A Novel Double-Stranded DNA Deaminase-Based and Transcriptional Activator-Assisted Nuclear and Mitochondrial Cytosine Base Editors with Expanded Target Compatibility and Enhanced Activity

Guo, Junfan; Yu, Wenxia; Li, Min; Chen, Hongyu; Liu, Jie; Xue, Xiaowen; Lin, Jianxiang; Huang, Shisheng; Shu, Wenjie; Huang, Xingxu; Liu, Zhen; Wang, Shengqi; Qiao, Yong

doi:10.2139/ssrn.4227259

Cited by 2 publications

(2 citation statements)

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“…Several recent studies have reported a functional characterization of enzymes from the DddA-like family [28][29][30] . One study 28 showed that deletion of SPKK-related motifs at the C-terminus of DddA abrogates its activity on dsDNA.…”

Section: Discussionmentioning

confidence: 99%

Discovery of novel DNA cytosine deaminase activities enables a nondestructive single-enzyme methylation sequencing method for base resolution high-coverage methylome mapping of cell-free and ultra-low input DNA

Vaisvila,

Johnson,

Yan

et al. 2023

Preprint

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Cytosine deaminases have important uses in the detection of epigenetic modifications and in genome editing. However, the range of applications of deaminases is limited by a small number of well characterized enzymes. To expand the toolkit of deaminases, we developed an in-vitro approach that bypasses a major hurdle with their severe toxicity in expression hosts. We systematically assayed the activity of 175 putative cytosine deaminases on an unprecedented variety of substrates with epigenetically relevant base modifications. We found enzymes with high activity on double- and single-stranded DNA in various sequence contexts including novel CpG-specific deaminases, as well as enzymes without sequence preference. We also report, for the first time, enzymes that do not deaminate modified cytosines. The remarkable diversity of cytosine deaminases opens new avenues for biotechnological and medical applications. Using a newly discovered non-specific, modification-sensitive double-stranded DNA deaminase, we developed a nondestructive single-enzyme 5-methylctyosine sequencing (SEM-seq) method. SEM-seq enables accurate, high-coverage, base-resolution methylome mapping of scarce biological material including clinically relevant cell-free DNA (cfDNA) and single-cell equivalent 10 pg input DNA. Using SEM-seq, we generated highly reproducible base-resolution 5mC maps, accounting for nearly 80% of CpG islands for a low input human cfDNA sample offering valuable information for identifying potential biomarkers for detection of early-stage cancer and other diseases. This streamlined protocol will enable robust, high-throughput, high-coverage epigenome profiling of challenging samples in research and clinical settings.

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Section: Discussionmentioning

confidence: 99%

Discovery of novel DNA cytosine deaminase activities enables a nondestructive single-enzyme methylation sequencing method for base resolution high-coverage methylome mapping of cell-free and ultra-low input DNA

Vaisvila,

Johnson,

Yan

et al. 2023

Preprint

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“…Programmable deaminases, which are composed of a custom DNA-binding protein and a nucleobase deaminase, enable mitochondrial DNA editing in a targeted manner 13, 18–25 . There are two types of programable deaminases that have been developed for organellar genome editing: cytosine base editors 13, 18, 20–24, 26 , including DddA-derived cytosine base editors (DdCBEs) and zinc finger deaminases, both of which induce C-to-T (= G-to-A on the other DNA strand) editing, and adenine base editors 19 , termed TALEDs, which catalyze A-to-G (= T-to-C) editing. These base editors bind to a target DNA site in the mitochondrial genome and deaminate cytosine or adenine to yield uracil or hypoxanthine, respectively, which is base-paired with adenine or cytosine, respectively, during DNA replication or repair.…”

Section: Mainmentioning

confidence: 99%

Engineering TALE-linked deaminases to facilitate precision adenine base editing in mitochondrial DNA

Cho,

Lim,

Hong

et al. 2023

Preprint

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DddA-derived cytosine base editors (DdCBEs) and transcription activator-like effector (TALE)-linked deaminases (TALEDs) catalyze targeted base editing of mitochondrial DNA (mtDNA) in eukaryotic cells, a method useful for modeling of mitochondrial genetic disorders and developing novel therapeutic modalities. Here, we report that A-to-G editing TALEDs but not C-to-T editing DdCBEs induce tens of thousands of transcriptome-wide off-target edits in human cells. To avoid these unwanted RNA edits, we engineered the substrate-binding site in TadA8e, the deoxy-adenine deaminase in TALEDs, and created TALED variants with fine-tuned deaminase activity. Our engineered TALED variants not only reduced RNA off-target edits by > 99% but also minimized off-target mtDNA mutations and bystander edits at a target site. Unlike wild-type versions, our TALED variants were not cytotoxic and did not cause developmental arrest of mouse embryos. As a result, we obtained mice with pathogenic mtDNA mutations, associated with Leigh disease, which showed reduced heart rates.

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A Novel Double-Stranded DNA Deaminase-Based and Transcriptional Activator-Assisted Nuclear and Mitochondrial Cytosine Base Editors with Expanded Target Compatibility and Enhanced Activity

Cited by 2 publications

References 0 publications

Discovery of novel DNA cytosine deaminase activities enables a nondestructive single-enzyme methylation sequencing method for base resolution high-coverage methylome mapping of cell-free and ultra-low input DNA

Discovery of novel DNA cytosine deaminase activities enables a nondestructive single-enzyme methylation sequencing method for base resolution high-coverage methylome mapping of cell-free and ultra-low input DNA

Engineering TALE-linked deaminases to facilitate precision adenine base editing in mitochondrial DNA

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