High-throughput functional variant screens via in vivo production of single-stranded DNA
Abstract
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. Additionally, 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.
- Authors:
-
[1];
[2];
[3];
[4];
[6];
[7]
- Department of Genetics, Harvard Medical School, Boston, MA 02115,, Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115,
- Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA 94143,
- Department of Genetics, Harvard Medical School, Boston, MA 02115,
- Department of Zoology, University of Warwick, Coventry CV4 7AL, United Kingdom,
- Research Laboratory of Electronics, Massachussetts Institute of Technology, Cambridge, MA 02139,
- Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA 94143,, Gladstone Institute of Data Science and Biotechnology, San Francisco, CA 94158
- Department of Genetics, Harvard Medical School, Boston, MA 02115,, Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115,, Research Laboratory of Electronics, Massachussetts Institute of Technology, Cambridge, MA 02139,
- Publication Date:
- Research Org.:
- Harvard Univ., Cambridge, MA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC)
- OSTI Identifier:
- 1780511
- Alternate Identifier(s):
- OSTI ID: 1849423
- Grant/Contract Number:
- FG02-02ER63445
- Resource Type:
- Published Article
- Journal Name:
- Proceedings of the National Academy of Sciences of the United States of America
- Additional Journal Information:
- Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 118 Journal Issue: 18; Journal ID: ISSN 0027-8424
- Publisher:
- Proceedings of the National Academy of Sciences
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; Science & Technology - Other Topics; genetic engineering; synthetic biology; functional genomics; antibiotic resistance; retron
Citation Formats
Schubert, Max G., Goodman, Daniel B., Wannier, Timothy M., Kaur, Divjot, Farzadfard, Fahim, Lu, Timothy K., Shipman, Seth L., and Church, George M. High-throughput functional variant screens via in vivo production of single-stranded DNA. United States: N. p., 2021.
Web. doi:10.1073/pnas.2018181118.
Schubert, Max G., Goodman, Daniel B., Wannier, Timothy M., Kaur, Divjot, Farzadfard, Fahim, Lu, Timothy K., Shipman, Seth L., & Church, George M. High-throughput functional variant screens via in vivo production of single-stranded DNA. United States. https://doi.org/10.1073/pnas.2018181118
Schubert, Max G., Goodman, Daniel B., Wannier, Timothy M., Kaur, Divjot, Farzadfard, Fahim, Lu, Timothy K., Shipman, Seth L., and Church, George M. Tue .
"High-throughput functional variant screens via in vivo production of single-stranded DNA". United States. https://doi.org/10.1073/pnas.2018181118.
@article{osti_1780511,
title = {High-throughput functional variant screens via in vivo production of single-stranded DNA},
author = {Schubert, Max G. and Goodman, Daniel B. and Wannier, Timothy M. and Kaur, Divjot and Farzadfard, Fahim and Lu, Timothy K. and Shipman, Seth L. and Church, George M.},
abstractNote = {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. Additionally, 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.},
doi = {10.1073/pnas.2018181118},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 18,
volume = 118,
place = {United States},
year = {Tue Apr 27 00:00:00 EDT 2021},
month = {Tue Apr 27 00:00:00 EDT 2021}
}
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Works referenced in this record:
Functional Genetic Variants Revealed by Massively Parallel Precise Genome Editing
journal, October 2018
- Sharon, Eilon; Chen, Shi-An A.; Khosla, Neil M.
- Cell, Vol. 175, Issue 2
λ Recombination and Recombineering
journal, January 2016
- Murphy, Kenan C.
- EcoSal Plus, Vol. 7, Issue 1
Cutadapt removes adapter sequences from high-throughput sequencing reads
journal, May 2011
- Martin, Marcel
- EMBnet.journal, Vol. 17, Issue 1
Precise Cas9 targeting enables genomic mutation prevention
journal, March 2018
- Chavez, Alejandro; Pruitt, Benjamin W.; Tuttle, Marcelle
- Proceedings of the National Academy of Sciences, Vol. 115, Issue 14
Drug concentration heterogeneity facilitates the evolution of drug resistance
journal, September 1998
- Kepler, T. B.; Perelson, A. S.
- Proceedings of the National Academy of Sciences, Vol. 95, Issue 20
Retrons and their applications in genome engineering
journal, October 2019
- Simon, Anna J.; Ellington, Andrew D.; Finkelstein, Ilya J.
- Nucleic Acids Research, Vol. 47, Issue 21
E. coli Genome Manipulation by P1 Transduction
journal, May 2014
- Thomason, Lynn C.; Costantino, Nina; Court, Donald L.
- Current Protocols in Molecular Biology, Vol. 79, Issue 1
Composability of regulatory sequences controlling transcription and translation in Escherichia coli
journal, August 2013
- Kosuri, Sriram; Goodman, Daniel B.; Cambray, Guillaume
- Proceedings of the National Academy of Sciences, Vol. 110, Issue 34
Search-and-replace genome editing without double-strand breaks or donor DNA
journal, October 2019
- Anzalone, Andrew V.; Randolph, Peyton B.; Davis, Jessie R.
- Nature, Vol. 576, Issue 7785
High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR–Cas9 in yeast
journal, May 2018
- Guo, Xiaoge; Chavez, Alejandro; Tung, Angela
- Nature Biotechnology, Vol. 36, Issue 6
Probing Cellular Processes with Oligo-Mediated Recombination and Using the Knowledge Gained to Optimize Recombineering
journal, March 2011
- Sawitzke, James A.; Costantino, Nina; Li, Xin-tian
- Journal of Molecular Biology, Vol. 407, Issue 1, p. 45-59
Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens
journal, December 2016
- Dixit, Atray; Parnas, Oren; Li, Biyu
- Cell, Vol. 167, Issue 7
Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants the Keio collection
journal, February 2006
- Baba, Tomoya; Ara, Takeshi; Hasegawa, Miki
- Molecular Systems Biology, Vol. 2, Article No. 2006.0008
A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria
journal, June 2016
- Peters, Jason M.; Colavin, Alexandre; Shi, Handuo
- Cell, Vol. 165, Issue 6
Genomically encoded analog memory with precise in vivo DNA writing in living cell populations
journal, November 2014
- Farzadfard, F.; Lu, T. K.
- Science, Vol. 346, Issue 6211
Programming cells by multiplex genome engineering and accelerated evolution
journal, July 2009
- Wang, Harris H.; Isaacs, Farren J.; Carr, Peter A.
- Nature, Vol. 460, Issue 7257, p. 894-898
Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation
journal, January 2019
- Oberacker, Phil; Stepper, Peter; Bond, Donna M.
- PLOS Biology, Vol. 17, Issue 1
Characterizing the portability of phage-encoded homologous recombination proteins
journal, January 2021
- Filsinger, Gabriel T.; Wannier, Timothy M.; Pedersen, Felix B.
- Nature Chemical Biology, Vol. 17, Issue 4
Precise, automated control of conditions for high-throughput growth of yeast and bacteria with eVOLVER
journal, June 2018
- Wong, Brandon G.; Mancuso, Christopher P.; Kiriakov, Szilvia
- Nature Biotechnology, Vol. 36, Issue 7
Multiplex Generation, Tracking, and Functional Screening of Substitution Mutants Using a CRISPR/Retron System
journal, April 2020
- Lim, Hyeonseob; Jun, Soyeong; Park, Minjeong
- ACS Synthetic Biology, Vol. 9, Issue 5
Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation
journal, October 2014
- Gilbert, Luke A.; Horlbeck, Max A.; Adamson, Britt
- Cell, Vol. 159, Issue 3
Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform
journal, February 2017
- Rock, Jeremy M.; Hopkins, Forrest F.; Chavez, Alejandro
- Nature Microbiology, Vol. 2, Issue 4
Precise Editing at DNA Replication Forks Enables Multiplex Genome Engineering in Eukaryotes
journal, November 2017
- Barbieri, Edward M.; Muir, Paul; Akhuetie-Oni, Benjamin O.
- Cell, Vol. 171, Issue 6
Rates and mechanisms of bacterial mutagenesis from maximum-depth sequencing
journal, June 2016
- Jee, Justin; Rasouly, Aviram; Shamovsky, Ilya
- Nature, Vol. 534, Issue 7609
Manipulating replisome dynamics to enhance lambda Red-mediated multiplex genome engineering
journal, August 2012
- Lajoie, M. J.; Gregg, C. J.; Mosberg, J. A.
- Nucleic Acids Research, Vol. 40, Issue 22
Escherichia coli rpoB Mutants Have Increased Evolvability in Proportion to Their Fitness Defects
journal, January 2010
- Barrick, J. E.; Kauth, M. R.; Strelioff, C. C.
- Molecular Biology and Evolution, Vol. 27, Issue 6
Synthetic biosensors for precise gene control and real-time monitoring of metabolites
journal, July 2015
- Rogers, Jameson K.; Guzman, Christopher D.; Taylor, Noah D.
- Nucleic Acids Research, Vol. 43, Issue 15
rpoB Mutations in Multidrug-Resistant Strains of Mycobacterium tuberculosis Isolated in Italy
journal, January 1999
- Pozzi, G.; Meloni, M.; Iona, E.
- Journal of Clinical Microbiology, Vol. 37, Issue 4
The MarR Repressor of the Multiple Antibiotic Resistance (mar) Operon in Escherichia coli: Prototypic Member of a Family of Bacterial Regulatory Proteins Involved in Sensing Phenolic Compounds
journal, May 1995
- Sulavik, Mark C.; Gambino, Laura F.; Miller, Paul F.
- Molecular Medicine, Vol. 1, Issue 4
High-Level dCas9 Expression Induces Abnormal Cell Morphology in Escherichia coli
journal, March 2018
- Cho, Suhyung; Choe, Donghui; Lee, Eunju
- ACS Synthetic Biology, Vol. 7, Issue 4
High-Order Epistasis in Catalytic Power of Dihydrofolate Reductase Gives Rise to a Rugged Fitness Landscape in the Presence of Trimethoprim Selection
journal, April 2019
- Tamer, Yusuf Talha; Gaszek, Ilona K.; Abdizadeh, Haleh
- Molecular Biology and Evolution, Vol. 36, Issue 7
Retrons, msDNA, and the bacterial genome
journal, January 2005
- Lampson, B. C.; Inouye, M.; Inouye, S.
- Cytogenetic and Genome Research, Vol. 110, Issue 1-4
Primerize-2D: automated primer design for RNA multidimensional chemical mapping
journal, January 2017
- Tian, Siqi; Das, Rhiju
- Bioinformatics, Vol. 33, Issue 9
Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants
journal, December 2003
- Costantino, N.; Court, D. L.
- Proceedings of the National Academy of Sciences, Vol. 100, Issue 26, p. 15748-15753
CRISPR /Cas9 recombineering‐mediated deep mutational scanning of essential genes in Escherichia coli
journal, March 2020
- Choudhury, Alaksh; Fenster, Jacob A.; Fankhauser, Reilly G.
- Molecular Systems Biology, Vol. 16, Issue 3
Tempo and mode of genome evolution in a 50,000-generation experiment
journal, August 2016
- Tenaillon, Olivier; Barrick, Jeffrey E.; Ribeck, Noah
- Nature, Vol. 536, Issue 7615
Improved bacterial recombineering by parallelized protein discovery
journal, May 2020
- Wannier, Timothy M.; Nyerges, Akos; Kuchwara, Helene M.
- Proceedings of the National Academy of Sciences, Vol. 117, Issue 24
The Molecular Diversity of Adaptive Convergence
journal, January 2012
- Tenaillon, O.; Rodriguez-Verdugo, A.; Gaut, R. L.
- Science, Vol. 335, Issue 6067, p. 457-461
BEDTools: a flexible suite of utilities for comparing genomic features
journal, January 2010
- Quinlan, Aaron R.; Hall, Ira M.
- Bioinformatics, Vol. 26, Issue 6
High-throughput bacterial functional genomics in the sequencing era
journal, October 2015
- Gray, Andrew N.; Koo, Byoung-Mo; Shiver, Anthony L.
- Current Opinion in Microbiology, Vol. 27
Bacterial Retrons Function In Anti-Phage Defense
journal, December 2020
- Millman, Adi; Bernheim, Aude; Stokar-Avihail, Avigail
- Cell, Vol. 183, Issue 6
PEAR: a fast and accurate Illumina Paired-End reAd mergeR
journal, October 2013
- Zhang, J.; Kobert, K.; Flouri, T.
- Bioinformatics, Vol. 30, Issue 5
Improving Lambda Red Genome Engineering in Escherichia coli via Rational Removal of Endogenous Nucleases
journal, September 2012
- Mosberg, Joshua A.; Gregg, Christopher J.; Lajoie, Marc J.
- PLoS ONE, Vol. 7, Issue 9
Design of a synthetic yeast genome
journal, March 2017
- Richardson, Sarah M.; Mitchell, Leslie A.; Stracquadanio, Giovanni
- Science, Vol. 355, Issue 6329
Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms
journal, May 2013
- van Opijnen, Tim; Camilli, Andrew
- Nature Reviews Microbiology, Vol. 11, Issue 7
A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species
journal, February 2016
- Nyerges, Ákos; Csörgő, Bálint; Nagy, István
- Proceedings of the National Academy of Sciences, Vol. 113, Issue 9
How do mutations at phenylalanine-153 and isoleucine-155 partially suppress the effects of the aspartate-27 .fwdarw. serine mutation in Escherichia coli dihydrofolate reductase?
journal, April 1993
- Dion, Amanda; Linn, Charles E.; Bradrick, Thomas D.
- Biochemistry, Vol. 32, Issue 13
Retroelement-Based Genome Editing and Evolution
journal, September 2018
- Simon, Anna J.; Morrow, Barrett R.; Ellington, Andrew D.
- ACS Synthetic Biology, Vol. 7, Issue 11
Multiplexed precision genome editing with trackable genomic barcodes in yeast
journal, May 2018
- Roy, Kevin R.; Smith, Justin D.; Vonesch, Sibylle C.
- Nature Biotechnology, Vol. 36, Issue 6
One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products
journal, May 2000
- Datsenko, K. A.; Wanner, B. L.
- Proceedings of the National Academy of Sciences, Vol. 97, Issue 12, p. 6640-6645
Design and synthesis of a minimal bacterial genome
journal, March 2016
- Hutchison, C. A.; Chuang, R. -Y.; Noskov, V. N.
- Science, Vol. 351, Issue 6280
Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides
journal, July 2010
- Warner, Joseph R.; Reeder, Philippa J.; Karimpour-Fard, Anis
- Nature Biotechnology, Vol. 28, Issue 8, p. 856-862
Spatiotemporal microbial evolution on antibiotic landscapes
journal, September 2016
- Baym, M.; Lieberman, T. D.; Kelsic, E. D.
- Science, Vol. 353, Issue 6304
MODEST: a web-based design tool for oligonucleotide-mediated genome engineering and recombineering
journal, May 2014
- Bonde, Mads T.; Klausen, Michael S.; Anderson, Mads V.
- Nucleic Acids Research, Vol. 42, Issue W1
Recovery of Phenotypes Obtained by Adaptive Evolution through Inverse Metabolic Engineering
journal, August 2012
- Hong, Kuk-Ki; Nielsen, Jens
- Applied and Environmental Microbiology, Vol. 78, Issue 21
Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate
journal, September 2010
- Mosberg, J. A.; Lajoie, M. J.; Church, G. M.
- Genetics, Vol. 186, Issue 3
Genomically Recoded Organisms Expand Biological Functions
journal, October 2013
- Lajoie, M. J.; Rovner, A. J.; Goodman, D. B.
- Science, Vol. 342, Issue 6156, p. 357-360
Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering
journal, December 2016
- Garst, Andrew D.; Bassalo, Marcelo C.; Pines, Gur
- Nature Biotechnology, Vol. 35, Issue 1, p. 48-55
Millstone: software for multiplex microbial genome analysis and engineering
journal, May 2017
- Goodman, Daniel B.; Kuznetsov, Gleb; Lajoie, Marc J.
- Genome Biology, Vol. 18, Issue 1
Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer
journal, February 1996
- Heim, Roger; Tsien, Roger Y.
- Current Biology, Vol. 6, Issue 2