Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.
Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.|Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 02138, USA.
Alexis C
AC
Komor
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.|Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 02138, USA.
Yongjoo B
YB
Kim
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.|Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 02138, USA.
Michael S
MS
Packer
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.|Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 02138, USA.
John A
JA
Zuris
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.|Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 02138, USA.
David R
DR
Liu
D000071479:APOBEC-1 Deaminase; D000818:Animals; D053327:Apolipoprotein E4 / Q000235:genetics; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000378:metabolism; D064113:CRISPR-Cas Systems; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D003562:Cytidine / Q000235:genetics*; D003564:Cytidine Deaminase / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D053837:DNA Cleavage; D004260:DNA Repair; D003850:Deoxyribonuclease I / Q000378:metabolism; D016158:Genes, p53 / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome / Q000235:genetics*; D006801:Humans; D054643:INDEL Mutation / Q000235:genetics; D051379:Mice; D017354:Point Mutation / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D013698:Templates, Genetic; D051981:Uracil-DNA Glycosidase / Q000037:antagonists & inhibitors; D014529:Uridine / Q000235:genetics*
D053327:Apolipoprotein E4; D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D003562:Cytidine; D004247:DNA; D003850:Deoxyribonuclease I; D051981:Uracil-DNA Glycosidase; D000071479:APOBEC-1 Deaminase; C000605089:APOBEC1 protein, human; C000605090:Apobec1 protein, mouse; D003564:Cytidine Deaminase; D014529:Uridine