cloned as described previously

Citation

Shalem, O., Sanjana, N.E., Hartenian, E., Shi, X., Scott, D.A., Mikkelsen, T.S., Heckl, D., Ebert, B.L., Root, D.E., Doench, J.G., and Zhang, F. (2014). 2013 Genome-scale CRISPR-Cas9 knockout screening in human cells Science 10.1126/science.1247005

To validate the sgRNAs constructs, DIV-3 hippocampal

primary neurons were infected with lentiviruses per 6hrs (MOI=5). Transduced

neurons were cultured to maturity (DIV17-DIV21) and then lysate for western

blotting analysis and genomic analysis.