Nuclear and Mitochondrial DNA Editing in Human Cells with Zinc Finger Deaminases

Overview

Journal Nat Commun

Specialty Biology

Date 2022 Jan 19

PMID 35042880

Authors

Kayeong Lim

Sung-Ik Cho

Jin-Soo Kim

Affiliations

Soon will be listed here.

Abstract

Base editing in nuclear DNA and mitochondrial DNA (mtDNA) is broadly useful for biomedical research, medicine, and biotechnology. Here, we present a base editing platform, termed zinc finger deaminases (ZFDs), composed of custom-designed zinc-finger DNA-binding proteins, the split interbacterial toxin deaminase DddA, and a uracil glycosylase inhibitor (UGI), which catalyze targeted C-to-T base conversions without inducing unwanted small insertions and deletions (indels) in human cells. We assemble plasmids encoding ZFDs using publicly available zinc finger resources to achieve base editing at frequencies of up to 60% in nuclear DNA and 30% in mtDNA. Because ZFDs, unlike CRISPR-derived base editors, do not cleave DNA to yield single- or double-strand breaks, no unwanted indels caused by error-prone non-homologous end joining are produced at target sites. Furthermore, recombinant ZFD proteins, expressed in and purified from E. coli, penetrate cultured human cells spontaneously to induce targeted base conversions, demonstrating the proof-of-principle of gene-free gene therapy.

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References

Urnov F, Miller J, Lee Y, Beausejour C, Rock J, Augustus S . Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature. 2005; 435(7042):646-51. DOI: 10.1038/nature03556. View

Kim H, Kim J . A guide to genome engineering with programmable nucleases. Nat Rev Genet. 2014; 15(5):321-34. DOI: 10.1038/nrg3686. View

Gonzalez B, Schwimmer L, Fuller R, Ye Y, Asawapornmongkol L, Barbas 3rd C . Modular system for the construction of zinc-finger libraries and proteins. Nat Protoc. 2010; 5(4):791-810. PMC: 2855653. DOI: 10.1038/nprot.2010.34. View

Miller J, Tan S, Qiao G, Barlow K, Wang J, Xia D . A TALE nuclease architecture for efficient genome editing. Nat Biotechnol. 2010; 29(2):143-8. DOI: 10.1038/nbt.1755. View

Kosicki M, Tomberg K, Bradley A . Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol. 2018; 36(8):765-771. PMC: 6390938. DOI: 10.1038/nbt.4192. View

Lee H, Kweon J, Kim E, Kim S, Kim J . Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases. Genome Res. 2011; 22(3):539-48. PMC: 3290789. DOI: 10.1101/gr.129635.111. View

Lee H, Lee S, Baek G, Kim A, Kang B, Seo H . Mitochondrial DNA editing in mice with DddA-TALE fusion deaminases. Nat Commun. 2021; 12(1):1190. PMC: 7895935. DOI: 10.1038/s41467-021-21464-1. View

Shin H, Wang C, Lee H, Yoo K, Zeng X, Kuhns T . CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome. Nat Commun. 2017; 8:15464. PMC: 5460021. DOI: 10.1038/ncomms15464. View

Kim D, Lim K, Kim S, Yoon S, Kim K, Ryu S . Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nat Biotechnol. 2017; 35(5):475-480. DOI: 10.1038/nbt.3852. View

10.

Mali P, Yang L, Esvelt K, Aach J, Guell M, DiCarlo J . RNA-guided human genome engineering via Cas9. Science. 2013; 339(6121):823-6. PMC: 3712628. DOI: 10.1126/science.1232033. View

11.

Adikusuma F, Piltz S, Corbett M, Turvey M, McColl S, Helbig K . Large deletions induced by Cas9 cleavage. Nature. 2018; 560(7717):E8-E9. DOI: 10.1038/s41586-018-0380-z. View

12.

Kim S, Lee M, Kim H, Kang M, Kim J . Preassembled zinc-finger arrays for rapid construction of ZFNs. Nat Methods. 2010; 8(1):7. DOI: 10.1038/nmeth0111-7a. View

13.

Miller J, Patil D, Xia D, Paine C, Fauser F, Richards H . Enhancing gene editing specificity by attenuating DNA cleavage kinetics. Nat Biotechnol. 2019; 37(8):945-952. DOI: 10.1038/s41587-019-0186-z. View

14.

Mok B, de Moraes M, Zeng J, Bosch D, Kotrys A, Raguram A . A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature. 2020; 583(7817):631-637. PMC: 7381381. DOI: 10.1038/s41586-020-2477-4. View

15.

Kang B, Yun J, Kim S, Shin Y, Ryu J, Choi M . Precision genome engineering through adenine base editing in plants. Nat Plants. 2018; 4(7):427-431. DOI: 10.1038/s41477-018-0178-x. View

16.

Gaj T, Guo J, Kato Y, Sirk S, Barbas 3rd C . Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nat Methods. 2012; 9(8):805-7. PMC: 3424280. DOI: 10.1038/nmeth.2030. View

17.

Ihry R, Worringer K, Salick M, Frias E, Ho D, Theriault K . p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nat Med. 2018; 24(7):939-946. DOI: 10.1038/s41591-018-0050-6. View

18.

Picardi E, DErchia A, Montalvo A, Pesole G . Using REDItools to Detect RNA Editing Events in NGS Datasets. Curr Protoc Bioinformatics. 2015; 49:12.12.1-12.12.15. DOI: 10.1002/0471250953.bi1212s49. View

19.

Liu J, Gaj T, Wallen M, Barbas 3rd C . Improved cell-penetrating zinc-finger nuclease proteins for precision genome engineering. Mol Ther Nucleic Acids. 2015; 4:e232. PMC: 4354341. DOI: 10.1038/mtna.2015.6. View

20.

Nakazato I, Okuno M, Yamamoto H, Tamura Y, Itoh T, Shikanai T . Targeted base editing in the plastid genome of Arabidopsis thaliana. Nat Plants. 2021; 7(7):906-913. PMC: 8289735. DOI: 10.1038/s41477-021-00954-6. View