Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector

Nucleic Acids Research
Ami M KabadiCharles A Gersbach

Abstract

Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significa...Continue Reading

References

Jan 13, 2016·Molecular Therapy : the Journal of the American Society of Gene Therapy·Morgan L Maeder, Charles A Gersbach
Oct 29, 2015·Critical Reviews in Biochemistry and Molecular Biology·Erik Serrao, Alan N Engelman
Sep 24, 2015·Wiley Interdisciplinary Reviews. Systems Biology and Medicine·Zehua BaoHuimin Zhao
Dec 29, 2015·Current Opinion in Biotechnology·Oleg Raitskin, Nicola J Patron
Jan 28, 2016·Scientific Reports·Xiaoxi LiuToru Takumi
Jan 27, 2016·Trends in Biotechnology·Barbara JusiakTimothy K Lu
Jan 11, 2016·Cell Stem Cell·William T HendriksChad A Cowan
Dec 31, 2015·Nature Methods·Mehmet Fatih BolukbasiScot A Wolfe
Dec 17, 2015·Scientific Reports·Shin YoshiokaKunihiko Naito
Aug 19, 2015·International Journal of Pharmaceutics·Justin S LaFountaineHugh D C Smyth
Feb 11, 2015·Biotechnology Journal·Florian Schmidt, Dirk Grimm
Apr 14, 2015·Biotechnology Journal·Lise Marie GravHelene Faustrup Kildegaard
Aug 12, 2015·Current Opinion in Neurology·Katrin Hollinger, Jeffrey S Chamberlain
Jun 11, 2015·Biotechnology Journal·Jae Seong LeeHelene Faustrup Kildegaard
May 6, 2016·Frontiers in Plant Science·Zafar IqbalMuhammad Shafiq
Aug 12, 2015·Nature Reviews. Genetics·Adrian D HaimovichFarren J Isaacs
May 25, 2015·Journal of Biotechnology·Xiquan LiangJonathan D Chesnut
Apr 29, 2015·Current Opinion in Virology·Paul Bg van ErpBlake Wiedenheft
Dec 3, 2014·Stem Cell Reports·Syandan ChakrabortyKam W Leong
May 23, 2015·Molecular Cell·Michael Boettcher, Michael T McManus
Apr 20, 2016·Critical Reviews in Biotechnology·Agnieszka Piatek, Magdy M Mahfouz
Feb 11, 2015·Nature Chemical Biology·Lauren R Polstein, Charles A Gersbach
Dec 21, 2014·Nucleic Acids Research·Richard MooreLeonidas Bleris
Apr 20, 2016·Journal of Virological Methods·Yong WuNing-Shao Xia
May 18, 2016·Progress in Retinal and Eye Research·Sandy S C HungAlex W Hewitt
Jun 9, 2016·Briefings in Functional Genomics·Marc ZuckermannJan Gronych
May 6, 2016·Annual Review of Chemical and Biomolecular Engineering·Christopher E Nelson, Charles A Gersbach
Jun 28, 2016·Briefings in Functional Genomics·Raul Torres-Ruiz, Sandra Rodriguez-Perales
May 6, 2016·Annual Review of Biophysics·Baohui ChenBo Huang
Oct 21, 2016·The Journal of Clinical Investigation·Jianyin LongFarhad R Danesh
Dec 6, 2016·Nature Biotechnology·Bernd ZetscheFeng Zhang
Dec 10, 2016·PloS One·Victoria B StockmanHarris H Wang
Apr 4, 2017·Molecular Therapy : the Journal of the American Society of Gene Therapy·Chaoran YinWenhui Hu
May 24, 2017·Nature Reviews. Urology·Shangqian WangYu Chen
May 27, 2017·Genome Biology·Gleb KuznetsovGeorge M Church

Citations

Aug 5, 2000·The Journal of Biological Chemistry·R R BeerliCarlos F Barbas
Jul 28, 2007·The Journal of Gene Medicine·Deming GouLin Liu
Apr 23, 2008·Current Protocols in Neuroscience·Patrick Salmon, Didier Trono
May 4, 2010·Molecular Therapy : the Journal of the American Society of Gene Therapy·Derek A Persons
Aug 4, 2010·Methods in Molecular Biology·Dmitry Y GuschinEdward J Rebar
Dec 16, 2011·Nucleic Acids Research·Pablo Perez-PiñeraCharles A Gersbach
Jun 30, 2012·Science·Martin JinekEmmanuelle Charpentier
Jul 24, 2012·Current Opinion in Chemical Biology·Pablo Perez-PiñeraCharles A Gersbach
Sep 12, 2012·Journal of the American Chemical Society·Lauren R Polstein, Charles A Gersbach
Jan 5, 2013·Science·Le CongFeng Zhang
Jan 5, 2013·Science·Prashant MaliGeorge M Church
Jan 31, 2013·Nature Biotechnology·Woong Y HwangJ Keith Joung
Jan 31, 2013·Nature Biotechnology·Seung Woo ChoJin-Soo Kim
Feb 5, 2013·Nature Methods·Pablo Perez-PiñeraCharles A Gersbach
Feb 7, 2013·ELife·Martin JinekJennifer A Doudna
Feb 12, 2013·Nature Methods·Morgan L MaederJ Keith Joung
Apr 3, 2013·Molecular Therapy : the Journal of the American Society of Gene Therapy·Mark J OsbornJakub Tolar
Apr 24, 2013·Integrative Biology : Quantitative Biosciences From Nano to Macro·Alireza SalmanzadehRafael V Davalos
May 15, 2013·Trends in Biotechnology·Thomas GajCarlos F Barbas
Jun 5, 2013·Molecular Therapy : the Journal of the American Society of Gene Therapy·David G OusteroutCharles A Gersbach
Jul 31, 2013·Nature Methods·Pablo Perez-PiñeraCharles A Gersbach
Jul 31, 2013·Nature Methods·Morgan L MaederJ Keith Joung
Aug 28, 2013·ACS Synthetic Biology·Fahim FarzadfardTimothy K Lu
Sep 10, 2013·Nature Biotechnology·Eric M MendenhallBradley E Bernstein
Oct 1, 2013·Nature Methods·Kevin M EsveltGeorge M Church
Dec 18, 2013·Science·Tim WangEric S Lander
Dec 18, 2013·Science·Ophir ShalemFeng Zhang

Related Concepts

RNA Polymerase III
Subfamily lentivirinae
Shuttle Vectors
Transcriptional Regulation
HEK293 Cells
Genome
CRISPR-Cas Systems
Transcription, Genetic
Promoter
Cell Engineering

Related Feeds

CRISPR Genome Editing & Therapy

CRISPR-Cas system enables the editing of genes to create or correct mutations. This feed focuses on the application of this system for gene editing and therapy in human diseases.

CRISPR (general)

Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). CRISPR-Cas system enables the editing of genes to create or correct mutations. Discover the latest research on CRISPR here.

CRISPR Ribonucleases Deactivation

CRISPR-Cas system enables the editing of genes to create or correct mutations. This feed focuses on mechanisms that underlie deactivation of CRISPR ribonucleases. Here is the latest research.

CRISPR for Genome Editing

Genome editing technologies enable the editing of genes to create or correct mutations. Clustered regularly interspaced short palindromic repeats (CRISPR) are DNA sequences in the genome that are recognized and cleaved by CRISPR-associated proteins (Cas). Here is the latest research on the use of CRISPR-Cas system in gene editing.