Fig. 1 | Nature Communications

Fig. 1

From: Multiplexed Cas9 targeting reveals genomic location effects and gRNA-based staggered breaks influencing mutation efficiency

Fig. 1

Overview of CRISPR-Cas9 assays in TRIP cell line and pools. a Barcoded TRIP reporter construct. b Clonal PGK-driven TRIP cell line with 36 IRs (left), and TRIP pool containing ~ 1k IRs with various promoters (right) - CMV, cMyc, Hoxb1, Nanog, Oct4, p53, PGK. Genomic location and expression of IRs were determined by DNA and RNA sequencing prior to Cas9 targeting of IR regions using different guides. Targeted DNA sequencing of IR regions was further used to characterize mutations arising from repair of Cas9-induced DSBs. (c) Cas9-guide RNA combinations used in independent assays. TRIP cell line was targeted using Cas9 complexes with sgRNA1, sgRNA2 or sgRNA3 (left). In TRIP pool assays, Cas9 was complexed with sgRNA2 or sgRNA3 (right). Knock-in of a single-stranded oligodeoxynuceotide (ssODN) was performed with sgRNA2

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