气候变化领域集成服务门户(CCPortal): High-throughput functional variant screens via in vivo production of single-stranded DNA

CCPortal

DOI	10.1073/pnas.2018181118
	High-throughput functional variant screens via in vivo production of single-stranded DNA
	Schubert M.G.; Goodman D.B.; Wannier T.M.; Kaur D.; Farzadfard F.; Lu T.K.; Shipman S.L.; Church G.M.
发表日期	2021
ISSN	00278424
卷号	118 期号:18
英文摘要	Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome. © 2021 National Academy of Sciences. All rights reserved.
英文关键词	Antibiotic resistance; Functional genomics; Genetic engineering; Retron; Synthetic biology
语种	英语
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/179692
作者单位	Department of Genetics, Harvard Medical School, Boston, MA 02115, United States; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, United States; Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA 94143, United States; Department of Zoology, University of Warwick, Coventry, CV4 7AL, United Kingdom; Research Laboratory of Electronics, Massachussetts Institute of Technology, Cambridge, MA 02139, United States; Gladstone Institute of Data Science and Biotechnology, San Francisco, CA 94158, United States
推荐引用方式 GB/T 7714	Schubert M.G.,Goodman D.B.,Wannier T.M.,et al. High-throughput functional variant screens via in vivo production of single-stranded DNA[J],2021,118(18).
APA	Schubert M.G..,Goodman D.B..,Wannier T.M..,Kaur D..,Farzadfard F..,...&Church G.M..(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).
MLA	Schubert M.G.,et al."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(2021).