Figure 1: The type II-B FnCas9 from Francisella novicida cleaves the target DNA in a staggered pattern to leave 4-nt 5′-overhangs. | Nature Communications

Figure 1: The type II-B FnCas9 from Francisella novicida cleaves the target DNA in a staggered pattern to leave 4-nt 5′-overhangs.

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

Figure 1

(a) Schematics of FnCas9 and SpCas9. BH, bridge helix; CTD, C-terminal domain; HNH, HNH nuclease domain; NLS, nuclear localization signal; RuvC-I-III, RuvC nuclease domain; REC, recognition lobe. (b) An EMX1 target on the purified DNA substrate used for cell-free cleavage assays. The protospacer is highlighted in purple and the PAM is underlined. The substrate was prepared by PCR from K562 genomic DNA. Blue triangles indicate cleavage positions by FnCas9 or SpCas9. (c) Run-off DNA sequencing on FnCas9 and SpCas9 cell-free cleavage products. The sequencing reads from a reverse primer show that FnCas9 cleaved the non-target strand 3–4 bp farther away from the PAM compared with the SpCas9 cleavage position. The sequencing reads from a forward primer show that both Cas9 nucleases cleaved the target strand at the same position. (d) Competitive ligation assays. dsDNA oligo inserts with compatible 3-nt or 4-nt 5′-overhangs without a 5′-phosphate group were ligated with FnCas9 or SpCas9 digested plasmid vectors. Inserts with 4-nt 5′-overhangs were predominant (87%) in recombinant plasmid DNA.

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