Enhanced proofreading governs CRISPR-Cas9 targeting accuracy
By Janice S. Chen, Y. S. Dagdas, B. Kleinstiver, Moira M. Welch, Alexander Sousa, Lucas B. Harrington, S. H. Sternberg, J. Keith Joung, A. Yildiz, Jennifer A. Doudna. 2017
1,070 citations recorded by the source corpus.
The RNA-guided CRISPR–Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity are unknown. Here, using single-molecule Förster resonance energy transfer experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we design a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.
Subfields: Molecular Biology
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Citations in the corpus
- A beginner (methodology): “The use of Cas9/gRNA protein-RNA particles (Kim 2014), and/or Cas9 variants such as Hypa-Cas9 (Chen et al., 2017) have been shown to reduce off-target effects with very little impact on on-target activity, Selection of edited cells – making precise alterations in the genome of cells is now…”