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Article ; Online: Small molecule regulated sgRNAs enable control of genome editing in E. coli by Cas9.

Iwasaki, Roman S / Ozdilek, Bagdeser A / Garst, Andrew D / Choudhury, Alaksh / Batey, Robert T

2020 Volume 11, Issue 1, Page(s) 1394

Abstract: CRISPR-Cas9 has led to great advances in gene editing for a broad spectrum of applications. To further the utility of Cas9 there have been efforts to achieve temporal control over its nuclease activity. While different approaches have focused on ... ...

Abstract	CRISPR-Cas9 has led to great advances in gene editing for a broad spectrum of applications. To further the utility of Cas9 there have been efforts to achieve temporal control over its nuclease activity. While different approaches have focused on regulation of CRISPR interference or editing in mammalian cells, none of the reported methods enable control of the nuclease activity in bacteria. Here, we develop RNA linkers to combine theophylline- and 3-methylxanthine (3MX)-binding aptamers with the sgRNA, enabling small molecule-dependent editing in Escherichia coli. These activatable guide RNAs enable temporal and post-transcriptional control of in vivo gene editing. Further, they reduce the death of host cells caused by cuts in the genome, a major limitation of CRISPR-mediated bacterial recombineering.
MeSH term(s)	CRISPR-Cas Systems ; Cloning, Molecular ; Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli/genetics ; Gene Editing/methods ; Gene Expression Regulation ; Nucleic Acid Conformation ; RNA, Guide, CRISPR-Cas Systems ; Theophylline
Chemical Substances	RNA, Guide, CRISPR-Cas Systems ; Theophylline (C137DTR5RG)
Language	English
Publishing date	2020-03-13
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-020-15226-8
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Article ; Online: Small molecule regulated sgRNAs enable control of genome editing in E. coli by Cas9

Roman S. Iwasaki / Bagdeser A. Ozdilek / Andrew D. Garst / Alaksh Choudhury / Robert T. Batey

Nature Communications, Vol 11, Iss 1, Pp 1-

2020 Volume 9

Abstract: Bacteria lack the same suite of CRISPR tools that have been developed for mammalian cells. Here, the authors link aptamers to sgRNAs to allow small molecule control of gene editing in E. coli. ...

Abstract	Bacteria lack the same suite of CRISPR tools that have been developed for mammalian cells. Here, the authors link aptamers to sgRNAs to allow small molecule control of gene editing in E. coli.
Keywords	Science ; Q
Language	English
Publishing date	2020-03-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Integrating CRISPR-Enabled Trackable Genome Engineering and Transcriptomic Analysis of Global Regulators for Antibiotic Resistance Selection and Identification in Escherichia coli.

Chen, Cong / Choudhury, Alaksh / Zhang, Shuanghong / Garst, Andrew D / Song, Xin / Liu, Xunli / Chen, Tao / Gill, Ryan T / Wang, Zhiwen

mSystems

2020 Volume 5, Issue 2

Abstract: It is important to expedite our understanding of antibiotic resistance to address the increasing numbers of fatalities and environmental pollution due to the emergence of antibiotic resistance and multidrug-resistant strains. Here, we combined the CRISPR- ...

Abstract	It is important to expedite our understanding of antibiotic resistance to address the increasing numbers of fatalities and environmental pollution due to the emergence of antibiotic resistance and multidrug-resistant strains. Here, we combined the CRISPR-enabled trackable genome engineering (CREATE) technology and transcriptomic analysis to investigate antibiotic tolerance in
Language	English
Publishing date	2020-04-21
Publishing country	United States
Document type	Journal Article
ISSN	2379-5077
ISSN	2379-5077
DOI	10.1128/mSystems.00232-20
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Article: A switch in time: detailing the life of a riboswitch.

Garst, Andrew D / Batey, Robert T

Biochimica et biophysica acta

2009 Volume 1789, Issue 9-10, Page(s) 584–591

Abstract: Riboswitches are non-protein coding RNA elements typically found in the 5' untranslated region (5'-UTR) of mRNAs that utilize metabolite binding to control expression of their own transcript. The RNA-ligand interaction causes conformational changes in ... ...

Abstract	Riboswitches are non-protein coding RNA elements typically found in the 5' untranslated region (5'-UTR) of mRNAs that utilize metabolite binding to control expression of their own transcript. The RNA-ligand interaction causes conformational changes in the RNA that direct the cotranscriptional folding of a downstream secondary structural switch that interfaces with the expression machinery. This review describes the structural themes common to the different RNA-metabolite complexes studied to date and conclusions that can be made regarding how these RNAs efficiently couple metabolite binding to gene regulation. Emphasis is placed on the temporal aspects of riboswitch regulation that are central to the function of these RNAs and the need to augment the wealth of data on metabolite receptor domains with further studies on the full regulatory element, particularly in the context of transcription.
MeSH term(s)	3' Untranslated Regions ; Animals ; Base Sequence ; Gene Expression Regulation ; Humans ; Ligands ; Molecular Conformation ; Molecular Sequence Data ; Nucleic Acid Conformation ; Open Reading Frames ; RNA/chemistry ; RNA/metabolism ; RNA, Messenger/metabolism ; Terminator Regions, Genetic ; Thermotoga maritima/metabolism ; Time Factors ; Transcription, Genetic
Chemical Substances	3' Untranslated Regions ; Ligands ; RNA, Messenger ; RNA (63231-63-0)
Language	English
Publishing date	2009-07-09
Publishing country	Netherlands
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Review
ZDB-ID	60-7
ISSN	1879-2596 ; 1879-260X ; 1872-8006 ; 1879-2642 ; 1879-2618 ; 1879-2650 ; 0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 1570-9639 ; 0925-4439 ; 1874-9399
ISSN (online)	1879-2596 ; 1879-260X ; 1872-8006 ; 1879-2642 ; 1879-2618 ; 1879-2650
ISSN	0006-3002 ; 0005-2728 ; 0005-2736 ; 0304-4165 ; 0167-4838 ; 1388-1981 ; 0167-4889 ; 0167-4781 ; 0304-419X ; 1570-9639 ; 0925-4439 ; 1874-9399
DOI	10.1016/j.bbagrm.2009.06.004
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Article ; Online: Deep scanning lysine metabolism in

Bassalo, Marcelo C / Garst, Andrew D / Choudhury, Alaksh / Grau, William C / Oh, Eun J / Spindler, Eileen / Lipscomb, Tanya / Gill, Ryan T

Molecular systems biology

2018 Volume 14, Issue 11, Page(s) e8371

Abstract: Our limited ability to predict genotype-phenotype relationships has called for strategies that allow testing of thousands of hypotheses in parallel. Deep scanning mutagenesis has been successfully implemented to map genotype-phenotype relationships at a ... ...

Abstract	Our limited ability to predict genotype-phenotype relationships has called for strategies that allow testing of thousands of hypotheses in parallel. Deep scanning mutagenesis has been successfully implemented to map genotype-phenotype relationships at a single-protein scale, allowing scientists to elucidate properties that are difficult to predict. However, most phenotypes are dictated by several proteins that are interconnected through complex and robust regulatory and metabolic networks. These sophisticated networks hinder our understanding of the phenotype of interest and limit our capabilities to rewire cellular functions. Here, we leveraged CRISPR-EnAbled Trackable genome Engineering to attempt a parallel and high-resolution interrogation of complex networks, deep scanning multiple proteins associated with lysine metabolism in
MeSH term(s)	CRISPR-Cas Systems ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Escherichia coli Proteins/genetics ; Escherichia coli Proteins/metabolism ; Gene Library ; Lysine/metabolism ; Metabolic Networks and Pathways ; Mutation ; Phenotype
Chemical Substances	Escherichia coli Proteins ; Lysine (K3Z4F929H6)
Language	English
Publishing date	2018-11-26
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2193510-5
ISSN	1744-4292 ; 1744-4292
ISSN (online)	1744-4292
ISSN	1744-4292
DOI	10.15252/msb.20188371
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Article ; Online: Insights into the regulatory landscape of the lysine riboswitch.

Garst, Andrew D / Porter, Ely B / Batey, Robert T

Journal of molecular biology

2012 Volume 423, Issue 1, Page(s) 17–33

Abstract: ... of the receptor for lysine (K(d)), as well as many of its analogs. However, above 400 μM [NTP], the concentration ...

Abstract	A prevalent means of regulating gene expression in bacteria is by riboswitches found within mRNA leader sequences. Like protein repressors, these RNA elements must bind an effector molecule with high specificity against a background of other cellular metabolites of similar chemical structure to elicit the appropriate regulatory response. Current crystal structures of the lysine riboswitch do not provide a complete understanding of selectivity as recognition is substantially mediated through main-chain atoms of the amino acid. Using a directed set of lysine analogs and other amino acids, we have determined the relative contributions of the polar functional groups to binding affinity and the regulatory response. Our results reveal that the lysine riboswitch has >1000-fold specificity for lysine over other amino acids. The aptamer is highly sensitive to the precise placement of the ε-amino group and relatively tolerant of alterations to the main-chain functional groups in order to achieve this specificity. At low nucleotide triphosphate (NTP) concentrations, we observe good agreement between the half-maximal regulatory activity (T(50)) and the affinity of the receptor for lysine (K(d)), as well as many of its analogs. However, above 400 μM [NTP], the concentration of lysine required to elicit transcription termination rises, moving into the riboswitch into a kinetic control regime. These data demonstrate that, under physiologically relevant conditions, riboswitches can integrate both effector and NTP concentrations to generate a regulatory response appropriate for global metabolic state of the cell.
MeSH term(s)	5' Untranslated Regions ; Binding Sites ; Crystallography, X-Ray ; Gene Expression Regulation, Bacterial ; Lysine/metabolism ; Models, Molecular ; Nucleic Acid Conformation ; Riboswitch ; Transcription, Genetic
Chemical Substances	5' Untranslated Regions ; Riboswitch ; Lysine (K3Z4F929H6)
Language	English
Publishing date	2012-07-03
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	80229-3
ISSN	1089-8638 ; 0022-2836
ISSN (online)	1089-8638
ISSN	0022-2836
DOI	10.1016/j.jmb.2012.06.038
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Article ; Online: Genomic Deoxyxylulose Phosphate Reductoisomerase (DXR) Mutations Conferring Resistance to the Antimalarial Drug Fosmidomycin in E. coli.

Pines, Gur / Oh, Eun Joong / Bassalo, Marcelo C / Choudhury, Alaksh / Garst, Andrew D / Fankhauser, Reilly G / Eckert, Carrie A / Gill, Ryan T

ACS synthetic biology

2018 Volume 7, Issue 12, Page(s) 2824–2832

Abstract: Sequence to activity mapping technologies are rapidly developing, enabling the generation and isolation of mutations conferring novel phenotypes. Here we used the CRISPR enabled trackable genome engineering (CREATE) technology to investigate the ... ...

Abstract	Sequence to activity mapping technologies are rapidly developing, enabling the generation and isolation of mutations conferring novel phenotypes. Here we used the CRISPR enabled trackable genome engineering (CREATE) technology to investigate the inhibition of the essential ispC gene in its native genomic context in Escherichia coli. We created a full saturation library of 33 sites proximal to the ligand binding pocket and challenged this library with the antimalarial drug fosmidomycin, which targets the ispC gene product, DXR. This selection is especially challenging since it is relatively weak in E. coli, with multiple naturally occurring pathways for resistance. We identified several previously unreported mutations that confer fosmidomycin resistance, in highly conserved sites that also exist in pathogens including the malaria-inducing Plasmodium falciparum. This approach may have implications for the isolation of resistance-conferring mutations and may affect the design of future generations of fosmidomycin-based drugs.
MeSH term(s)	Aldose-Ketose Isomerases/genetics ; Aldose-Ketose Isomerases/metabolism ; Antimalarials/metabolism ; Antimalarials/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Drug Resistance/drug effects ; Escherichia coli/chemistry ; Escherichia coli/metabolism ; Fosfomycin/analogs & derivatives ; Fosfomycin/metabolism ; Fosfomycin/pharmacology ; Genetic Engineering/methods ; Mutation ; Plasmids/genetics ; Plasmids/metabolism ; Plasmodium falciparum/drug effects
Chemical Substances	Antimalarials ; Fosfomycin (2N81MY12TE) ; fosmidomycin (5829E3D9I9) ; 1-deoxy-D-xylulose 5-phosphate reductoisomerase (EC 1.1.1.267) ; Aldose-Ketose Isomerases (EC 5.3.1.-)
Language	English
Publishing date	2018-12-07
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	2161-5063
ISSN (online)	2161-5063
DOI	10.1021/acssynbio.8b00219
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Article ; Online: Iterative genome editing of Escherichia coli for 3-hydroxypropionic acid production.

Liu, Rongming / Liang, Liya / Choudhury, Alaksh / Bassalo, Marcelo C / Garst, Andrew D / Tarasava, Katia / Gill, Ryan T

Metabolic engineering

2018 Volume 47, Page(s) 303–313

Abstract: Synthetic biology requires strategies for the targeted, efficient, and combinatorial engineering of biological sub-systems at the molecular level. Here, we report the use of the iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method for ... ...

Abstract	Synthetic biology requires strategies for the targeted, efficient, and combinatorial engineering of biological sub-systems at the molecular level. Here, we report the use of the iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method for the rapid construction of combinatorially modified genomes. We coupled this genome engineering strategy with high-throughput phenotypic screening and selections to recursively engineer multiple traits in Escherichia coli for improved production of the platform chemical 3-hydroxypropionic acid (3HP). Specifically, we engineered i) central carbon metabolism, ii) 3HP synthesis, and (iii) 3HP tolerance through design, construction and testing of ~ 162,000 mutations across 115 genes spanning global regulators, transcription factors, and enzymes involved in 3HP synthesis and tolerance. The iCREATE process required ~ 1 month to perform 13 rounds of combinatorial genome modifications with targeted gene knockouts, expression modification by ribosomal binding site (RBS) engineering, and genome-level site-saturation mutagenesis. Specific mutants conferring increased 3HP titer, yield, and productivity were identified and then combined to produce 3HP at a yield and concentration ~ 60-fold higher than the wild-type strain.
MeSH term(s)	Escherichia coli/genetics ; Escherichia coli/metabolism ; Gene Editing ; Genome, Bacterial ; Lactic Acid/analogs & derivatives ; Lactic Acid/biosynthesis
Chemical Substances	Lactic Acid (33X04XA5AT) ; hydracrylic acid (C4ZF6XLD2X)
Language	English
Publishing date	2018-04-14
Publishing country	Belgium
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	1470383-x
ISSN	1096-7184 ; 1096-7176
ISSN (online)	1096-7184
ISSN	1096-7176
DOI	10.1016/j.ymben.2018.04.007
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Article ; Online: Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering.

Liu, Rongming / Liang, Liya / Garst, Andrew D / Choudhury, Alaksh / Nogué, Violeta Sànchez I / Beckham, Gregg T / Gill, Ryan T

Metabolic engineering

2018 Volume 47, Page(s) 10–20

Abstract: Strain engineering for industrial production requires a targeted improvement of multiple complex traits, which range from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome ... ...

Abstract	Strain engineering for industrial production requires a targeted improvement of multiple complex traits, which range from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method to engineer rapid glucose and xylose co-consumption and tolerance to hydrolysate inhibitors in E. coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target ~40,000 mutations across 30 genes. These libraries included global and high-level regulators that regulate global gene expression, transcription factors that play important roles in genome-level transcription, enzymes that function in the sugar transport system, NAD(P)H metabolism, and the aldehyde reduction system. Specific mutants that conferred increased growth in mixed sugars and hydrolysate tolerance conditions were isolated, confirmed, and evaluated for changes in genome-wide expression levels. We tested the strain with positive combinatorial mutations for 3-hydroxypropionic acid (3HP) production under high furfural and high acetate hydrolysate fermentation, which demonstrated a 7- and 8-fold increase in 3HP productivity relative to the parent strain, respectively.
MeSH term(s)	Escherichia coli/genetics ; Escherichia coli/metabolism ; Gene Editing/methods ; Metabolic Engineering/methods ; Mutagenesis
Language	English
Publishing date	2018-03-29
Publishing country	Belgium
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	1470383-x
ISSN	1096-7184 ; 1096-7176
ISSN (online)	1096-7184
ISSN	1096-7176
DOI	10.1016/j.ymben.2018.02.007
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Article ; Online: Multiplex navigation of global regulatory networks (MINR) in yeast for improved ethanol tolerance and production.

Liu, Rongming / Liang, Liya / Choudhury, Alaksh / Garst, Andrew D / Eckert, Carrie A / Oh, Eun Joong / Winkler, James / Gill, Ryan T

Metabolic engineering

2018 Volume 51, Page(s) 50–58

Abstract: Multiplex navigation of global regulatory networks (MINR) is an approach for combinatorially reprogramming gene expression to manipulate complex phenotypes. We designed, constructed, and mapped MINR libraries containing 43,020 specific mutations in 25 ... ...

Abstract	Multiplex navigation of global regulatory networks (MINR) is an approach for combinatorially reprogramming gene expression to manipulate complex phenotypes. We designed, constructed, and mapped MINR libraries containing 43,020 specific mutations in 25 regulatory genes expected to perturb the yeast regulatory network. We selected growth competition experiments for library mutants conferring increased ethanol and/or glucose tolerance. We identified specific mutants that not only possessed improved ethanol and/or glucose tolerance but also produced ethanol at concentrations up to 2-fold higher than those produced by the wild-type strain. We further determined that mutations increasing ethanol tolerance were transferable to a diploid industrial yeast strain. The facile construction and mapping of 43,020 designer regulatory mutations provide a roadmap for how to access and engineer complex phenotypes in future synthetic biology and broader efforts.
MeSH term(s)	CRISPR-Cas Systems ; Ethanol/metabolism ; Ethanol/pharmacology ; Fermentation ; Gene Expression ; Gene Library ; Gene Regulatory Networks ; Metabolic Engineering/methods ; Mutation ; Plasmids/genetics ; Saccharomyces cerevisiae/drug effects ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism
Chemical Substances	Ethanol (3K9958V90M)
Language	English
Publishing date	2018-07-17
Publishing country	Belgium
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	1470383-x
ISSN	1096-7184 ; 1096-7176
ISSN (online)	1096-7184
ISSN	1096-7176
DOI	10.1016/j.ymben.2018.07.007
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