AUTHOR=Dong Shujie , Qin Yinping Lucy , Vakulskas Christopher A. , Collingwood Michael A. , Marand Mariam , Rigoulot Stephen , Zhu Ling , Jiang Yaping , Gu Weining , Fan Chunyang , Mangum Anna , Chen Zhongying , Yarnall Michele , Zhong Heng , Elumalai Sivamani , Shi Liang , Que Qiudeng TITLE=Efficient Targeted Mutagenesis Mediated by CRISPR-Cas12a Ribonucleoprotein Complexes in Maize JOURNAL=Frontiers in Genome Editing VOLUME=3 YEAR=2021 URL=https://www.frontiersin.org/journals/genome-editing/articles/10.3389/fgeed.2021.670529 DOI=10.3389/fgeed.2021.670529 ISSN=2673-3439 ABSTRACT=

Recent advances in the development of CRISPR-Cas genome editing technologies have made it possible to perform targeted mutagenesis and precise gene replacement in crop plants. CRISPR-Cas9 and CRISPR-Cas12a are two main types of widely used genome editing systems. However, when CRISPR-Cas12a editing machinery is expressed from a transgene, some chromosomal targets encountered low editing frequency in important crops like maize and soybean. Here, we report efficient methods to directly generate genome edited lines by delivering Cas12a-gRNA ribonucleoprotein complex (RNP) to immature maize embryos through particle bombardment in an elite maize variety. Genome edited lines were obtained at ~7% frequency without any selection during regeneration via biolistic delivery of Cas12a RNP into immature embryos. Strikingly, the gene editing rate was increased to 60% on average and up to 100% in some experiments when the Cas12a RNP was co-delivered with a PMI selectable marker gene cassette and the induced callus cultures were selected with mannose. We also show that use of higher activity Cas12a mutants resulted in improved editing efficiency in more recalcitrant target sequence. The advances described here provide useful tools for genetic improvement of maize.