Fig. 3: A CRISPR-Cas9 paired-guide RNA strategy can force targeted distal poly(A) site usage. | Nature Communications

Fig. 3: A CRISPR-Cas9 paired-guide RNA strategy can force targeted distal poly(A) site usage.

From: Multiplexed screening reveals how cancer-specific alternative polyadenylation shapes tumor growth in vivo

Fig. 3

A Schematic of CRISPR-Cas9 paired-guide RNA (pgRNA) approach to excise proximal poly(A) sites and force use of distal poly(A) sites. B Diagram of Sap30l terminal exon with two distinct 3′ UTRs which vary depending on use of a proximal or distal poly(A) signal. Schematic of two distinct sets of proximal poly(A) KO (pKO) pgRNAs designed to delete the proximal poly(A) signal in Sap30l, annotated as Sap30l pKO1 and pKO2. C Genotyping PCR of polyclonal B16-F10 Cas9-expressing cells treated with a non-targeting control (NTC) or one of two distinct Sap30l pKO pgRNAs, with the calculated percent of signal showing DNA excision (representative gel from n = 3 biological replicates). D Read coverage plots of Poly(A)-seq completed for Cas9-expressing B16-F10 cells treated with either a control pgRNA (gray), Sap30l pKO1 (green), or Sap30l pKO1 (blue). Schematic illustrates the Sap30l terminal exon and 3′ UTR with the annotated poly(A) signal sequences (PAS). E Box plots of the percent distal isoform (pdi) usage per cell line quantified from the nested RT-PCR of the Sap30l 3′ UTR for each monoclonal cell line grouped by genotype (WT, heterozygous or homozygous deletion of the proximal poly(A) signal). Data reflects six, seven, and five distinct monoclonal lines with wild-type, heterozygous, or homozygous deletion of the proximal poly(A) signal sequence, respectively. P value from a two-sided Wilcoxon rank-sum test. F Box plots of Sap30l mRNA abundance per cell line measured by q-RT-PCR for each monoclonal cell line grouped by genotype (WT, heterozygous or homozygous deletion of the proximal poly(A) signal). Data reflects six, seven, and five distinct monoclonal lines with wild-type, heterozygous, or homozygous deletion of the proximal poly(A) signal sequence, respectively. P value from a two-sided Wilcoxon rank-sum test. G Scatter plot and Pearson correlation of Sap30l percent distal isoform usage versus Sap30l mRNA abundance. Gray error band represents 95% confidence interval. P value from a Pearson correlation.

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