Targeted DNA transposition in vitro using a dCas9-transposase fusion protein
Shivam Bhatt,Ronald Chalmers +1 more
TLDR
An in vitro mechanistic study that demonstrates efficient Cas9 targeting of the mariner transposon Hsmar1 and shows that the transposase moiety behaved normally and was proficient for integration in vitro and in Escherichia coli.Abstract:
Homology-directed genome engineering is limited by transgene size. Although DNA transposons are more efficient with large transgenes, random integrations are potentially mutagenic. Here we present an in vitro mechanistic study that demonstrates efficient Cas9 targeting of the mariner transposon Hsmar1. Integrations were unidirectional and tightly constrained to one side of the sgRNA binding site. Further analysis of the nucleoprotein intermediates demonstrated that the transposase and Cas9 moieties can bind their respective substrates independently or in concert. Kinetic analysis of the reaction in the presence of the Cas9 target-DNA revealed a delay between first and second strand cleavage at the transposon end. This step involves a significant conformational change that may be hindered by the properties of the interdomainal linker. Otherwise, the transposase moiety behaved normally and was proficient for integration in vitro and in Escherichia coli. Specific integration into the lacZ gene in E. coli was obscured by a high background of random integrations. Nevertheless, Cas9 is an attractive candidate for transposon-targeting because it has a high affinity and long dwell-time at its target site. This will facilitate a future optogenetic strategy for the temporal control of integration, which will increase the ratio of targeted to untargeted events.read more
Citations
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Journal ArticleDOI
CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering.
Phuc Leo H. Vo,Carlotta Ronda,Sanne E. Klompe,Ethan E. Chen,Christopher Acree,Harris H. Wang,Samuel H. Sternberg +6 more
TL;DR: A substantially improved version of the Tn7-like transposon from Vibrio cholerae is introduced, which uses a Type I-F CRISPR-Cas system for programmable, RNA-guided transposition and establishes INTEGRATE as a versatile tool for multiplexed, kilobase-scale genome engineering.
Posted ContentDOI
CRISPR RNA-guided integrases for high-efficiency and multiplexed bacterial genome engineering
Phuc Leo H. Vo,Carlotta Ronda,Sanne E. Klompe,Ethan E. Chen,Christopher Acree,Harris H. Wang,Samuel H. Sternberg +6 more
TL;DR: This work establishes INTEGRATE as a versatile and portable tool that enables multiplex and kilobase-scale genome engineering and develops an accessible computational algorithm for guide RNA design.
Journal Article
Excision of the Drosophila Mariner Transposon Mos1: Comparison with Bacterial Transposition and V(D)J Recombination.: Comparison with Bacterial Transposition and V(D)J Recombination.
Angela Dawson,David J. Finnegan +1 more
TL;DR: The mechanism by which mariner, a eukaryotic transposable element, performs DNA cleavage is examined and it is shown that the nontransferred strand is cleaved initially, unlike prokaryotictransposons which cleave the transferred strand first.
Journal ArticleDOI
Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases
Matthew T. N. Yarnall,Eleonora I. Ioannidi,Cian Schmitt-Ulms,Rohan N. Krajeski,Justin K. Lim,Lukas Villiger,Wenyuan Zhou,Kaiyi Jiang,Sofya K. Garushyants,Nathan Roberts,Liyang Zhang,Christopher A. Vakulskas,John A. Walker,Anastasia P. Kadina,Adrianna E Zepeda,Kevin Holden,Hong-Yu Ma,Jun Xie,Guangping Gao,Lander Foquet,Greg Bial,Sara K. Donnelly,Yoshinari Miyata,Daniel R. Radiloff,Jordana M. Henderson,Andrew Ujita,Omar O. Abudayyeh,Jonathan S. Gootenberg +27 more
Journal ArticleDOI
A highly soluble Sleeping Beauty transposase improves control of gene insertion
Irma Querques,Andreas Mades,Cecilia Zuliani,Csaba Miskey,Miriam Alb,Esther Grueso,Markus Machwirth,Tobias Rausch,Hermann Einsele,Zoltán Ivics,Michael Hudecek,Orsolya Barabas +11 more
TL;DR: It is demonstrated that hsSB can be delivered with transposon DNA to genetically modify cell lines and embryonic, hematopoietic and induced pluripotent stem cells (iPSCs), overcoming uncontrolled transposase activity.
References
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Book
Experiments in molecular genetics
TL;DR: Molecular Genetics (Biology): An Overview | Sciencing Experimental in Molecular Genetics Experiments in molecular genetics (1972 edition) | Open ...
Journal ArticleDOI
Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation
Luke A. Gilbert,Max A. Horlbeck,Britt Adamson,Jacqueline E. Villalta,Yuwen Chen,Evan H. Whitehead,Carla P. Guimaraes,Barbara Panning,Hidde L. Ploegh,Michael C. Bassik,Lei S. Qi,Martin Kampmann,Jonathan S. Weissman +12 more
TL;DR: This work identifies rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators to endogenous genes via endonuclease-deficient Cas9, which enable modulation of gene expression over a ∼1,000-fold range.
Journal ArticleDOI
Highly efficient endogenous human gene correction using designed zinc-finger nucleases
Fyodor D. Urnov,Jeffrey C. Miller,Ya-Li Lee,Christian Beauséjour,Jeremy M. Rock,Sheldon Augustus,Andrew C. Jamieson,Matthew H. Porteus,Philip D. Gregory,Michael C. Holmes +9 more
TL;DR: It is shown that zinc-finger nucleases designed against an X-linked severe combined immune deficiency mutation in the IL2Rγ gene yielded more than 18% gene-modified human cells without selection, raising the possibility of strategies based on zinc- finger nucleases for the treatment of disease.
Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation
Luke A. Gilbert,Max A. Horlbeck,Britt Adamson,Jacqueline E. Villalta,Yuwen Chen,Evan H. Whitehead,Carla P. Guimaraes,Barbara Panning,Michael C. Bassik,Lei S. Qi,Martin Kampmann,Jonathan S. Weissman,Hidde L. Ploegh +12 more
TL;DR: In this article, the authors identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRisPRa) to endogenous genes via endonuclease-deficient Cas9.
Journal ArticleDOI
Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system
TL;DR: A Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase to promoter sequences or as a transcription terminator by blocking the running RNAP is described.
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