RNA-guided programmable nucleases from CRISPR systems generate precise breaks in DNA or RNA at specified positions. In cells, this activity can lead to changes in DNA sequence or RNA transcript abundance. Base editing is a newer genome-editing approach that uses components from CRISPR systems together with other enzymes to directly install point mutations into cellular DNA or RNA without making double-stranded DNA breaks. DNA base editors comprise a catalytically disabled nuclease fused to a nucleobase deaminase enzyme and, in some cases, a DNA glycosylase inhibitor. RNA base editors achieve analogous changes using components that target RNA. Base editors directly convert one base or base pair into another, enabling the efficient installation of point mutations in non-dividing cells without generating excess undesired editing by-products. In this Review, we summarize base-editing strategies to generate specific and precise point mutations in genomic DNA and RNA, highlight recent developments that expand the scope, specificity, precision and in vivo delivery of base editors and discuss limitations and future directions of base editing for research and therapeutic applications.
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D.R.L. acknowledges support from Defense Advanced Research Projects Agency (DARPA) HR0011-17-2-0049; the Ono Pharma Foundation; US National Institutes of Health (NIH) RM1 HG009490, R01 EB022376, U01 AI142756 and R35 GM118062; and Howard Hughes Medical Institute (HHMI). H.A.R. is supported by the Kilpatrick Educational fund from the Chemistry and Chemical Biology Department, Harvard University. The authors thank J.K. Joung, F. Zhang, A. Raguram, W.-H. Yeh, T. Huang, K. Zhao and W. Tang for their helpful comments.
D.R.L. is a consultant and co-founder of Beam Therapeutics, Editas Medicine and Pairwise Plants, which are all companies that use genome-editing technologies. H.A.R. declares no competing financial interests. Both authors have no competing non-financial interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- Guide RNA
Short RNA sequence comprising a scaffold for binding to the necessary CRISPR-associated (Cas) enzyme and a variable spacer region that defines the target site for the enzyme. In natural CRISPR systems, the guide RNA is often made of two molecules of RNA with complementarity. Engineered ‘single-guide’ RNAs connecting the two natural guide RNA components are often accepted by Cas enzymes.
- Protospacer adjacent motif
(PAM). A small region of nucleotides in the target DNA sequence adjacent to the sequence specified by a guide RNA. The PAM is not specified in the guide RNA, but CRISPR-associated (Cas) enzymes do not bind or cleave a sequence unless they are next to the appropriate PAM.
- Cas9 nickase
A catalytically disabled mutant of a Cas9 enzyme that is able to create a single-stranded DNA break but not a double-stranded DNA break.
- Activity window
The region of DNA or RNA, typically defined by the number of nucleotides from the protospacer adjacent motif (PAM), in which a particular base editor acts to induce efficient point mutations. The activity window for most base editors is approximately four to five nucleotides wide.
A region in a guide RNA of 15–25 nucleotides in length that specifies the target RNA or DNA locus.
- Proximal off-target editing
Unwanted editing of bases that occurs outside of the activity window but is found nearby (for example, 100 nucleotides upstream or downstream of) the target site.
- Distal off-target editing
Unwanted editing of bases residing in locations of the genome or transcriptome unrelated to (for example, >100 nucleotides away from) the target site of the base editor.
A class 2, type VI RNA-guided RNase from the CRISPR system. Variants from several species have been characterized. It catalyses site-specific cleavage of single-stranded RNA.
- Wobble position
The third nucleotide in a codon.
- Base-editing product purity
The term used to describe the spectrum of mutations induced by a particular base-editing technology. Low product purity occurs when a target base is mutated to bases other than the desired point mutation or when small insertions or deletions are generated in addition to the desired edit; for example, C-to-G or C-to-A edits, rather than the desired C-to-T edit, from a cytosine base editor.
An RNA-guided endonuclease variant isolated from the CRISPR system of Streptococcus pyogenes. It catalyses site-specific cleavage of double-stranded DNA at sites with an NGG protospacer adjacent motif (PAM).
An RNA-guided endonuclease variant isolated from the CRISPR system of Staphylococcus aureus. It catalyses site-specific cleavage of double-stranded DNA at sites with an NNGRRT protospacer adjacent motif (PAM).
- Bystander editing
Editing of a non-target base that resides in the activity window of a particular base editor and guide RNA. Bystander editing occurs in addition to editing of the target base.
A class 2, type V RNA-guided endonuclease from the CRISPR system. Variants from several species have been characterized. It catalyses site-specific cleavage of double-stranded DNA at sites with a TTTV protospacer adjacent motif (where V is A, C or G).
A state in which two or more cell populations with distinct genotypes are present in the same organism and derived from a single fertilized egg.
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Rees, H.A., Liu, D.R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet 19, 770–788 (2018). https://doi.org/10.1038/s41576-018-0059-1
Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain
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