The Rise of the CRISPR/Cpf1 System for Efficient Genome Editing in Plants

Abstract

Cpf1, an endonuclease of the class 2 CRISPR family, fills the gaps that were previously faced in the world of genome engineering tools, which include the TALEN, ZFN, and CRISPR/Cas9. Other simultaneously discovered nucleases were not able to carry out re-engineering at the same region due to the loss of a target site after first-time engineering. Cpf1 acts as a dual nuclease, functioning as an endoribonuclease to process crRNA and endodeoxyribonuclease to cleave target sequences and generate double-stranded breaks. Additionally, Cpf1 allows for multiplexed genome editing, as a single crRNA array transcript can target multiple loci in the genome. The CRISPR/Cpf1 system enables gene deletion, insertion, base editing, and locus tagging in monocot as well as in dicot plants with fewer off-target effects. This tool has been efficiently demonstrated into tobacco, rice, soybean, wheat, etc. This review covers the development and applications of Cpf1 mediated genome editing technology in plants.

Keywords: CRISPR; Cas9; Cpf1; NHEJ; TALEN; ZFN; crRNA; endoribonuclease.

FIGURE 1

Schematic diagram of delivery of CRISPR/Cpf1 vector into plant cells and the mechanism action of editing. Upper panel depicts single construct and individual vectors carrying Cpf1 and gRNA. LB: left border; T1: Target one; AtU6: Small RNA promoter; CaMV: constitutive promoter, regulates Cpf1; and RB: right border. The delivery of these vectors into plant tissue is usually done by Agrobacterium, gene gun, and PEG mediated co-transformation. The black circle represents the nucleus where Cpf1 and gRNA are expressed. The lower panel depicts the mechanism of editing at a target site within DNA.

FIGURE 2

Mechanism of Cpf1-cys4-mediated multiplex genome editing. Construct carrying Cpf1, CRISPR system Yersinia (Csy4), and various gRNAs. The yellow arrow indicates the promoter; the Cpf1 gene is indicated by sky blue; T means the terminator; and green square boxes represent Csy4 spacers. The delivery and integration of construct into the plant genome leads to the transcription and translation of Cpf1 (sky blue oval) and Csy4 endonuclease (brown circle) within the cell. Csy4 endonuclease acts on spacers (green) and separate individual gRNA1, 2, 3, and so on. Furthermore, these individuals gRNAs bind to target their respective sites within the plant genome, where Cpf1 endonuclease creates double-stranded breaks.

FIGURE 3

Mechanism of Cpf1-glytRNA-mediated multiplex genome editing. The construct consists of the Cpf1 gene and multiple tRNA-gRNA units. Each gRNA (blue rectangle) containing a plant-specific tRNA spacer (black color). The delivery and integration of construct into plant genome leads to expression of Cpf1 and tRNA-gRNA primary transcript. The primary transcript carries gRNA along with tRNA (black cloverleaf structure), which is cleaved by endogenous RNase P and RNase Z (labeled as the yellow and green circle, respectively). The separated individual gRNAs direct Cpf1 to their respective targets within the plant genome.