Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation

Abstract

Type II CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA–induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

Functional annotation of cis-regulatory elements in human cells by dCas9/sgRNA

“…Since the basepaired target of sgC-185 is only 10 bp away from the N2 element that is TNFα-responsive ( Figure 1E), the latter result suggests that the dCas9/sgRNA complex exhibits a rather localized, ~40-nt cis-element-targeting resolution (20-nt paired bases plus 10 flanking nucleotides on either side) in live cells. Such results are in general agreement with the spatial details of CRISPR/Cas9 foot-printing data in vitro [9].…”

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

“…Point mutations were introduced by Gibson assembly or around-the-horn PCR and verified by DNA sequencing. Proteins were purified as described 23 , with the following modifications: after Ni-NTA affinity purification and overnight TEV cleavage at 4 °C, proteins were purified over an MBPTrap HP column connected to a HiTrap Heparin HP column for cation exchange chromatography. The final gel filtration step (Superdex 200) was carried out in elution buffer containing 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5% (v/v) glycerol and 1 mM TCEP.…”

“…The amplified PCR product was extracted with phenol:chloroform:isoamyl alcohol and served as the DNA template for sgRNA transcription reactions, which were performed as described 24 . DNA oligonucleotides and 5′end biotinylated DNAs (Supplementary Table 3) were synthesized commercially (Integrated DNA Technologies), and DNA duplexes were prepared and purified by native PAGE as described 23 .…”

Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition

“…CRISPR-Cas9 has been extensively used for genome editing in various cell types and organisms [11,12]. A series of structural studies of Streptococcus pyogenes Cas9 (SpyCas9) and its orthologs have revealed the detailed intermolecular interactions, as well as the conformational changes among different substrate-bound states [13][14][15][16][17][18].…”