Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

By Alexis C. Komor, Yongjoo B. Kim, M. Packer, John A. Zuris, David R. Liu. 2016

4,662 citations recorded by the source corpus.

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.

Subfields: Molecular Biology

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Citations in the corpus

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity (2018, positive): “To evaluate C•G-to-T•A base editing activity of xCas9 variants, we substituted SpCas9 with xCas9 3.7 and 3.6 in the third-generation (BE3) base editor architecture 4 .”
CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture. (2019, background): “The cytosine base-editor (CBE) system, which consists of a cytidine deaminase fused with an nCas9 (D10A) and a uracil glycosylase inhibitor, converts targeted cytosine to uracil in genomic DNA (63) (Figure 3a).”
Genome Editing—Principles and Applications for Functional Genomics Research and Crop Improvement (2017, background): “A) substitution, without DSBs or exogenous donor DNA (Komor et al., 2016).”
Current Challenges of iPSC-Based Disease Modeling and Therapeutic Implications (2019, background): “Zinc-finger nucleases, transcription activator-like effector nucleases (TALENS), clustered regularly interspaced short palindromic repeats (CRISPR) associated (Cas) systems-9 based gene or base editing (whereby cytidine deaminase converts cytidine to uridine without the need for double strand break ”
RNA Editing with CRISPR-Cas13 (2017, background): “the formation of double-strand break induced indels (7, 8).”
Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions (2017, background): “Our third-generation base editor, BE3, converts C:G base pairs to T:A base pairs, including disease-relevant point mutations, in a variety of cell lines with higher efficiency and lower indel frequency than what can be achieved using other genome editing methods 1 .”
CRISPR technology: A decade of genome editing is only the beginning (2023, background): “Beyond traditional CRISPR-induced knockouts involving double-stranded DNA cutting, base editing (11)—which uses Cas effectors fused to enzymes that alter the chemical nature of DNAbases—has provided a successful strategy to generate site-specific and precise point mutations without DSBs, eliminating”