An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities

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From: Nature Biotechnology(Vol. 36, Issue 10)
Publisher: Nature Publishing Group
Document Type: Report
Length: 8,021 words
Lexile Measure: 1730L

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Author(s): Jason M Gehrke [1, 2]; Oliver Cervantes [1]; M Kendell Clement [1, 3]; Yuxuan Wu [4]; Jing Zeng [4]; Daniel E Bauer [4]; Luca Pinello [1, 3]; J Keith Joung (corresponding author) [1, 3]

Base editor technology, which uses CRISPR-Cas9 to direct cytidine deaminase enzymatic activity to specific genomic loci, enables the highly efficient introduction of precise cytidine-to-thymidine DNA alterations [1, 2, 3, 4, 5, 6]. However, existing base editors create unwanted C-to-T alterations when more than one C is present in the enzyme's five-base-pair editing window. Here we describe a strategy for reducing bystander mutations using an engineered human APOBEC3A (eA3A) domain, which preferentially deaminates cytidines in specific motifs according to a TCR>TCY>VCN hierarchy. In direct comparisons with the widely used base editor 3 (BE3) fusion in human cells, our eA3A-BE3 fusion exhibits similar activities on cytidines in TC motifs but greatly reduced editing on cytidines in other sequence contexts. eA3A-BE3 corrects a human [beta]-thalassemia promoter mutation with much higher (>40-fold) precision than BE3. We also demonstrate that eA3A-BE3 shows reduced mutation frequencies on known off-target sites of BE3, even when targeting promiscuous homopolymeric sites.

In contrast to gene-editing nucleases [7, 8, 9], base editors do not require double-strand breaks or exogenous donor DNA templates, and they induce lower levels of unwanted variable-length insertion/deletion mutations (indels) [1, 2, 10], but their ability to edit all Cs within their editing window can potentially have deleterious effects. Mutations in the cytidine deaminase enzyme can shorten the length of the editing window and thereby partially address this limitation but these base editor variants still do not discriminate among multiple cytidines within the narrowed window and also possess a more limited targeting range [11].

To engineer base editors with greater precision within the editing window, we leveraged the natural diversity of cytidine deaminases to identify one with greater sequence specificity than the rat APOBEC1 (rAPO1) deaminase present in the widely used BE3 architecture. BE3 consists of a Streptococcus pyogenes Cas9 nuclease, bearing a mutation that converts it into a nickase (nCas9), fused to rAPO1 and a uracil glycosylase inhibitor (UGI) (Fig. 1a). We replaced rAPO1 in BE3 with the human APOBEC3A (A3A) cytidine deaminase to create A3A-BE3 (Fig. 1a). We used A3A because previous in vitro studies showed preferential deamination of cytidines in a T C R motif (where R = A/G) [12, 13, 14]. To test the precision of A3A-BE3, we used a guide RNA (gRNA) targeted to a single integrated EGFP reporter gene in human U2OS cells, which bears both a cognate motif (TCG) and a non-cognate bystander (GCT) motif within its expected editing window. Surprisingly, A3A-BE3 did not preferentially edit the cytidine in the TCG motif over the GCT motif (Fig. 1b).

We hypothesized that the lack of expected sequence preference by A3A-BE3 on the EGFP site might have been due to the increased proximity of A3A secondary to its recruitment to that site. We envisioned that sequence selectivity might be restored by reducing the non-specific binding of A3A...

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Gale Document Number: GALE|A572704122