Efficient Targeted Mutagenesis Mediated by CRISPR-Cas12a Ribonucleoprotein Complexes in Maize.

Shujie Dong,Yaping Jiang,Michael A Collingwood,Michele Yarnall,Zhongying Chen,Christopher A Vakulskas,Weining Gu,Liang Shi,Qiudeng Que,Ling Zhu,Chunyang Fan,Heng Zhong,Yinping Lucy Qin,Mariam Marand,Anna Mangum,Stephen Rigoulot,Sivamani Elumalai

doi:10.3389/fgeed.2021.670529

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.

Highlights

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas)adaptive immune systems are widely distributed in nature to defend bacteria from invasion of phages and other mobile genetic elements, such as plasmids and transposons (Sapranauskas et al, 2011; Hille et al, 2018)
The results show that some genomic targets such as Bx9TS2 can be edited efficiently using AsCas12a ribonucleoprotein complex (RNP)
It is remarkable that one of the tested CRISPR RNA (crRNA) resulted in 3.42% reads with edits, suggesting that AsCas12a RNP has been delivered to many of the surface cells and it is very active in editing the Bx9 gene sequence

Summary

Introduction

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas)adaptive immune systems are widely distributed in nature to defend bacteria from invasion of phages and other mobile genetic elements, such as plasmids and transposons (Sapranauskas et al, 2011; Hille et al, 2018). Maize Editing With Cas12a RNP contrast to Cas, Cas12a (Cpf1) has several distinct features such as T-rich protospacer-adjacent motif (PAM), a guide crRNA without the need for trans-activating RNA (tracrRNA), creation of sticky ends, and ability to self-process crRNA in addition to the DNA nuclease activity (Zetsche et al, 2015; Bayat et al, 2018) These RNA-guided Cas nucleases (RGNs) scan the genome to search for target DNA sequences complementary to the gRNA and generate a DNA double-strand break at the target sequence if there is a proper protospacer adjacent motif (PAM) present. The resulting chromosomal break is repaired by the host DNA repair machineries, either through nonhomologous end joining (NHEJ) or homology-directed repair (HDR) (Chakrabarti et al, 2019)

Methods

Results

Conclusion