Shuta Kurokawa, Hafizur Rhaman, Naoshi Yamanaka, Chisato Ishizaki, Shaikhul Islam, Tsuyoshi Aiso, Shunya Hirata, Mayuka Yamamoto, Kappei Kobayashi, Hidetaka Kaya

Plant and Cell Physiology, pcab123, https://doi.org/10.1093/pcp/pcab123

Published: 04 August 2021

Abstract

The CRISPR/Cas9 system is now commonly employed for genome editing in various plants such as Arabidopsis, rice, and tobacco. In general, in genome editing of the Arabidopsis genome, the SpCas9 and guide RNA genes are introduced into the genome by the floral dip method. Mutations induced in the target sequence by SpCas9 are confirmed after selecting transformants by screening the T₁ seed population. The advantage of this method is that genome-edited plants can be isolated easily. However, mutation efficiency in Arabidopsis using SpCas9 is not as high as that achieved in rice and tobacco, which are subjected to a tissue culture step. In this study, we compared four promoters and found that the parsley UBIQITIN promoter is highly active in Arabidopsis meristem tissue. Furthermore, we examined whether a simple heat treatment could improve mutation efficiency in Arabidopsis. Just one heat treatment at 37 °C for 24 hours increased the mutation efficiency at all four target sites from 3% to 42%, 43% to 62%, 54% to 75%, and 89 to 91%, respectively, without detectable off-target mutations. We recommend heat treatment of plate-grown plants at 37 °C for 24 hours as a simple method to increase the efficiency of CRISPR/Cas9-mediated mutagenesis in Arabidopsis.

See: https://academic.oup.com/pcp/advance-article-abstract/doi/10.1093/pcp/pcab123/6340079