PARA: A New Platform for the Rapid Assembly of gRNA Arrays for Multiplexed CRISPR Technologies

被引：11

作者：

Yuan, Guoliang ^{[1
,2
]}

Martin, Stanton ^{[1
,2
]}

Hassan, Md Mahmudul ^{[1
,2
,3
]}

Tuskan, Gerald A. ^{[1
,2
]}

Yang, Xiaohan ^{[1
,2
]}

机构：

[1] Oak Ridge Natl Lab, Biosci Div, POB 2009, Oak Ridge, TN 37831 USA

[2] Oak Ridge Natl Lab, Ctr Bioenergy Innovat, POB 2009, Oak Ridge, TN 37831 USA

[3] Patuakhali Sci & Technol Univ, Dept Genet & Plant Breeding, Dumki 8602, Patuakhali, Bangladesh

来源：

CELLS | 2022年 / 11卷 / 16期

关键词：

gRNA array; multiplexed CRISPR; genome editing; assembly method; Golden Gate assembly; PARA; web tool;

D O I：

10.3390/cells11162467

中图分类号：

Q2 [细胞生物学];

学科分类号：

071009 ; 090102 ;

摘要：

Multiplexed CRISPR technologies have great potential for pathway engineering and genome editing. However, their applications are constrained by complex, laborious and time-consuming cloning steps. In this research, we developed a novel method, PARA, which allows for the one-step assembly of multiple guide RNAs (gRNAs) into a CRISPR vector with up to 18 gRNAs. Here, we demonstrate that PARA is capable of the efficient assembly of transfer RNA/Csy4/ribozyme-based gRNA arrays. To aid in this process and to streamline vector construction, we developed a user-friendly PARAweb tool for designing PCR primers and component DNA parts and simulating assembled gRNA arrays and vector sequences.

引用

页数：11

共 19 条

[1] A versatile modular vector system for rapid combinatorial mammalian genetics [J].

Albers, Joachim ;

Danzer, Claudia ;

Rechsteiner, Markus ;

Lehmann, Holger ;

Brandt, Laura P. ;

Hejhal, Tomas ;

Catalano, Antonella ;

Busenhart, Philipp ;

Goncalves, Ana Filipa ;

Brandt, Simone ;

Bode, Peter K. ;

Bode-Lesniewska, Beata ;

Wild, Peter J. ;

Frew, Ian J. .

JOURNAL OF CLINICAL INVESTIGATION, 2015, 125 (04) :1603-1619

[2] A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants [J].

Cermak, Tomas ;

Curtin, Shaun J. ;

Gil-Humanes, Javier ;

Cegan, Radim ;

Kono, Thomas J. Y. ;

Konecna, Eva ;

Belanto, Joseph J. ;

Starker, Colby G. ;

Mathre, Jade W. ;

Greenstein, Rebecca L. ;

Voytas, Daniel F. .

PLANT CELL, 2017, 29 (06) :1196-1217

[3] Multiplexed CRISPR/Cas9 Genome Editing and Gene Regulation Using Csy4 in Saccharomyces cerevisiae [J].

Ferreira, Raphael ;

Skrekas, Christos ;

Nielsen, Jens ;

David, Florian .

ACS SYNTHETIC BIOLOGY, 2018, 7 (01) :10-15

[4] Self- processing of ribozyme- flanked RNAs into guide RNAs in vitro and in vivo for CRISPR- mediated genome editing [J].

Gao, Yangbin ;

Zhao, Yunde .

JOURNAL OF INTEGRATIVE PLANT BIOLOGY, 2014, 56 (04) :343-349

[5] Phytozome: a comparative platform for green plant genomics [J].

Goodstein, David M. ;

Shu, Shengqiang ;

Howson, Russell ;

Neupane, Rochak ;

Hayes, Richard D. ;

Fazo, Joni ;

Mitros, Therese ;

Dirks, William ;

Hellsten, Uffe ;

Putnam, Nicholas ;

Rokhsar, Daniel S. .

NUCLEIC ACIDS RESEARCH, 2012, 40 (D1) :D1178-D1186

[6] Construct design for CRISPR/Cas-based genome editing in plants [J].

Hassan, Md Mahmudul ;

Zhang, Yingxiao ;

Yuan, Guoliang ;

De, Kuntal ;

Chen, Jin-Gui ;

Muchero, Wellington ;

Tuskan, Gerald A. ;

Qi, Yiping ;

Yang, Xiaohan .

TRENDS IN PLANT SCIENCE, 2021, 26 (11) :1133-1152

[7] Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector [J].

Kabadi, Ami M. ;

Ousterout, David G. ;

Hilton, Isaac B. ;

Gersbach, Charles A. .

NUCLEIC ACIDS RESEARCH, 2014, 42 (19)

[8] Highly multiplexed genome engineering using CRISPR/Cas9 gRNA arrays [J].

Kurata, Morito ;

Wolf, Natalie K. ;

Lahr, Walker S. ;

Weg, Madison T. ;

Kluesner, Mitchell G. ;

Lee, Samantha ;

Hui, Kai ;

Shiraiwa, Masano ;

Webber, Beau R. ;

Moriarity, Branden S. .

PLOS ONE, 2018, 13 (09)

[9] CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing [J].

Labun, Kornel ;

Montague, Tessa G. ;

Krause, Maximilian ;

Cleuren, Yamila N. Torres ;

Tjeldnes, Hakon ;

Valen, Eivind .

NUCLEIC ACIDS RESEARCH, 2019, 47 (W1) :W171-W174

[10] Multiplexed CRISPR technologies for gene editing and transcriptional regulation [J].

McCarty, Nicholas S. ;

Graham, Alicia E. ;

Studena, Lucie ;

Ledesma-Amaro, Rodrigo .

NATURE COMMUNICATIONS, 2020, 11 (01)

← 1 2 →