Publisher Correction: Structural basis for mismatch surveillance by CRISPR–Cas9

Bravo, Jack P.K.; Liu, Mu‐Sen; Hibshman, Grace; Dangerfield, Tyler L.; Jung, Kyungseok; McCool, Ryan S.; Johnson, Kenneth A.; Taylor, David W.

doi:10.1038/s41586-022-04655-8

Cited by 5 publications

(2 citation statements)

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“…4e). Similar REC2 and REC3 rearrangements have previously been reported 40,41 . The sgRNA P2-P4 rearrangement seems to be analogous to the REC2 rearrangement from the 8nt to 10nt conformations of Sp.Cas9, in which REC2 widens to allow REC3 to position itself for PAM distal R-loop recognition 32 .…”

Section: Cas9d Domain Rearrangements Upon Dna Recognition Provide Mul...supporting

confidence: 88%

DNA targeting by compact Cas9d and its resurrected ancestor

Ocampo,

Bravo,

Dangerfield

et al. 2024

Preprint

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The type II-A CRISPR effector SpCas9 has gained widespread popularity as an efficient and programmable genome editing tool. However, much remains to be known about novel compact variants that may overcome some limitations of current systems (1,2). Recently, alternative CRISPR-Cas systems with highly compact nucleases capable of genome editing in mammalian cells have been discovered through metagenomic analysis of uncultivated microbes, including Cas9d (a type II-D CRISPR-Cas effector) (3). Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in multiple functional states, revealing a stepwise process of DNA targeting involving a conformational switch in a REC2 domain insertion. Our structures provide insights into the intricately folded guide RNA which acts as a structural scaffold to anchor small, flexible protein domains and facilitate DNA target recognition. We find that the sgRNA can be truncated by up to ~25% yet still retain activity in vivo. We also show that despite preferentially targeting an NGG PAM, Cas9d exhibits a unique mechanism for PAM recognition. Finally, we identify the first Cas9d smaller than 800 amino acids exhibiting robust nuclease activity in mammalian cells. Using ancestral sequence reconstruction, we demonstrate that it is possible to generate compact nucleases capable of efficient genome editing by expanding the diversity of Cas9d families. Collectively, our results provide mechanistic insights into the evolution and DNA targeting of diverse type II CRISPR-Cas systems, providing a molecular blueprint for future rational re-engineering of minimal RNA-guided DNA nucleases.

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Section: Cas9d Domain Rearrangements Upon Dna Recognition Provide Mul...supporting

confidence: 88%

DNA targeting by compact Cas9d and its resurrected ancestor

Ocampo,

Bravo,

Dangerfield

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

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“…The affinity eluted protein was mixed with ion-exchange beads (SP Sepharose Fast Flow, GE Healthcare) equilibrated with buffer C (20 mM Tris-HCl, pH 8.0, and 0.15 M NaCl) and the protein was eluted by buffer D (20 mM Tris-HCl, pH 8.0, and 1 M NaCl). SpCas9, SpRY, Superfi-Cas9, AaCas12b and Cas14a1 and were purified essentially by following the purification methods described earlier with some modifications 4 , 79 , 86 , 87 . The concentration of purified proteins was measured by the Pierce BCA protein assay kit (Thermo Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics

Acharya,

Ansari,

Kumar Das

et al. 2024

Nat Commun

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The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.

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