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.
Highlights
The original BE3 cytosine base editor (CBE) comprised the rat APOBEC1 deaminase fused to the N-terminal end of a Cas9 nickase (Cas9n D10A [1])
Most MagnEdit complexes failed to stimulate editing beyond these background levels or those caused by a non-interacting blue fluorescent protein (BFP)-Cas9n control (Fig 1C)
This study describes a fundamentally different approach to single base editing through the use of non-covalent interactions to “attract” a DNA cytosine deaminase to a single target cytosine
Summary
The original BE3 cytosine base editor (CBE) comprised the rat APOBEC1 deaminase fused to the N-terminal end of a Cas nickase (Cas9n D10A [1]). This technology is prone to a number of off-target effects, including RNA editing [3, 4], random genomic DNA editing [5, 6, 7, 8], and most frequently target-adjacent editing [1, 2, 5, 9, 10] The latter problem is due predominantly, if not exclusively, to deamination of single-stranded DNA cytosines located adjacent to the desired target cytosine in the same gRNA-displaced R-loop.
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