Genome Editing of Golden SNP-Carrying Lycopene Epsilon-Cyclase (LcyE) Gene Using the CRSPR-Cas9/HDR and Geminiviral Replicon System in Rice

Abstract

Lycopene epsilon-cyclase (LcyE) is a key enzyme in the carotenoid biosynthetic pathway of higher plants. Using the CRSPR/Cas9 and the geminiviral replicon, we optimized a method for targeted mutagenesis and golden SNP replacement of the LcyE gene in rice. We have exploited the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene via homology-directed repair (HDR). Mutagenesis experiments performed on the Donggin variety achieved precise modification of the LcyE loci with an efficiency of up to 90%. In HDR experiments, our target was the LcyE allele (LcyE-H523L) derived from anther culture containing a golden SNP replacement. The phenotype of the homologous recombination (HR) mutant obtained through the geminiviral replicon-based template delivery system was tangerine color, and the frequency was 1.32% of the transformed calli. In addition, the total carotenoid content of the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines was 6.8–9.6 times higher than that of the wild-type (WT) calli, respectively. The reactive oxygen species content was lower in the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines. These results indicate that efficient HDR can be achieved in the golden SNP replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits.

Phenotypic characterization of pre-harvest sprouting resistance mutants generated by the CRISPR/Cas9-geminiviral replicon system in rice

“…In addition, we increased HDR efficiency by heat treatment at 42°C for 12 hr after infecting rice calli with Agrobacterium tumefaciens during transformation experiments ( Supplementary Table 2 ). In this experiment, the frequency of HDR was 2.6%, which was twice as high as in our previous report ( 37 ) and much higher than the frequency of 0.66% in tomato ( 51 ). The difference of the present study from the previous experiment was that heat stress was performed for 12 hours after Agrobacterium infection.…”

“…It has been recently reported that HDR efficiency can be improved using a CRISPR/Cas9-geminiviral replicon delivery system ( 33 - 36 ). In a previous experiment, we performed an HDR experiment using the CRISPR/Cas9-geminiviral replicon system in rice calli and found that the HDR frequency was 1.32% ( 37 ). Here, we used the CRISPR/Cas9-geminiviral replicon system to generate HDR mutant lines with a replacement of one region SNP (C/T) and a 6-bp insertion (GGT/GGTGGCGGC) in the OsERF1 gene for PHS resistance in rice.…”

Development and validation of SSR markers related to flower color based on full-length transcriptome sequencing in Chrysanthemum

“…Additionally, LCYE and PSY are two key genes in the carotenoid metabolic pathway 13 . The mutant of OsLCYE gene had higher carotenoid content than wild-type plants under salt stress 30 . PSY catalyzes the first step of the carotenoid biosynthetic pathway and is an important rate-limiting enzyme of carotenoid biosynthesis 48 .…”

“…LYCOPENE EPSILON CYCLASE ( LCYE ) and PHYTOENE SYNTHASE ( PSY ) are two key genes involved in carotenoid metabolism 13 . In rice, the mutation of OsLCYE gene would increase total carotenoid content and decrease ROS accumulation under salt stress 30 . In tomato, SlBBX20 could activate the expression of SlPSY1 , which caused the accumulation of carotenoids and gave dark green color to the fruits and leaves 31 .…”

Review of the Use of Animal Models of Human Polycystic Kidney Disease for the Evaluation of Experimental Therapeutic Modalities

“…To elucidate the pathogenesis of PKD and test the efficacy of the candidate drugs, spontaneous and genetically modified laboratory animal models are the primary source of development information. The revelation of the nucleotide sequences of PKD1 , PKD2 , and PKHD1 has led to the development of various genetically modified animals, including transgenic animals with a foreign gene inserted, knockout (KO) animals with a specific gene disrupted, conditional KO (CKO) animals with cells forming a specific organ intentionally damaged or genetically disrupted at specific locations, knock-in animal models with hypomorphic genetic mutations, and genome-editing animal models that have been modified using new techniques such as the CRISPR/Cas9 method [ 16 , 17 , 18 , 19 ]. This study introduces the use of animal models in research on renal cystic disease and characterizes different animal model strains.…”