Abstract
Although CRISPR/Cas9 technology has created a renaissance in genome engineering, particularly for gene knockout generation, methods to introduce precise single base changes are also highly desirable. The covalent fusion of a DNA-editing enzyme such as APOBEC to a Cas9 nickase complex has heightened hopes for such precision genome engineering. However, current cytosine base editors are prone to undesirable off-target mutations, including, most frequently, target-adjacent mutations. Here, we report a method to "attract" the DNA deaminase, APOBEC3B, to a target cytosine base for specific editing with minimal damage to adjacent cytosine bases. The key to this system is fusing an APOBEC-interacting protein (not APOBEC itself) to Cas9n, which attracts nuclear APOBEC3B transiently to the target site for editing. Several APOBEC3B interactors were tested and one, hnRNPUL1, demonstrated proof-of-concept with successful C-to-T editing of episomal and chromosomal substrates and lower frequencies of target-adjacent events.
© 2020 McCann et al.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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APOBEC Deaminases / genetics
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CRISPR-Associated Protein 9 / genetics
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CRISPR-Cas Systems / genetics
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Clustered Regularly Interspaced Short Palindromic Repeats / genetics
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Cytidine Deaminase / genetics*
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Cytosine / chemistry
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DNA / genetics
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Deoxyribonuclease I / genetics
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Gene Editing / methods*
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Genetic Engineering / methods*
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Genome / genetics
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Heterogeneous-Nuclear Ribonucleoproteins / genetics*
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Heterogeneous-Nuclear Ribonucleoproteins / metabolism
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Minor Histocompatibility Antigens / genetics*
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Nuclear Proteins / genetics*
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Nuclear Proteins / metabolism
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Proof of Concept Study
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Transcription Factors / genetics*
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Transcription Factors / metabolism
Substances
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HNRNPUL1 protein, human
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Heterogeneous-Nuclear Ribonucleoproteins
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Minor Histocompatibility Antigens
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Nuclear Proteins
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Transcription Factors
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Cytosine
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DNA
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CRISPR-Associated Protein 9
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Deoxyribonuclease I
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APOBEC Deaminases
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APOBEC3B protein, human
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Cytidine Deaminase