Applications of CRISPR/Cas9 as New Strategies for Short Breeding to Drought Gene in Rice

Jae-Ryoung Park; Eun-Gyeong Kim; Yoon-Hee Jang; Rahmatullah Jan; Muhammad Farooq; Mohammad Ubaidillah; Kyung-Min Kim

doi:10.3389/fpls.2022.850441

Applications of CRISPR/Cas9 as New Strategies for Short Breeding to Drought Gene in Rice

Front Plant Sci. 2022 Feb 24:13:850441. doi: 10.3389/fpls.2022.850441. eCollection 2022.

Authors

Jae-Ryoung Park^{1

2}, Eun-Gyeong Kim¹, Yoon-Hee Jang¹, Rahmatullah Jan¹, Muhammad Farooq¹, Mohammad Ubaidillah³, Kyung-Min Kim¹

Affiliations

¹ Division of Plant Biosciences, School of Applied Biosciences, College of Agriculture and Life Science, Kyungpook National University, Daegu, South Korea.
² Crop Breeding Division, National Institute of Crop Science, Rural Development Administration, Wanju, South Korea.
³ Department of Agronomy, Faculty of Agriculture, Jember University, Jember, Indonesia.

Abstract

Recent unpredictable climate change is the main reason for the decline in rice yield. In particular, drought stress is a major constraint in reducing yield and quality for rice at rainfed agriculture areas, such as Asia and South America. CRISPR/Cas9 provides an effective solution for gene function study and molecular breeding due to specific editing of targeted genome sequences. In addition, CRISPR/Cas9 application can significantly reduce the time required to develop new cultivars with improved traits compared to conventional complex and time-consuming breeding. Here, drought-induced gene Oryza sativa Senescence-associated protein (OsSAP) was edited by CRISPR/Cas9. To investigate the possible role of OsSAP in drought stress, genome-editing plants were subjected to drought stress until the soil moisture content reached 20%, and the reactive oxygen species (ROS) scavenging efficiency of genome-editing plants were decreased. When the genome-editing plants were subjected to drought stress, survival rate, shoot length, root length, content of chlorophyll number of tiller, and 1,000-grain weight decreased, and more H₂O₂ and O₂ ^- were detected in leaves. In addition, expression levels of several critical stress-related transcription factors were decreased in the OsSAP genome-editing plant. These results suggest that OsSAP function as a positive regulator during drought stress response in rice. We analyzed the expression of OsSAP and Cas9 in T₀ and T₁ plants as well as T₂ seeds. As the course of generation advancement progressed, Cas9 expression remained stable or weakened but the OsSAP expression was continuously removed from the T₀ plant. The coefficient of variation (CV) in both T₁ plants and T₂ seeds was lower than 5%. Overall, our results suggest that CRISPR/Cas9 could be a novel and important tool for efficiently generating specific and inheritable targeted genome editing in rice, with short breeding cycles.

Keywords: OsSAP; abiotic stress; agricultural trait; generation; reactive oxygen species.