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.
Bibliographical noteFunding Information:
We thank David Liu for providing BE3 and University of Minnesota Center for Genome Engineering colleagues for helpful comments. These studies were supported in part by a grant from the National Cancer Institute (P01-CA234228 to RS Harris). Salary support for JL McCann was provided in part by the National Science Foundation Graduate Research Fellowship (Grant Number 00039202) and for DJ Salamango from the University of Minnesota Craniofacial Research Training (MinnCResT) program (T90-DE022732) and National Institute of Allergy and Infectious Disease K99/R00 (K99-AI147811). RS Harris is the Margaret Harvey Schering Land Grant Chair for Cancer Research, a Distinguished University McKnight Professor and an Investigator of the Howard Hughes Medical Institute.