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

Cells. 2022 Aug 9;11(16):2467. doi: 10.3390/cells11162467.

Authors

Guoliang Yuan^{1

2}, Stanton Martin^{1

2}, Md Mahmudul Hassan^{1

2

3}, Gerald A Tuskan^{1

2}, Xiaohan Yang^{1

2}

Affiliations

¹ Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
² The Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
³ Department of Genetics and Plant Breeding, Patuakhali Science and Technology University, Dumki, Patuakhali 8602, Bangladesh.

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.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

CRISPR-Cas Systems / genetics
Clustered Regularly Interspaced Short Palindromic Repeats* / genetics
Gene Editing
Polymerase Chain Reaction
RNA, Guide, CRISPR-Cas Systems* / genetics

Substances

RNA, Guide, CRISPR-Cas Systems

Grants and 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.