New Human Chromosomal Sites with "Safe Harbor" Potential for Targeted Transgene Insertion.

Human Gene Therapy
Stefan PellenzRaymond Monnat

Abstract

This study identified 35 new sites for targeted transgene insertion that have the potential to serve as new human genomic "safe harbor" sites (SHS). SHS potential for these 35 sites, located on 16 chromosomes, including both arms of the human X chromosome, and for the existing human SHS AAVS1, hROSA26, and CCR5 was assessed using eight different desirable, widely accepted criteria for SHS verifiable with human genomic data. Three representative newly identified sites on human chromosomes 2 and 4 were then experimentally validated by in vitro and in vivo cleavage-sensitivity tests, and analyzed for population-level and cell line-specific sequence variants that might confound site targeting. The highly ranked site on chromosome 4 (SHS231) was further characterized by targeted homology-dependent and -independent transgene insertion and expression in different human cell lines. The structure and fidelity of transgene insertions at this site were confirmed, together with analyses that demonstrated stable expression and function of transgene-encoded proteins, including fluorescent protein markers, selectable marker cassettes, and Cas9 protein variants. SHS-integrated transgene-encoded Cas9 proteins were shown to be capable of introdu...Continue Reading

References

Aug 1, 1987·Molecular and Cellular Biology·C Chen, H Okayama
Sep 15, 1967·International Journal of Cancer. Journal International Du Cancer·J Pontén, E Saksela
Apr 15, 1997·Proceedings of the National Academy of Sciences of the United States of America·B P ZambrowiczP Soriano
Oct 10, 1998·Journal of Virology·T DullL Naldini
Aug 31, 2002·Cell·Astrid R W SchröderFrederic Bushman
Aug 18, 2004·PLoS Biology·Rick S MitchellFrederic D Bushman
Dec 16, 2006·Current Gene Therapy·Michele P Calos
Nov 27, 2007·Nature Biotechnology·Stefan IrionGordon Keller
Feb 16, 2010·Molecular and Cellular Biology·Jeannine R Larocque, Maria Jasin
Dec 2, 2011·Nature Reviews. Cancer·Michel SadelainFrederic D Bushman
Feb 4, 2012·Cold Spring Harbor Protocols·Andrea L Szymczak-WorkmanDario A A Vignali
Feb 22, 2012·Science·Daniel G MacArthurChris Tyler-Smith
Aug 22, 2012·Briefings in Bioinformatics·Robert M KuhnW James Kent
Sep 4, 2012·Nature Methods·Michael T CertoAndrew M Scharenberg
Sep 8, 2012·Nature·UNKNOWN ENCODE Project Consortium
Nov 7, 2012·Nature·UNKNOWN 1000 Genomes Project ConsortiumGil A McVean
Jan 5, 2013·Science·Le CongFeng Zhang
Jan 15, 2013·Hernia : the Journal of Hernias and Abdominal Wall Surgery·A Montgomery
May 30, 2013·Nature Genetics·UNKNOWN GTEx Consortium
Jul 23, 2013·Nature Biotechnology·Patrick D HsuFeng Zhang
Oct 8, 2013·Cold Spring Harbor Perspectives in Biology·Maria Jasin, Rodney Rothstein
Feb 11, 2014·Methods in Molecular Biology·Marlene Belfort, Richard P Bonocora
May 16, 2014·Nature Reviews. Molecular Cell Biology·Jörg RenkawitzStefan Jentsch
Oct 2, 2014·The Journal of Clinical Investigation·Rajat M Gupta, Kiran Musunuru
Mar 3, 2015·Nature Methods·Alejandro ChavezGeorge M Church
Oct 4, 2015·Nature·UNKNOWN 1000 Genomes Project ConsortiumGonçalo R Abecasis
Feb 13, 2016·Molecular Therapy : the Journal of the American Society of Gene Therapy·Eirini P Papapetrou, Axel Schambach
Nov 17, 2016·Nature·Keiichiro SuzukiJuan Carlos Izpisua Belmonte
Dec 14, 2016·Proceedings of the National Academy of Sciences of the United States of America·Michael P PhelpsEleanor Y Chen
Mar 24, 2017·Nucleic Acids Research·Kenneth A MatreyekDouglas M Fowler
May 26, 2018·Nucleic Acids Research·Aaron K WongOlga G Troyanskaya

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Citations

May 23, 2019·Human Genetics·Minyoung Lee, Hyongbum Kim
Apr 25, 2020·Frontiers in Physiology·Madeleine Iafrate, Gilbert O Fruhwirth
Jul 16, 2020·American Journal of Respiratory Cell and Molecular Biology·Reka Lorincz, David T Curiel
Jan 27, 2021·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Lena GoshayeshiHesam Dehghani

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Methods Mentioned

BETA
gene-trap
acetylation
PCR
electrophoresis
transfection
flow cytometry
reverse transcription PCR

Software Mentioned

CLC Workbench Primer Design Tool
BLAST
CRISPR Design Tools
mCreI
CLC Workbench Alignment tool
ImageJ
CLC Main Workbench CLCBio
TALEN Targeter
CLC Bio

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