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

Guoliang Yuan; Stanton Martin; Md Mahmudul Hassan; Gerald A. Tuskan; Xiaohan Yang

doi:10.3390/cells11162467

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

Guoliang Yuan, Stanton Martin, Md Mahmudul Hassan, Gerald A. Tuskan, Xiaohan Yang

Research output: Contribution to journal › Article › peer-review

11 Scopus citations

Abstract

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.

Original language	English
Article number	2467
Journal	Cells
Volume	11
Issue number	16
DOIs	https://doi.org/10.3390/cells11162467
State	Published - Aug 2022

Funding

This work was supported by the Center for Bioenergy Innovation, a US DOE Bioenergy Research Center supported by the Biological and Environmental Research program. The authors declare no conflict of interest. This manuscript has been authored by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the US Department of Energy (DOE). The US government retains and the publisher, by accepting the article for publication, acknowledges that the US government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for US government purposes. DOE will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan ( http://energy.gov/downloads/doe-public-access-plan (accessed on 17 June 2022)).

Funders	Funder number
U.S. Department of Energy
Biological and Environmental Research
Center for Bioenergy Innovation

Keywords

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

Access to Document

10.3390/cells11162467

Cite this

@article{5cfefb74c0264b99ab8a32ab07903746,

title = "PARA: A New Platform for the Rapid Assembly of gRNA Arrays for Multiplexed CRISPR Technologies",

abstract = "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.",

keywords = "Golden Gate assembly, PARA, assembly method, gRNA array, genome editing, multiplexed CRISPR, web tool",

author = "Guoliang Yuan and Stanton Martin and Hassan, {Md Mahmudul} and Tuskan, {Gerald A.} and Xiaohan Yang",

note = "Publisher Copyright: {\textcopyright} 2022 by the authors.",

year = "2022",

month = aug,

doi = "10.3390/cells11162467",

language = "English",

volume = "11",

journal = "Cells",

issn = "2073-4409",

publisher = "MDPI",

number = "16",

}

TY - JOUR

T1 - PARA

T2 - A New Platform for the Rapid Assembly of gRNA Arrays for Multiplexed CRISPR Technologies

AU - Yuan, Guoliang

AU - Martin, Stanton

AU - Hassan, Md Mahmudul

AU - Tuskan, Gerald A.

AU - Yang, Xiaohan

PY - 2022/8

Y1 - 2022/8

N2 - 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.

AB - 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.

KW - Golden Gate assembly

KW - PARA

KW - assembly method

KW - gRNA array

KW - genome editing

KW - multiplexed CRISPR

KW - web tool

UR - http://www.scopus.com/inward/record.url?scp=85136580864&partnerID=8YFLogxK

U2 - 10.3390/cells11162467

DO - 10.3390/cells11162467

M3 - Article

C2 - 36010544

AN - SCOPUS:85136580864

SN - 2073-4409

VL - 11

JO - Cells

JF - Cells

IS - 16

M1 - 2467

ER -

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

Abstract

Funding

Keywords

Access to Document

Other files and links

Fingerprint

Cite this