Structural basis of stringent PAM recognition by CRISPR-C2c1 in complex with sgRNA

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems function as adaptive immune systems in bacteria^1,2, which are used to defend against phages and invading nucleic acids. The CRISPR-Cas systems are broadly grouped into two classes: Class 1 systems contain a multi-subunit protein complex, whereas Class 2 systems use a single effector protein, as exemplified by the well-studied Cas9³. Cas9 is an RNA-guided endonuclease, which targets and cleaves DNA bearing complementary sequences to the guide RNA. Protospacer adjacent motif (PAM) recognition by Cas9 and crRNA:tracrRNA complex is a critical prerequisite for substrate DNA melting and guide RNA:target DNA heteroduplex formation^4,5. Both catalytically active and inactive Cas9, combined with a single-strand guide RNA (sgRNA), have been widely used as programmable systems for various genetic manipulations^6,7.

The sgRNA in our structure consists of a guide segment (C1-U19), a repeat segment (C(−1)-G(−13)), a tetraloop (C(−14)-U(−17)), an anti-repeat segment (C(−18)-A(−24), and U(−57)-G(−61)), and three stem loops (stem loops 1-3) (Figure 1D and 1E). The guide segment and 19 nucleotides of the target DNA strand (dG(1′)-dA(19′)) form the guide:target heteroduplex, whereas the other 9 nucleotides of the target DNA strand (dG(−1′)-dA(−9′)) and the non-target DNA strand (dC(−1^*)-dT(−9^*)) form a PAM-containing duplex (PAM duplex) (Figure 1D and 1E; “′” indicates nucleotide in the target DNA strand and “^*” indicates nucleotide in the non-target DNA strand).

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