Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions

By Y. B. Kim, Alexis C. Komor, Jonathan M. Levy, M. Packer, K. Zhao, David R. Liu. 2017

705 citations recorded by the source corpus.

Base editing induces single-nucleotide changes in the DNA of living cells using a fusion protein containing a catalytically defective Streptococcus pyogenes Cas9, a cytidine deaminase, and an inhibitor of base excision repair. This genome editing approach has the advantage that it does not require formation of double-stranded DNA breaks or provision of a donor DNA template. Here we report the development of five C to T (or G to A) base editors that use natural and engineered Cas9 variants with different protospacer-adjacent motif (PAM) specificities to expand the number of sites that can be targeted by base editing 2.5-fold. Additionally, we engineered base editors containing mutated cytidine deaminase domains that narrow the width of the editing window from ∼5 nucleotides to as little as 1–2 nucleotides. We thereby enabled discrimination of neighboring C nucleotides, which would otherwise be edited with similar efficiency, and doubled the number of disease-associated target Cs able to be corrected preferentially over nearby non-target Cs.

Subfields: Molecular Biology

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In Molecular Biology

1. Gregory A. Newby
2. H. Rees
3. Aditya Raguram
4. Jordan L. Doman
5. David R. Liu
6. Caixia Gao

Citations in the corpus

• Evolved Cas9 variants with broad PAM compatibility and high DNA specificity (2018, positive): “Overall, these results indicate that xCas9 variants are compatible with the BE3 architecture, and enable cytidine base editing of target sites that cannot be accessed by SpCas9–BE3 or, with the exception of NGA PAM sites 33 , by any other previously reported base editors.”
• Evaluation and Minimization of Cas9-Independent Off-Target DNA Editing by Cytosine Base Editors (2020, positive): “We created APOBEC1 variants W90Y+R126E (YE1), W90Y+R132E (YE2), R126E+R132E (EE), and W90Y +R126E+R132E (YEE) to narrow the on-target base editing window 31 .”
• Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage (2017, neutral): “2 variants and challenged the resulting TadA*1.2–dCas9 mutants in bacteria with higher concentrations of chloramphenicol than were used in round 1 (Supplementary Tables 7 and 8).”