Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

Abstract

SHORT ABSTRACT CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9. LONG ABSTRACT The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

RHO binding to FAM65A regulates Golgi reorientation during cell migration

“…CRISPR pSpCas9 (BB)-2A-Puro plasmid (pX459) was obtained from Addgene (plasmid ID 48139). The following 20-mer guide sequences were cloned into the sgRNA site of pX459, as described in Bauer et al (2015), to generate specific CRISPR plasmids: 5ʹ-GTGTACACGGCGCTGAAGCG-3ʹ (FAM65A), 5ʹ-CAGATAGGATCCATAATATT-3ʹ (MST3) and 5ʹ-TTGGACAGCCACCGGCGAGT-3ʹ (MST4).…”

EHMT1 and EHMT2 inhibition induces fetal hemoglobin expression

“…18,19 Briefly, sgRNA-specifying oligos were chosen using publicly available online tools (supplemental Table 1 and supplemental Figure 1; see the Blood Web site). 20 Oligos were phosphorylated, annealed, and cloned into pSpCas9(BB) (pX330; Addgene plasmid ID: 42230) using a Golden Gate Assembly strategy.…”

Identification of a novel distal regulatory element of the humanNeuroglobingene by the chromosome conformation capture approach

“…(34) was modified to delete the putative regulatory Element I detected by 3C. Instead of electroporation, we performed transfection by using Lipofectamine 3000 (Thermo Fisher Scientific).…”