RNA-guided retargeting of Sleeping Beauty transposition in human cells.

ELife
Adrian KovačZoltán Ivics

Abstract

An ideal tool for gene therapy would enable efficient gene integration at predetermined sites in the human genome. Here we demonstrate biased genome-wide integration of the Sleeping Beauty (SB) transposon by combining it with components of the CRISPR/Cas9 system. We provide proof-of-concept that it is possible to influence the target site selection of SB by fusing it to a catalytically inactive Cas9 (dCas9) and by providing a single guide RNA (sgRNA) against the human Alu retrotransposon. Enrichment of transposon integrations was dependent on the sgRNA, and occurred in an asymmetric pattern with a bias towards sites in a relatively narrow, 300 bp window downstream of the sgRNA targets. Our data indicate that the targeting mechanism specified by CRISPR/Cas9 forces integration into genomic regions that are otherwise poor targets for SB transposition. Future modifications of this technology may allow the development of methods for specific gene insertion for precision genetic engineering.

References

Apr 28, 1995·The Journal of Biological Chemistry·E W Englander, B H Howard
Feb 23, 2000·The Journal of Clinical Investigation·T H Bestor
May 11, 2000·Proceedings of the National Academy of Sciences of the United States of America·D Szüts, M Bienz
Aug 31, 2000·Journal of Molecular Biology·Z IzsvákR H Plasterk
Aug 31, 2002·Cell·Astrid R W SchröderFrederic Bushman
May 6, 2003·Science·Matthew H Porteus, David Baltimore
Aug 18, 2004·PLoS Biology·Rick S MitchellFrederic D Bushman
Nov 3, 2004·Genome Research·Alkes L PricePavel A Pevzner
Jan 25, 2005·Journal of Molecular Biology·Geyi LiuPerry B Hackett
Mar 4, 2005·Molecular and Cellular Biology·Stephen R YantMark A Kay
Mar 16, 2006·Proceedings of the National Academy of Sciences of the United States of America·Oliver WaliskoZoltán Ivics
Jul 25, 2006·Nature·Eran SegalJonathan Widom
Mar 9, 2007·Nucleic Acids Research·Stephen R YantMark A Kay
Apr 12, 2007·Molecular Therapy : the Journal of the American Society of Gene Therapy·Zoltán IvicsZsuzsanna Izsvák
Mar 21, 2008·The EMBO Journal·Rupak MitraNancy L Craig
Apr 17, 2008·Cancer Research·Harjeet SinghLaurence J N Cooper
Oct 7, 2008·Genome Research·E Andrew BennettScott E Devine
May 30, 2009·Nature Methods·Zoltán IvicsZsuzsanna Izsvák
Jun 10, 2009·Bioinformatics·Heng LiUNKNOWN 1000 Genome Project Data Processing Subgroup
Dec 8, 2009·Nucleic Acids Research·Xiaofeng FengSean D Colloms
Feb 10, 2010·Archivum Immunologiae Et Therapiae Experimentalis·David Escors, Karine Breckpot
May 19, 2010·BMC Genomics·Yoshiaki TanakaKenta Nakai
Jul 8, 2010·Molecular Therapy : the Journal of the American Society of Gene Therapy·Xin HuangXianzheng Zhou
Aug 19, 2010·Nature Reviews. Genetics·Fyodor D UrnovPhilip D Gregory
Sep 17, 2010·Nature·Marina Cavazzana-CalvoPhilippe Leboulch
Apr 7, 2011·Molecular Therapy : the Journal of the American Society of Gene Therapy·Brian MoldtJacob Giehm Mikkelsen
Apr 19, 2011·Nature Reviews. Genetics·Federico Mingozzi, Katherine A High
Jul 7, 2011·Molecular Therapy : the Journal of the American Society of Gene Therapy·Claudia KettlunMatthew H Wilson
Jul 9, 2011·Nature Biotechnology·Dirk HockemeyerRudolf Jaenisch
Mar 6, 2012·Nature Methods·Ben Langmead, Steven L Salzberg
Apr 24, 2012·Nucleic Acids Research·Ismahen AmmarZoltán Ivics

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Citations

Nov 20, 2020·Chemical Science·Anna V Leopold, Vladislav V Verkhusha
Jan 27, 2021·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Lena GoshayeshiHesam Dehghani
Apr 4, 2021·International Journal of Molecular Sciences·Katarzyna M TerlikowskaSławomir J Terlikowski
Oct 30, 2021·Frontiers in Genome Editing·Georges BlattnerGiandomenico Turchiano

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Methods Mentioned

BETA
electrophoresis
cleavage assay
PCR
PCRs
Assay
transfection
dot blot
transfections

Software Mentioned

samtools
SeqLogo
genomation
TIDE
ImageJ
GraphPad QuickCalcs
Bowtie2
R
Fiji
R Development Core Team

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