High-throughput functional variant screens via in vivo production of single-stranded DNA
Name
pnas.2018181118.pdf
Description
Published version
Size
1.29 MB
Format
Adobe PDF
Checksum (MD5)
0c11ef6b123956ec7ca972409f475045
Author(s)
Schubert, Max G
Goodman, Daniel B
Wannier, Timothy M
Kaur, Divjot
Farzadfard, Fahim
Lu, Timothy K
Shipman, Seth L
Church, George M
Date Issued
2021
Journal
Proceedings of the National Academy of Sciences of the United States of America
Publisher
Proceedings of the National Academy of Sciences
Citation
Schubert, Max G, Goodman, Daniel B, Wannier, Timothy M, Kaur, Divjot, Farzadfard, Fahim et al. 2021. "High-throughput functional variant screens via in vivo production of single-stranded DNA." Proceedings of the National Academy of Sciences of the United States of America, 118 (18).
Version
Final published version
Abstract
Significance
We report a methodology for the pooled construction of mutants bearing precise genomic sequence variations and multiplex phenotypic characterization of these mutants using next-generation sequencing (NGS). Unlike existing techniques depending on CRISPR-Cas–directed genomic breaks for genome editing, this strategy instead uses single-stranded DNA produced by a retron element for recombineering. This enables libraries of millions of elements to be constructed and offers relaxed design constraints which permit natural DNA or random variation to be used as inputs.
We report a methodology for the pooled construction of mutants bearing precise genomic sequence variations and multiplex phenotypic characterization of these mutants using next-generation sequencing (NGS). Unlike existing techniques depending on CRISPR-Cas–directed genomic breaks for genome editing, this strategy instead uses single-stranded DNA produced by a retron element for recombineering. This enables libraries of millions of elements to be constructed and offers relaxed design constraints which permit natural DNA or random variation to be used as inputs.
MIT Department
Massachusetts Institute of Technology. Research Laboratory of Electronics
Terms of Use
Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1073/PNAS.2018181118
