AUTHOR=Liang Zhen , Wu Yuqing , Ma Lingling , Guo Yingjie , Ran Yidong 

TITLE=Efficient Genome Editing in Setaria italica Using CRISPR/Cas9 and Base Editors

JOURNAL=Frontiers in Plant Science

VOLUME=12

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.815946

DOI=10.3389/fpls.2021.815946

ISSN=1664-462X

ABSTRACT=<p>The genome editing toolbox based on CRISPR/Cas9 has brought revolutionary changes to agricultural and plant scientific research. With the development of stable genetic transformation protocols, a highly efficient genome editing system for foxtail millet (<italic>Setaria italica</italic>) is required. In the present study, we use the CRISPR/Cas9 single- and multi-gene knockout system to target the <italic>SiFMBP</italic>, <italic>SiDof4</italic>, <italic>SiBADH2</italic>, <italic>SiGBSS1</italic>, and <italic>SiIPK1</italic> genes in the foxtail millet protoplasts to screen out highly efficient targeted sgRNAs. Then, we recovered homozygous mutant plants with most of the targeted genes through an <italic>Agrobacterium</italic>-mediated genetic transformation of foxtail millet. The mutagenesis frequency in the T<sub>0</sub> generation was as high as 100%, and it was passed stably on to the next generation. After screening these targeted edited events, we did not detect off-target mutations at potential sites. Based on this system, we have achieved base editing successfully using two base editors (CBE and ABE) to target the <italic>SiALS</italic> and <italic>SiACC</italic> genes of foxtail millet. By utilizing CBE to target the <italic>SiALS</italic> gene, we created a homozygous herbicide-tolerant mutant plant. The current system could enhance the analysis of functional genomics and genetic improvement of foxtail millet.</p>