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. 2017 Jul 6;67(1):139-147.e2.
doi: 10.1016/j.molcel.2017.04.019. Epub 2017 Jun 6.

Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1

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Free PMC article

Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1

Hiroshi Nishimasu et al. Mol Cell. .
Free PMC article

Abstract

The RNA-guided Cpf1 nuclease cleaves double-stranded DNA targets complementary to the CRISPR RNA (crRNA), and it has been harnessed for genome editing technologies. Recently, Acidaminococcus sp. BV3L6 (AsCpf1) was engineered to recognize altered DNA sequences as the protospacer adjacent motif (PAM), thereby expanding the target range of Cpf1-mediated genome editing. Whereas wild-type AsCpf1 recognizes the TTTV PAM, the RVR (S542R/K548V/N552R) and RR (S542R/K607R) variants can efficiently recognize the TATV and TYCV PAMs, respectively. However, their PAM recognition mechanisms remained unknown. Here we present the 2.0 Å resolution crystal structures of the RVR and RR variants bound to a crRNA and its target DNA. The structures revealed that the RVR and RR variants primarily recognize the PAM-complementary nucleotides via the substituted residues. Our high-resolution structures delineated the altered PAM recognition mechanisms of the AsCpf1 variants, providing a basis for the further engineering of CRISPR-Cpf1.

Keywords: CRISPR-Cas system; Cas12a; Cpf1; crystal structure; protospacer adjacent motif.

Figures

Figure 1
Figure 1. In vitro cleavage activities of WT AsCpf1 and AsCpf1 variants
(A and B) PAM specificities of the RVR (A) and RR (B) variants. The AsCpf1-crRNA complex (100 nM) was incubated at 37°C for 5 min with a linearized plasmid target with the different PAMs. The favorable PAMs for the RVR (TATA) and RR (TCCC) variants are boxed in red. The substituted nucleotides are colored red. (C) Fourth PAM nucleotide preferences of WT AsCpf1 and the RVR and RR variants. The AsCpf1-crRNA complex (100 nM) was incubated at 37°C for 5 min with a linearized plasmid target with the TTTN PAMs. The substituted nucleotides are colored red. See also Figures S1 and S2.
Figure 2
Figure 2. Overall structure of the AsCpf1 variant
(A) Domain organization of AsCpf1. BH, bridge helix. (B) Nucleotide sequences of the crRNA and the target DNA. The PAM nucleotides are TATA and TCCA in the RVR and RR variant structures, respectively. The disordered nucleotides in the variant structures are surrounded by dashed lines. TS, target DNA strand; NTS, non-target DNA strand. (C) Superimposition of the crystal structures of WT AsCpf1 (Yamano et al., 2016) (PDB: 5B43) (colored as in A and B) and the RVR (orange) and RR (purple) variants. (D) PAM duplex in the WT AsCpf1 structure (Yamano et al., 2016) (PDB: 5B43). The substituted residues are shown as stick models.
Figure 3
Figure 3. PAM recognition by WT AsCpf1 and AsCpf1 variants
(A and B) TTTA PAM recognition by WT AsCpf1 (Yamano et al., 2016) (PDB: 5B43). (C and D) TATA PAM recognition by the RVR variant. (E and F) TCCA PAM recognition by the RR variant. The interactions with the nucleotides at the first and second PAM positions are shown in (A), (C) and (E). The interactions with the nucleotides at the third PAM position are shown in (B), (D) and (F). In (C)–(F), the substituted residues are highlighted by red labels. In (C) and (E), water molecules are depicted by red spheres. See also Figure S3.
Figure 4
Figure 4. Structural differences between WT AsCpf1 and AsCpf1 variants
(A) Conformational differences in the PAM duplexes in the structures of WT AsCpf1 (PDB: 5B43) (stereo view). (B) Structural differences in Arg542 (S542R) between the RVR and RR variants (stereo view). (C) Structural rearrangements around Arg552 (N552R) in the RVR variant (stereo view). A water molecule is shown as a sphere. In (A)–(C), WT AsCpf1 and the RVR and RR variants are colored gray, orange and purple, respectively. See also Figure S4.

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