DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

By S. H. Sternberg, S. Redding, Martin Jinek, E. Greene, Jennifer A. Doudna. 2014

1,788 citations recorded by the source corpus.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA–DNA base-pairing to target foreign DNA in bacteria. Cas9–guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9–RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9–RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9–RNA. Competition assays provide evidence that DNA strand separation and RNA–DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

Subfields: Molecular Biology

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Citations in the corpus

Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation (2014, background): “Single-molecule and biochemical experiments underscore the singular importance of PAMbinding in both DNA interrogation and cleavage by Cas9 (42).”
CRISPR-Cas12a: Functional overview and applications (2020, background): “[37] Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA.”
Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing (2014, background): “Two sequence requirements typically restrict the targeting range of a dCas9 monomer: the requirement for a PAM sequence of 5 ′ -NGG that is specified by dCas9 14 and a requirement for a G nucleotide at the 5 ′ end of the gRNA imposed by the use of a U6 promoter in most expression vectors.”
CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity (2014, background): “Evidence for this model comes from studies of Cas9 targeting (Sternberg et al., 2014) (see below).”
Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems (2016, background): “Base pairing between a 12 nucleotide seed sequence of the guide RNA and the target DNA strand [92] drives further stepwise destabilization of the target DNA duplex and directional formation of the guide-RNA–target-DNA heteroduplex [121].”
Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells (2014, neutral): “The position of the seed region next to PAM is consistent with previous observations that base pairing near PAM is critical for targeting 6,8,16,33 , but the seeds we identified for 3 of the 4 sgRNAs are shorter than those previously reported; seed lengths of 8-13 nucleotides have been described as…”
Concerning RNA-guided gene drives for the alteration of wild populations (2014, background): “, 2009) if supplied with a shortened guide RNA that directs it to bind and block transcription but not cut (Bikard et al., 2013; Sternberg et al., 2014).”
Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 (2016, neutral): “Biochemical and structural studies indicate that the sgRNA participates in step-wise association with DNA, suggesting that localized thermodynamic properties may also be useful 37,38 .”