Journal article

Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing

Minh Thuan Nguyen Tran, Mohd Khairul Nizam Mohd Khalid, Qi Wang, Jacqueline KR Walker, Grace E Lidgerwood, Kimberley L Dilworth, Leszek Lisowski, Alice Pebay, Alex W Hewitt

Nature Communications | NATURE RESEARCH | Published : 2020

Abstract

Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaC..

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Grants

Awarded by National Health and Medical Research Council (NHMRC)


Funding Acknowledgements

This work was supported by a National Health and Medical Research Council (NHMRC) Senior Research Fellowship (A.P., 1154389) and a Practitioner Fellowship (A.W.H., APP1103329), the Australian Research Council Special Research Initiative in Stem Cell Science (Stem Cells Australia), NHMRC project grant, Vector and Genome Engineering Facility, and the Australian Medical Research Future Fund. We are grateful for scripts used in the identification of off-target base editing in RNA-seq data provided by Sowmya Iyer. We thank G.S. Liu, A.L. Cook, K. Fairfax, and F. Patterson for their assistance with cell culture and DNA extraction. In addition, we thank R. KC for his assistance with western blots and J. Marthick for the maintenance of the Illumina MiSeq machine.