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. 2017 Aug 17;67(4):633-645.e3.
doi: 10.1016/j.molcel.2017.06.035. Epub 2017 Aug 3.

Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1

Takashi Yamano  1 , Bernd Zetsche  2 , Ryuichiro Ishitani  1 , Feng Zhang  2 , Hiroshi Nishimasu  3 , Osamu Nureki  4
Affiliations

Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1

Takashi Yamano et al. Mol Cell. .

Abstract

The RNA-guided Cpf1 (also known as Cas12a) nuclease associates with a CRISPR RNA (crRNA) and cleaves the double-stranded DNA target complementary to the crRNA guide. The two Cpf1 orthologs from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) have been harnessed for eukaryotic genome editing. Cpf1 requires a specific nucleotide sequence, called a protospacer adjacent motif (PAM), for target recognition. Besides the canonical TTTV PAM, Cpf1 recognizes suboptimal C-containing PAMs. Here, we report four crystal structures of LbCpf1 in complex with the crRNA and its target DNA containing either TTTA, TCTA, TCCA, or CCCA as the PAM. These structures revealed that, depending on the PAM sequences, LbCpf1 undergoes conformational changes to form altered interactions with the PAM-containing DNA duplexes, thereby achieving the relaxed PAM recognition. Collectively, the present structures advance our mechanistic understanding of the PAM-dependent, crRNA-guided DNA cleavage by the Cpf1 family nucleases.

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

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Figures

Figure 1
Figure 1. DNA cleavage activities of LbCpf1 and AsCpf1
(A) In vitro DNA cleavage activities of LbCpf1 and AsCpf1. The Cpf1-crRNA complex (50 nM) was incubated at 37°C for the indicated time with a linearized plasmid target with the TTTA PAM. S, substrate; P, product. (B and C) PAM specificities of LbCpf1 and AsCpf1. The Cpf1-crRNA complex (50 nM) was incubated at 37°C for 5 min with a linearized plasmid target with the different PAMs. (D) In vivo cleavage activities of LbCpf1 and AsCpf1. Indel frequencies for 42 endogenous target sites with the different PAMs were measured in mammalian cells. Data are shown as mean ± s.e.m (n = 3). In (B)–(D), the canonical PAM is boxed in red, and the substituted nucleotides are colored red.
Figure 2
Figure 2. Structure of the LbCpf1-crRNA-target DNA complex
(A) Domain organization of LbCpf1. BH, bridge helix. (B) Schematic of the crRNA and its target DNA. TS, target DNA strand; NTS, non-target DNA strand. (C) Crystal structure of LbCpf1 in complex with the crRNA and its target DNA. (D) Structure of the crRNA and its target DNA. (E) Binding of Mg2+ ions to the crRNA. The bound Mg2+ ions and water molecules are indicated by gray and green spheres, respectively. Hydrogen bonds are shown as dashed lines. See Figures S1 and S2.
Figure 3
Figure 3. Comparison between the binary and ternary complex structures of LbCpf1
(A) Crystal structure of the LbCpf1-crRNA complex (PDB: 5ID6) (Dong et al., 2016). (B) Superimposition of the LbCpf1-crRNA-target DNA complex (colored) and the LbCpf1-crRNA complex (blue). Structural changes are indicated by orange arrows. (C–E) Interactions between the REC and NUC lobes in the LbCpf1 binary complex (C), the LbCpf1 ternary complex (D), and the AsCpf1 ternary complex (PDB: 5B43) (Yamano et al., 2016) (E). Hydrogen bonds are shown as dashed lines. See Figure S3.
Figure 4
Figure 4. Comparison between the ternary complex structures of LbCpf1 and AsCpf1
(A) Crystal structure of the LbCpf1-crRNA-DNA complex (PDB: 5B43) (Yamano et al., 2016). (B) Superimposition of the LbCpf1-crRNA-DNA complex (colored) and the AsCpf1-crRNA-DNA complex (blue) (C and D) RNA-DNA heteroduplex recognition by LbCpf1 (C) and AsCpf1 (D). (E and F) RuvC and Nuc domains of LbCpf1 (E) and AsCpf1 (F). See Figures S4, S5 and S6.
Figure 5
Figure 5. Canonical PAM recognition mechanism
(A) PAM-duplex binding in the LbCpf1 ternary complex. (B) Schematic of the PAM-duplex recognition by LbCpf1. (C) Superimposition of the PAM duplexes in LbCpf1 and AsCpf1 (PDB: 5B43) (Yamano et al., 2016) onto the B-form DNA duplex (stereo view). (D–F) Recognition of dA(−2):dT(−2*) (D), dA(−3):dT(−3*) (E), and dA(−4):dT(−4*) (F) by LbCpf1. (G–I) Recognition of dA(−2):dT(−2*) (G), dA(−3):dT(−3*) (H), and dA(−4):dT(−4*) (I) by AsCpf1. In (D–I), hydrogen bonds are shown as dashed lines.
Figure 6
Figure 6. Comparison between the TTTA, TCTA, TCCA and CCCA complex structures
(A) Superimposition of the TCTA (orange) and TTTA (colored) complexes. (B) Superimposition of the TCCA (green) and TTTA (colored) complexes. (C) Superimposition of the CCCA (blue) and TTTA (colored) complexes. (D) Superimposition of the PAM duplexes in the TTTA, TCTA, TCCA and CCCA complex structures onto the B-form DNA duplex (stereo view). See Figure S7.
Figure 7
Figure 7. Non-canonical PAM recognition mechanism
(A–D) The mFO – DFC omit electron density maps for Lys595 and the key nucleotides in the TTTA (A), TCTA (B), TCCA (C) and CCCA (D) complexes (gray, contoured at 5.0σ). In (B) and (C), the electron density map contoured at 2.0σ (blue) is also shown for Lys595. (E–H) Recognitions of the TTTA (E), TCTA (F), TCCA (G) and CCCA (H) PAMs. Hydrogen bonds are shown as dashed lines. See Figure S7.
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