Journal: Nature biotechnology

Article Title: Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR-Cas9 library.

doi: 10.1038/nbt.3715

Figure Lengend Snippet: Figure 1. Lentivirally-delivered paired-guide RNAs create large-fragment deletion with high efficiency in Cas9 stably expressing human cells. (a) Structures of the lentiviral plasmids expressing paired guide RNAs (pgRNAs). The U6 promoter(s) and gRNA coding sequences were cloned into an LL3.7 lentiviral backbone. Amplified DNA fragments encoding customized pgRNAs were ligated into the lentiviral backbone with U6 promoters (U62) or only one mutual U6 promoter (U61) using the Golden Gate method. (b,c) pgRNA vectors were delivered into human cells that express Cas9 through lentivirus. Large-fragment deletions induced by pgRNAs targeting the CSPG4 gene were identified by PCR. Six pairs of gRNAs that produced large-fragment deletions from 2–4.5 kb were chosen (b) and primers L1/R1 were used for the genomic PCR reactions (b,c). All infected Huh7.5OC cells were enriched by FACS and incubated for 6 days. The letter U62 or U61 represents two tandem ways in Fig. 1a and the control is a pair of gRNAs with one targeting the CSPG4 locus and the other targeting AAVS1 region. (d) Quantification for the efficiency of large- fragment deletions using genomic PCR over the course of time post transduction. The pgRNAs (3+3′ in Fig. 1c, generating 3.5-kb deletion by design) were delivered into Huh7.5OC cells through lentiviral infection, and genomic DNA was extracted from different time points as indicated (upper). The primers L2/R2 corresponding to sequences flanking pgRNA targeting sites (b) were used for quantification, and primers L3/R3 corresponding to sequences farther away from the targeting sites (b) were used for normalization. Primer sequences are listed in Supplementary Table 11. Images were analysed using ImageJ software and data are presented as the mean ± s.d. (n = 3) (below). (e) DNA sequencing analysis of large-fragment deletions in the human CSPG4 locus targeted by pgRNAs (3+3′) from pooled cells 3 weeks post infection (Fig. 1d). Partial sequences of targeted genes containing the two gRNAs’ targeting regions are labelled in red and the shaded nucleotides represent the PAM sequences. Dashes indicate deletions.

Article Snippet: Plasmid construction The lentiviral pgRNA-expressing vector was constructed by cloning the human U6 promoter, ccdB cassette and gRNA scaffold into pLL3.7 (Addgene, Inc.) by replacing its original U6 promoter8.

Techniques: Stable Transfection, Expressing, Clone Assay, Amplification, Produced, Infection, Incubation, Control, Transduction, Software, DNA Sequencing