Abstract
As Cas9-mediated cleavage requires both protospacer and protospacer adjacent motif (PAM) sequences, it is impossible to employ the CRISPR/Cas9 system to directly edit genomic sites without available PAM sequences nearby. Here, we optimized the CRISPR/Cas9 system and developed an innovative two-step strategy for efficient genome editing of any sites, which did not rely on the availability of PAM sequences. An antibiotic resistance cassette was employed as both a positive and a negative selection marker. By integrating the optimized two-plasmid CRISPR/Cas system and donor DNA, we achieved gene insertion and point mutation with high efficiency in Escherichia coli, and importantly, obtained clean mutants with no other unwanted mutations. Moreover, genome editing of essential genes was successfully achieved using this approach with a few modifications. Therefore, our newly developed method is PAM-independent and can be used to edit any genomic loci, and we hope this method can also be used for efficient genome editing in other organisms.
References
May 10, 1994·Proceedings of the National Academy of Sciences of the United States of America·L JanosiA Kaji
Jun 1, 2000·Proceedings of the National Academy of Sciences of the United States of America·K A Datsenko, B L Wanner
Aug 5, 2005·Microbiology·Alexander BolotinS Dusko Ehrlich
Mar 11, 2006·BioTechniques·B Karsten TischerNikolaus Osterrieder
Dec 11, 2007·Journal of Bacteriology·Hélène DeveauSylvain Moineau
Jun 24, 2008·Nucleic Acids Research·Byung Jo YuSun Chang Kim
Sep 4, 2012·Applied Microbiology and Biotechnology·Björn PetersArmin Ehrenreich
Jan 5, 2013·Science·Le CongFeng Zhang
Jan 31, 2013·Nature Biotechnology·Woong Y HwangJ Keith Joung
Jan 31, 2013·Nature Biotechnology·Wenyan JiangLuciano A Marraffini
Feb 7, 2013·ELife·Martin JinekJennifer Doudna
Mar 5, 2013·Cell·Lei S QiWendell A Lim
Aug 21, 2013·Applied Microbiology and Biotechnology·David KostnerArmin Ehrenreich
Dec 27, 2013·Nucleic Acids Research·Hailong WangA Francis Stewart
Sep 11, 2014·ACS Synthetic Biology·Zehua BaoHuimin Zhao
Feb 1, 2015·Applied and Environmental Microbiology·Yu JiangSheng Yang
Mar 7, 2015·FEMS Yeast Research·Robert MansJean-Marc G Daran
May 24, 2015·Applied and Environmental Microbiology·Michael E PyneC Perry Chou
Jul 5, 2015·Metabolic Engineering·Yifan LiXueming Zhao
Apr 14, 2016·ACS Synthetic Biology·Marcelo C BassaloRyan T Gill
Apr 27, 2016·ACS Synthetic Biology·Yi WangHans P Blaschek
May 3, 2016·Journal of Biological Engineering·Damien Biot-Pelletier, Vincent J J Martin
Jun 14, 2016·ACS Synthetic Biology·Shi-Yuan LiJin Wang
Dec 3, 2016·Microbial Cell Factories·Dongdong ZhaoChanghao Bi
Citations
Dec 2, 2017·Scientific Reports·Dongdong ZhaoChanghao Bi
Jan 14, 2018·Applied and Environmental Microbiology·Kaifeng LiShouwen Chen
Jun 5, 2018·Biotechnology Journal·Takayuki ArazoeKeiji Nishida
Mar 25, 2019·Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences·Gayetri Ramachandran, David Bikard
Sep 11, 2019·Applied Microbiology and Biotechnology·Chaoyong HuangYi-Xin Huo
Aug 15, 2020·Applied Microbiology and Biotechnology·Chaoyong HuangYi-Xin Huo
Oct 26, 2018·Parasites & Vectors·Muhammad MajeedMona Saleh
May 6, 2020·Infection and Drug Resistance·Pourya GholizadehHossein Samadi Kafil
Oct 26, 2018·Frontiers in Microbiology·Xiang AoYingying Guo
May 26, 2020·BMC Microbiology·Francesca Romana CianfanelliDirk Bumann
Jun 26, 2020·World Journal of Microbiology & Biotechnology·Atieh Hashemi
Oct 24, 2020·The ISME Journal·Jake N BarberMichael J McDonald
Mar 7, 2021·Microorganisms·Mouna HamelSophie Alexandra Baron
Jun 15, 2021·EcoSal Plus·Nicholas Backes, Gregory J Phillips
Nov 23, 2021·Applied Microbiology and Biotechnology·Yali KongWei-Min Zhang