Figure 5: Analysis of target binding and cleavage by the type II-C Cas9 from Campylobacter jejuni (CjCas9) in K562 cells. | Nature Communications

Figure 5: Analysis of target binding and cleavage by the type II-C Cas9 from Campylobacter jejuni (CjCas9) in K562 cells.

From: Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting

Figure 5

(a) CjCas9 and SpdCas9 targets in the human POR and AAVS1 loci. The AAVS1 target had previously been determined to be cleavable by CjCas9, while the three CjCas9 targets in POR had previously been determined to be uncleavable in K562 cells. Targets are indicated by bars and PAMs are highlighted in purple (SpdCas9) and dark blue (CjCas9). (b) Schematic of target binding assays by chromatin immunoprecipitation (ChIP) and droplet digital PCR (ddPCR). CjCas9 was converted to a catalytically dead Cas9 (CjdCas9) with D8A and H559A double mutations and tagged at the N terminus with a 3XFLAG epitope (FLAG-CjdCas9). (c) FLAG-CjdCas9 target binding activities on the AAVS1 target and POR target 1 with or without the assistance of SpdCas9 binding at proximal locations. The sgRNA numbers correspond to the target numbers in a (n=3 biological replicates; error bars show mean±s.d.). (d) CjCas9 cleavage activities (% indels) on the three POR targets with or without the assistance of SpdCas9. The sgRNA numbers correspond to the target numbers in a. Data are representative of three independent experiments. M, wide-range DNA markers; ND, not determined.

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