Person: Sousa, Alexander
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Publication Enhanced proofreading governs CRISPR-Cas9 targeting accuracy
(2017) Chen, Janice S.; Dagdas, Yavuz S.; Kleinstiver, Benjamin; Welch, Moira M.; Sousa, Alexander; Harrington, Lucas B.; Sternberg, Samuel H.; Joung, J. Keith; Yildiz, Ahmet; Doudna, Jennifer A.The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1–4. 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 were unknown5–9. Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state10 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 designed 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.
Publication Search-and-Replace Genome Editing Without Double-Strand Breaks or Donor DNA
(Springer Science and Business Media LLC, 2019-10-21) Anzalone, Andrew; Randolph, Peyton B.; Davis, Jessie R.; Sousa, Alexander; Koblan, Luke; Levy, Jonathan; Chen, Peter; Wilson, Christopher; Newby, Gregory; Raguram, Aditya; Liu, David R.