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Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity.

ABSTRACT: Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination. Compared to commonly used CBEs, TadA-derived cytosine base editors (TadCBEs) offer similar or higher on-target activity, smaller size and substantially lower Cas-independent DNA and RNA off-target editing activity. We also identified a TadA dual base editor (TadDE) that performs equally efficient cytosine and adenine base editing. TadCBEs support single or multiplexed base editing at therapeutically relevant genomic loci in primary human T cells and primary human hematopoietic stem and progenitor cells. TadCBEs expand the utility of CBEs for precision gene editing.

SUBMITTER: Neugebauer ME

PROVIDER: S-EPMC10188366 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity.

Neugebauer Monica E ME Hsu Alvin A Arbab Mandana M Krasnow Nicholas A NA McElroy Amber N AN Pandey Smriti S Doman Jordan L JL Huang Tony P TP Raguram Aditya A Banskota Samagya S Newby Gregory A GA Tolar Jakub J Osborn Mark J MJ Liu David R DR

Nature biotechnology 20221110 5

Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that a ...[more]

PMID: 36357719

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Project description:BackgroundBase editors (BEs) display diverse applications in a variety of plant species such as Arabidopsis, rice, wheat, maize, soybean, and cotton, where they have been used to mediate precise base pair conversions without the collateral generation of undesirable double-stranded breaks (DSB). Studies of single-nucleotide polymorphisms (SNPs) underpinning plant traits are still challenging, particularly in polyploidy species where such SNPs are present in multiple copies, and simultaneous modification of all alleles would be required for functional analysis. Allotetraploid cotton has a number of homoeologous gene pairs located in the A and D sub-genomes with considerable SNPs, and it is desirable to develop adenine base editors (ABEs) for efficient and precise A-to-G single-base editing without DSB in such complex genome.ResultsWe established various ABE vectors based on different engineered adenosine deaminase (TadA) proteins fused to Cas9 variants (dCas9, nCas9), enabling efficient A to G editing up to 64% efficiency on-target sites of the allotetraploid cotton genome. Comprehensive analysis showed that GhABE7.10n exhibited the highest editing efficiency, with the main editing sites specifically located at the position A5 (counting the PAM as positions 21-23). Furthermore, DNA and RNA off-target analysis of cotton plants edited with GhABE7.10n and GhABE7.10d by whole genome and whole-transcriptome sequencing revealed no DNA off-target mutations, while very low-level RNA off-target mutations were detected. A new base editor, namely GhABE7.10dCpf1 (7.10TadA + dCpf1), that recognizes a T-rich PAM, was developed for the first time. Targeted A-to-G substitutions generated a single amino acid change in the cotton phosphatidyl ethanolamine-binding protein (GhPEBP), leading to a compact cotton plant architecture, an ideotype for mechanized harvesting of modern cotton production.ConclusionsOur data illustrate the robustness of adenine base editing in plant species with complex genomes, which provides efficient and precise toolkit for cotton functional genomics and precise molecular breeding.

Dataset Information

Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity.

Publications

Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity.

Similar Datasets

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