Effective editing for lysophosphatidic acid acyltransferase 2/5 in allotetraploid rapeseed (Brassica napus L.) using CRISPR-Cas9 system

Abstract

Background Brassica napus is one of the most important oilseed crops, and can supply considerable amounts of edible oil as well as provide raw materials for the production of biodiesel in the biotechnology industry. Lysophosphatidic acid acyltransferase (LPAT), a key enzyme in the Kennedy pathway, catalyses fatty acid chains into 3-phosphoglycerate and promotes further production of oil in the form of triacylglycerol. However, because B. napus is an allotetraploid with two subgenomes, the precise genes which involved in oil production remain unclear due to the intractability of efficiently knocking out all copies with high genetic redundancy. Therefore, a robust gene editing technology is necessary for gene function analysis. Results An efficient gene editing technology was developed for the allotetraploid plant B. napus using the CRISPR-Cas9 system. Previous studies showed poor results in either on-target or off-target activity in B. napus. In the present study, four single-gRNAs and two multi-gRNAs were deliberately designed from the conserved coding regions of BnLPAT2 which has seven homologous genes, and BnLPAT5, which has four homologous genes. The mutation frequency was found to range from 17 to 68%, while no mutation was observed in the putative off-target sites. The seeds of the Bnlpat2/Bnlpat5 mutant were wizened and showed enlarged oil bodies, disrupted distribution of protein bodies and increased accumulation of starch in mature seeds. The oil content decreased, with an average decrease of 32% for Bnlpat2 lines and 29% for Bnlpat5 lines in single-gRNA knockout lines, and a decline of 24% for Bnlpat2 mutant lines (i.e., g123) and 39% for Bnlpat2/Bnlpat5 double mutant lines (i.e., g134) in multi-gRNA knockout lines. Conclusions Seven BnLPAT2 homologous genes and four BnLPAT5 homologous genes were cleaved completely using the CRISPR-Cas9 system, which indicated that it is effective for editing all homologous genes in allotetraploid rapeseed, despite the relatively low sequence identities of both gene families. The size of the oil bodies increased significantly while the oil content decreased, confirming that BnLPAT2 and BnLPAT5 play a role in oil biosynthesis. The present study lays a foundation for further oil production improvement in oilseed crop species.

Elevating seed oil content in a polyploid crop by induced mutations in SEED FATTY ACID REDUCER genes

“…In less than two years, stably inheriting homozygous T 3 winter‐type mutant lines could be selected. As shown in previous studies (Yang et al , 2018; Zhang et al , 2019), we also found T 1 plants with more than two alleles (haplotypes) at one locus as clear evidence for chimerism. A possible explanation is the low activity of the Cas9 nuclease caused by partial transgene silencing in the BnSFAR5 T 1 plant, which carries multiple transgene insertions.…”

CRISPR/Cas: A powerful tool for gene function study and crop improvement

“…Grains with high amylose content and resistant starch has human health benefits. CRISPR/Cas9 genome editing shows that knockout of pat2/5 genes resulted in increased accumulation of starch in the mature seeds of allotetraploid rapeseed [124] . Sun and colleagues (2018) employed CRISPR/Cas technology to successfully knockout the starch branching enzyme (SBE) gene SBEIIb and generated genome-edited rice with high content of amylose, in which up to 25% of amylose was generated with higher proportion of long chain in debranched amylopectin in rice seeds [125] .…”

The Use of Genetic and Gene Technologies in Shaping Modern Rapeseed Cultivars (Brassica napus L.)

“…By overexpressing the fatty acid elongase gene ( fae1 ) simultaneously with the lysophosphatidic acid acyltransferase gene from Limnanthes douglasii (Ld-LPAAT), erucic acid synthesis was predominant over PUFA synthesis in competitive elongation to the triaclyglycerol backbone in transgenic lines, which resulted in an increase in erucic acid content of up to 72% and a PUFA content as low as 6% [ 13 ]. The roles of LPAAT genes of B. napus , BnLPAT2 and BnLPAT5, in regulating oil biosynthesis has been recently confirmed by targeted mutations using Cas9 with single-gRNAs and multi-sgRNAs whereby the resulted Bnlat2 and Bnlat5 mutants showed decreased oil content and enlarged oil bodies [ 33 ]. Cytoplasmic genomes are also attracting more interest as the new approach for maximizing oil content in canola [ 90 ].…”

“…Limitations in gene pool diversity [ 31 , 32 ], and the time and laborious constraints in canola breeding can now be overcome by using genetic modification [ 32 ] and genome editing technologies whereby transgenes or mutations can be directly introduced into plants, providing additional ways to investigate gene functions in biological processes [ 33 , 34 , 35 , 36 ]. The most commonly applied genome editing tool in eukaryotes in the last 8 years, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system [ 37 , 38 ], is gaining more interest compared to conventional genetic modification methods due to its high efficiency in targeted nucleotide modification and generation of a transgene-free end-product [ 39 ].…”