Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease

By C. Anders, O. Niewoehner, A. Duerst, Martin Jinek. 2014

1,241 citations recorded by the source corpus.

The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5′-NGG-3′ PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA–DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.

Subfields: Genetics, Molecular Biology

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Citations in the corpus

New CRISPR-Cas systems from uncultivated microbes (2016, background): “Active DNA-targeting CRISPR-Cas systems use 2 to 4 nt protospacer-adjacent motifs (PAMs) located next to target sequences for self versus non-self discrimination 19,20 .”
Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System (2015, background): “sgRNA is displayed with its secondary structure (51).”
Delivering CRISPR: a review of the challenges and approaches (2018, background): “…interrogated DNA sequence matches the crRNA target sequence, the HNH nuclease domain of the Cas9 nuclease lobe cleaves the target strand while the RuvC-like nuclease domain of the Cas9 a-helical lobe cleaves the non-target strand (Anders et al., 2014; Jinek et al., 2014; Nishimasu et al., 2015).”
Identifying and visualizing functional PAM diversity across CRISPR-Cas systems (2016, background): “One drawback of gene repression is the lack of nuclease activity associated with fully functioning systems, potentially missing PAM-dependent allosteric changes that drive DNA cleavage or Cas3 recruitment (Anders et al., 2014; Hochstrasser et al., 2014).”
Crystal structure of Cpf1 in complex with guide RNA and target DNA (2016, background): “The conserved, negatively charged residues Asp908, Glu993, and Asp1263 form an active site similar to that of the Cas9 RuvC domain (Nishimasu et al., 2014; Anders et al., 2014) (Figure 6B).”
In Silico Predictive Modeling of CRISPR/Cas9 guide efficiency (2015, background): “There is a body of related literature concerning computational approaches for designing small interfering RNA (siRNAs) 17,18 , wherein properties of siRNA sequences are encoded into features for predicting efficiency, including sequence-based features such as GC content, melting temperature of the…”