Abstract
Genome editing technology represented by CRISPR-Cas9 had been widely used in many biological fields such as gene function analysis, gene therapy, and crop improvement. However, in the face of the complexity of the eukaryotic genome, the CRISPR-Cas9 genome editing tools have shown an unstable editing efficiency with large variability at different target sites. It was important to further improve the editing efficiency of the CRISPR-Cas9 system among the whole genome. In this study, based on the previous single transcription unit genome editing system (STU-SpCas9), using the ubiquitin-associated domain (UBA) to enhance the stability of Cas9 protein, we constructed three Cas9-UBA fusion systems (SpCas9-SD01, SpCas9-SD02, and SpCas9-SD03). Four different target sites of rice OsPDS, OsDEP1 and OsROC5 genes were chosen to evaluate the genome editing efficiency in rice protoplasts and stable transformed rice plants. The results showed that the fusion of UBA domains did not affect the cleavage mode of Cas9 protein, and effectively increase the editing efficiency of STU-SpCas9 at the target sites. This new CRISPR-Cas9-UBA system provided a new strategy and tool for improving the genome editing efficiency of CRISPR-Cas9 in plants.
Highlights
The CRISPR-Cas9 system has been the most widely used genome editing technology for gene function analysis, gene therapy, and crop improvement in eukaryotic species because of its simple construction, high efficiency, and low cost (Li et al, 2013; Nekrasov et al, 2013; Shan et al, 2013; Liang et al, 2017; Lowder et al, 2018; Zhou et al, 2019)
To investigate whether Cas9 proteins fused with different ubiquitin-associated domain (UBA) stable domains (SD) had editing activities, the three Cas9-UBA systems were used and compared to the STU-Cas9 system (Figure 1A)
The deletion majority ranged from 1 to 3 bp in size, and 1 bp deletions were the most predominant deletion type (Figure 2C, Supplementary Figure S1B). These results indicated that the fusion of the UBA domain does not influence the cleavage mode of Cas9 protein, and they were completely consistent with our previous report that the NHEJ repair outcomes are largely dictated by the sequence composition of the target sites but not the expression systems (Tang et al, 2016, 2019)
Summary
The CRISPR-Cas system has been the most widely used genome editing technology for gene function analysis, gene therapy, and crop improvement in eukaryotic species because of its simple construction, high efficiency, and low cost (Li et al, 2013; Nekrasov et al, 2013; Shan et al, 2013; Liang et al, 2017; Lowder et al, 2018; Zhou et al, 2019). Numerous CRISPR-Cas tools have been developed to achieve targeted mutagenesis, base editing, precise editing by homologydirected repair (HDR) and transcriptional regulation in plants (Chen et al, 2019; Zhang et al, 2019). The CRISPR-Cas system still has some shortcomings such as off-target effects, ineffectiveness at some genomic sites, considerably variable editing efficiency, etc. The editing frequencies (insertion/deletion, indel) at different target sites are quite variable. The efficiency of some target sites was as high as 90–100%, while the others were less than 1% (Liu et al, 2017, 2019; Ding et al, 2019; Mao et al, 2019). It is desirable to develop an ideal CRISPR-Cas system with sustained high activity at whole genome target sites
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