Efficient Genome Editing in Populus Using CRISPR/Cas12a.

Yi An,Juan Du,Mengzhu Lu,Chun Wang,Ya Geng,Junguang Yao,Chunxiang Fu

doi:10.3389/fpls.2020.593938

Abstract

The ability to create targeted mutations using clustered regularly inter-spaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 in support of forest tree biotechnology is currently limited. CRISPR/Cas12a is a novel CRISPR effector protein that not only broadens the CRISPR/Cas targeting range but also enables the generation of large-fragment deletions. In this study, a CRISPR/Cas12a system was evaluated for the induction of targeted mutations in the woody tree poplar (Populus alba × Populus glandulosa). Three Cas12a nucleases, namely, AsCas12a (Acidaminococcus sp. BV3L6), LbCas12a (Lachnospiraceae bacterium ND2006), and FnCas12a (Francisella tularensis subsp. novicidain U112), were used. We knocked out multiple targets of the phytoene desaturase gene 8 (PDS) using the CRISPR/Cas12a genome-targeting system, and the results indicated that the AsCas12a system is the most efficient. We further optimized the co-cultivation temperature after Agrobacterium-mediated transformation from 22 to 28°C to increase the Cas12a nuclease editing efficiency in poplar. AsCas12a showed the highest mutation efficiency, at 70%, and the majority of editing sites were composed of large-fragment deletions. By using this simple and high-efficiency CRISPR/Cas12a system, multiple targets can be modified to obtain multigene simultaneous knockout mutants in tree species, which will provide powerful tools with which to facilitate genetic studies of forest trees.

Highlights

Populus species are of economic and ecological value for paper, timber, construction materials, and biofuel production (Jansson and Douglas, 2007)
To test whether the clustered regularly inter-spaced short palindromic repeats (CRISPR)/Cas12a system functions in the editing of multiple target sites in poplar 84K, five conserved target sites with the identical target sequences in allelic genes, as well as 5 -TTTV-3 protospacer-adjacent motif (PAM) located in three exons of phytoene desaturase gene 8 (PagPDS) gene from 84 K, were chosen (Figure 1A)
Because Cas12a has the ability to process its own CRISPR RNA and the mature crRNA is sufficient for gene editing, 21 bp mature direct repeat (DR) (DRs of LbCas12a) with five 24 bp guide sequences were ligated in tandem

Summary

Introduction

Populus species are of economic and ecological value for paper, timber, construction materials, and biofuel production (Jansson and Douglas, 2007). Genome editing facilitates the process of generating mutations in target genes and shows great potential in expediting improvements in plant health and productivity (White et al, 2007; Strauss et al, 2019). A synthetic single-guide RNA (sgRNA), composed of a CRISPR RNA (crRNA) and a trans-activating small RNA, guides Cas to target genes and generates double-stranded DNA breaks, which can lead to gene mutations that result from non-homologous end-joining repair. Cas12a, a type V CRISPR effector, recognizes a thymidine-rich protospacer-adjacent motif (PAM) and induces cohesive double-stranded breaks at the target site guided by a single crRNA (Zetsche et al, 2015). Because Cas12a generates staggered DSBs away from the seed region, it may promote repeated cleavage and extensive end processing, promoting the efficiency of CRISPR/Cpf homology directed repair (HDR)mediated mutations (Wolter and Puchta, 2019; Li et al, 2020)

Methods

Results

Conclusion