Journal
SCIENCE ADVANCES
Volume 6, Issue 29, Pages -Publisher
AMER ASSOC ADVANCEMENT SCIENCE
DOI: 10.1126/sciadv.aba1773
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Funding
- Robert A. Welch Foundation [C-1952, C-1559]
- NIH [HL151545]
- Rice University Creative Ventures Fund
- NSF [CHE-1664218, PHY-1427654]
- National Natural Science Foundation of China [31922048]
- Agricultural Science and Technology Innovation Program
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Cytosine base editors (CBEs) enable efficient cytidine-to-thymidine (C-to-T) substitutions at targeted loci without double-stranded breaks. However, current CBEs edit all Cs within their activity windows, generating undesired bystander mutations. In the most challenging circumstance, when a bystander C is adjacent to the targeted c, existing base editors fail to discriminate them and edit both Cs.To improve the precision of CBE, we identified and engineered the human APOBEC3G (A3G) deaminase; when fused to the Cas9 nickase, the resulting A3G-BEs exhibit selective editing of the second C in the 5'-CC-3' motif in human cells. Our A3G-BEs could install a single disease-associated C-to-T substitution with high precision. The percentage of perfectly modified alleles is more than 6000-fold for disease correction and more than 600-fold for disease modeling compared with BE4max. On the basis of the two-cell embryo injection method and RNA sequencing analysis, our A3G-BEs showed minimum genome- and transcriptome-wide off-target effects, achieving high targeting fidelity.
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