Nuclear and mitochondrial DNA editing in human cells with zinc finger deaminases

Kayeong Lim,Sung-Ik Cho,Jin-Soo Kim

doi:10.1038/s41467-022-27962-0

Kayeong Lim, Sung-Ik Cho + Show 1 more

Open Access

https://doi.org/10.1038/s41467-022-27962-0

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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 DddAtox, 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.

Highlights

Base editing in nuclear DNA and mitochondrial DNA is broadly useful for biomedical research, medicine, and biotechnology
Base editing enables C-to-T or A-to-G conversions in cell lines[3,7,8], animals[19,32,33], and plants[20,21,34], allowing researchers to study the functional effects of singlenucleotide polymorphisms (SNPs) and to correct disease-causing point mutations for therapeutic applications
CRISPR-derived base editors are composed of catalytically-impaired Cas[9] or Cas12a variants as DNA-binding units and single-strand DNA-specific deaminases originated from rat, sea lamprey, or E. coli[7,8,9,35], whereas DdCBEs are composed of transcription activator-like effector (TALE) DNA-binding arrays and double-strand DNA-specific DddAtox[3]

Summary

Introduction

Base editing in nuclear DNA and mitochondrial DNA (mtDNA) is broadly useful for biomedical research, medicine, and biotechnology. We present a base editing platform, termed zinc finger deaminases (ZFDs), composed of custom-designed zinc-finger DNAbinding proteins, the split interbacterial toxin deaminase DddAtox, 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. A growing list of tools have been reported for genome editing in eukaryotic cells and organisms, a broadly useful method in research and medicine, which include, but are not limited to, zinc finger nucleases (ZFNs)[1], transcription activator-like effector (TALE) nucleases (TALENs)[2], TALElinked split interbacterial deaminase toxin DddA-derived cytosine base editors (a.k.a. DdCBEs)[3], CRISPR-Cas[94–6], and catalytically-impaired Cas9-linked deaminases (a.k.a. base editors)[7,8,9].

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