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| Status |
Public on Oct 05, 2023 |
| Title |
Discovery of diverse DNA cytosine deaminases enables a single-enzyme method for base resolution methylation detection |
| Organisms |
Escherichia coli; Homo sapiens |
| Experiment type |
Other
|
| Summary |
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 their substrate preference. 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 screened 175 putative cytosine deaminases, primarily from bacteria, and found enzymes with strong activity on double- and single-stranded DNA in various sequence contexts, including some without any sequence constraints. We also found enzymes that do not deaminate modified cytosines. The remarkable diversity of cytosine deaminases opens new avenues for biotechnological and medical applications. As a demonstration, we developed a single-enzyme methylation sequencing (SEM-seq) method for 5-methylcytosine detection using a novel non-specific, modification-sensitive double-stranded DNA deaminase, MsddA. SEM-seq generated accurate base-resolution maps of 5-methylcytosine in human genome samples including cell free DNA and samples of 10 pg DNA, equivalent to single cell input. This simple and efficient protocol has the potential to allow high-throughput epigenome profiling of scarce biological material.
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| Overall design |
DNA sequencing of deaminated double-stranded and single-stranded E.coli genomic DNA plus other control DNAs with various DNA modification types. 2 replicates were conducted for each deaminase tested.
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| Web link |
https://www.biorxiv.org/content/10.1101/2023.06.29.547047v1
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| Contributor(s) |
Vaisvila R, Johnson S, Yan B, Sun Z |
| Citation missing |
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| Submission date |
Jun 01, 2023 |
| Last update date |
Jan 31, 2024 |
| Contact name |
Zhiyi Sun |
| E-mail(s) |
sunz@neb.com
|
| Organization name |
New England Biolabs
|
| Department |
Research
|
| Street address |
240 County Road
|
| City |
Ipswich |
| State/province |
MASSACHUSETTS |
| ZIP/Postal code |
01938 |
| Country |
USA |
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| Platforms (3) |
| GPL14548 |
Illumina HiSeq 2000 (Escherichia coli) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
| GPL32081 |
NextSeq 2000 (Escherichia coli) |
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| Samples (478)
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| Relations |
| BioProject |
PRJNA978603 |
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