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

Journal Article · 09 August 2022 · Cells

DOI: https://doi.org/10.3390/cells11162467 · OSTI ID:1883861

Yuan, Guoliang  ^[1]; Martin, Stanton ^[1]; Hassan, Md Mahmudul  ^[2]; Tuskan, Gerald A.  ^[1]; Yang, Xiaohan  ^[1]

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1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Patuakhali Science and Technology University, Dumki (Bangladesh)

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.

• Research Organization: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER)
• Grant/Contract Number: AC05-00OR22725
• OSTI ID: 1883861
• Journal Information: Cells, Journal Name: Cells Journal Issue: 16 Vol. 11; ISSN 2073-4409
• Publisher: MDPICopyright Statement
• Country of Publication: United States
• Language: English