Comparative Study between the CRISPR/Cpf1 (Cas12a) and CRISPR/Cas9 Systems for Multiplex Gene Editing in Maize

Chongzhi Gong,Rentao Song,Weiwei Qi,Shengchan Huang

doi:10.3390/agriculture11050429

Chongzhi Gong, Rentao Song + Show 2 more

Open Access

https://doi.org/10.3390/agriculture11050429

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Journal: Agriculture	Publication Date: May 10, 2021
Citations: 12	License type: CC BY 4.0

Affiliation: China Agricultural University, Shanghai University

Abstract

Although the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been proved to be an efficient multiplex gene editing system in maize, it was still unclear how CRISPR/Cpf1 (Cas12a) system would perform for multiplex gene editing in maize. To this end, this study compared the CRISPR/Cpf1 system and CRISPR/Cas9 system for multiplex gene editing in maize. The bZIP transcription factor Opaque2 (O2) was used as the target gene in both systems. We found that in the T0 and T1 generations, the CRISPR/Cpf1 system showed lower editing efficiency than the CRISPR/Cas9 system. However, in the T2 generation, the CRISPR/Cpf1 system generated more types of new mutations. While the CRISPR/Cas9 system tended to edit within the on-target range, the CRISPR/Cpf1 system preferred to edit in between the targets. We also found that in the CRISPR/Cpf1 system, the editing efficiency positively correlated with the expression level of Cpf1. In conclusion, the CRISPR/Cpf1 system offers alternative choices for target-site selection for multiplex gene editing and has acceptable editing efficiency in maize and is a valuable alternative choice for gene editing in crops.

Highlights

Gene-editing technologies can efficiently create mutations in targeted genes, which are important in basic and applied biological research
Multiple CRISPR RNA (crRNA) were separated by a simple short direct repeat (DR)-based unit for the clustered regularly interspaced short palindromic repeats (CRISPR)/Cpf1 system, and multiple guide RNAs (gRNAs) were separated by a tRNA-based unit for the CRISPR/CRISPRassociated protein 9 (Cas9) system (Figure 1a)
Our results showed that the editing efficiency based on multiple gRNAs of the CRISPR/Cas9 system was superior to that of multiple crRNAs of the CRISPR/Cpf1 system

Summary

Introduction

Gene-editing technologies can efficiently create mutations in targeted genes, which are important in basic and applied biological research. The CRISPR/Cas system, among many gene editing technologies, is one of the most popular systems due to its simple process of vector construction and high editing efficiency [1,2,3,4,5,6]. CRISPR/Cas is a functionally prioritized editing system from Streptococcus pyogenes (Sp) and has been successively applied to endogenous genome editing in multiple organisms [6,7,8,9,10,11,12]; it is a natural one-unit system and cannot target multiple targets without modifications [2,3]. Multiplex gene editing with the CRISPR/Cas system is of great value, as it is expected to greatly facilitate crop genome engineering and precision breeding [2,13,14]. The tRNA-based system was thoroughly tested from a single transcript with high editing efficiency in crops [18,19,20]

Results

Discussion

Conclusion