Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9

Abstract

SUMMARYThermostable CRISPR-Cas9 enzymes could improve genome editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold higher genome editing levels. Cryo-EM structures of the wildtype and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome editing activity.

Atomistic Tuning of the GeoCas9 Recognition Lobe Modulates Allosteric Motions and Guide RNA Interactions

“…7,22 A similar dynamic modulation of Geo Cas9 may fine-tune its DNA cleavage, which has been demonstrated within the Geo HNH nuclease 29 and wedge (WED) domains. 26 We also assessed the ps-ns fluctuations of Geo Rec2 variants (a negligible contribution to the WT Geo Rec2 dynamic profile) and calculated order parameters from R 1 , R 2 and 1 H-[ 15 N] NOE relaxation measurements ( Figure 3A, S8 ). Bond vector fluctuations on the ps-ns timescale are only locally altered, thus the mutation-induced reshuffling of these dynamics is negligible (< Δ S 2 > ≤ 0.1) and suggests that, like WT Geo Rec2, ps-ns motion arises primarily from global tumbling in solution.…”

“…NMR and MD studies of high-specificity Sp Cas9 variants (HF-1, Hypa, and Evo, each with distinct mutations in the Sp Rec3 subdomain) reveal universal structural and dynamic variations in regions of Sp Rec3 that interface with the RNA;DNA hybrid. Notably, a recently published variant, i Geo Cas9, 26 demonstrated enhanced genome-editing capabilities in HEK293T cells with eight mutations, though none in the Rec2 subdomain. This study highlighted the functional adaptability of i Geo Cas9 under low magnesium conditions, a trait beneficial in mammalian cells, distinguishing it from WT Geo Cas9.…”

“…Despite NMR advancements in perdeuteration, 40 transverse relaxation-optimized spectroscopy (TROSY), 41 and sparse isotopic labeling, 42 per-residue dynamics underlying allosteric signaling in large multi-domain proteins such as Geo Cas9 (∼126 kDa) have remained challenging to characterize. A novel cryo-EM structure of Geo Cas9 26 will facilitate the merging of future NMR studies with molecular dynamics simulations that we have previously used to report on RNP dynamics and atomic level networks of communication. The identification of additional (or synergistic) allosteric hotspots within Geo Rec using an integrated workflow will help to further resolve the balance between structural stability and flexibility in Geo Cas9, leading to new insight into targeted manipulation of RNA affinity and enhanced variants.…”

“…Off-target DNA cleavage by Cas9 remains an area of intense study and substantial effort from various groups has gone into mitigating such effects. 18,21,[26][27][28] Indeed, many high-specificity SpCas9 variants contain mutations within SpRec3 that increase the threshold for its conformational activation, reducing the propensity for HNH to sample its active state in the presence of off-target DNA sequences. 18,19,28 Studies of flexibility within Rec itself, as well as its gRNA interactions in the presence of mutations, are therefore essential for connecting biophysical properties to function and specificity in related Cas9s.…”