An Efficient Marker Gene Excision Strategy Based on CRISPR/Cas9-Mediated Homology-Directed Repair in Rice.

Jiantao Tan,Yang Xue,Yanchang Zhao,Yaxi Wang,Shuifu Chen,Yao-Guang Liu,Qinlong Zhu,Zhansheng Lin,Yuyu Luo

doi:10.3390/ijms23031588

Abstract

In order to separate transformed cells from non-transformed cells, antibiotic selectable marker genes are usually utilized in genetic transformation. After obtaining transgenic plants, it is often necessary to remove the marker gene from the plant genome in order to avoid regulatory issues. However, many marker-free systems are time-consuming and labor-intensive. Homology-directed repair (HDR) is a process of homologous recombination using homologous arms for efficient and precise repair of DNA double-strand breaks (DSBs). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system is a powerful genome editing tool that can efficiently cause DSBs. Here, we isolated a rice promoter (Pssi) of a gene that highly expressed in stem, shoot tip and inflorescence, and established a high-efficiency sequence-excision strategy by using this Pssi to drive CRISPR/Cas9-mediated HDR for marker free (PssiCHMF). In our study, PssiCHMF-induced marker gene deletion was detected in 73.3% of T0 plants and 83.2% of T1 plants. A high proportion (55.6%) of homozygous marker-excised plants were obtained in T1 progeny. The recombinant GUS reporter-aided analysis and its sequencing of the recombinant products showed precise deletion and repair mediated by the PssiCHMF method. In conclusion, our CRISPR/Cas9-mediated HDR auto-excision method provides a time-saving and efficient strategy for removing the marker genes from transgenic plants.

Highlights

In order to effectively separate rare, transformed cells from non-transformed cells, selectable marker genes conferring antibiotic or herbicide resistance are widely employed in plant genetic transformation
We found that the relative expression levels of OsSRABB compared with OsActin1 (39.66-fold in shoot apical meristem (SAM) and 4.9–63.33-fold in inflorescences) (Figure S2) are higher than that compared with another reference gene OsUFC1 (5.17-fold in SAM and 1.32–50.75-fold in inflorescences) in transcriptome database (Figure 1a)
These results suggested that the OsUFC1 expresses more strongly and stably than OsActin1 in SAM and inflorescences, so we chose the OsUFC1 as a reference gene for qRT-PCR analysis in this study

Summary

Introduction

In order to effectively separate rare, transformed cells from non-transformed cells, selectable marker genes conferring antibiotic or herbicide resistance are widely employed in plant genetic transformation. The Cre/loxP site-specific recombination system is the most widely applied auto-excision system, and uses heat-shock inducible, chemically-regulated, or tissue-specific promoters to control the expression of Cre [2,8,9,10]. The downside of these methods is that they are applicable to sexually reproducing species and require time-consuming breeding. Inducible Cre may result in considerable toxicity to rapidly proliferating cells [11] and a 34-bp loxP recombination site remains in the genome as a transgenic marker [12]

Methods

Results

Conclusion