Development of efficient targeted insertion mediated by CRISPR-Cas12a and homology-directed repair in maize

Brenden Barco^1*Shujie Dong^1*Yuki Matsuba¹Ashley Crook¹Ruiji Xu¹Yingxiao Zhang¹Chengjin Zhang¹Ryan Carlin¹Kevin Potter¹Stephen B Rigoulot¹Jeongmoo Park¹Erin M Seaberry^1,2Allison Parrish¹Sivamani Elumalai¹Samson P Nalapalli¹Craig Schuller¹Anna Prairie¹Anna Mangum¹Kangfeng Mei^1,3Hao Wu¹Melissa Murray¹Kristin Setliff¹Francine Johnson¹Dawn Mcnamara¹Ling Zhu¹Mark Rose¹Weining Gu¹Hao Hu¹Yuanji Zhang¹Yaping Jiang¹Wenling Wang¹Guozhu Tang¹Lizhao Geng¹Jianping Xu¹Wan Shi¹Jason Nichols¹Timothy Joseph Kelliher^1,4Liang Shi¹Ian Jepson¹Qiudeng Que¹

• ¹Syngenta, Basel, Switzerland
• ²Primary Products Ingredients Americas LLC (Primient), Schaumburg, United States
• ³Meiwenti AgBio Ops and IP Solutions, Cary, United States
• ⁴NC State University, Raleigh, United States

Targeted insertion (TIN) of transgenic trait cassettes has the potential to reduce timeline and cost for GM product development and commercialization. Precise genome engineering has made remarkable progress over the last several decades, particularly with the use of site-directed nucleases as core editing machinery. However, there are still many critical factors that can impact TIN efficiency including insertion site selection, nuclease optimization and expression, donor vector design, gene delivery, and stable event regeneration. Here, we develop workflows for target site sequence identification and gRNA screening for CRISPR-Cas12a system and demonstrate its successful application for TIN in maize with donor sequences up to 10 kilobase pairs (kb) in size. We first prioritize genomic regions for inserting transgenes in silico using bioinformatics tools and then tested gRNA performance using a leaf protoplast transient assay. Despite its known low efficiency, we identify homology-directed repair (HDR) as the preferential pathway for directing targeted insertions of large sequence in immature embryos and demonstrated double-junction integrations at a rate of up to 4%. We further apply a molecular analysis workflow using large amplicon TaqMan assays and nanopore sequencing for streamlined identification and characterization of high-quality insertion events with intact large inserts. Analysis of TIN events across generations suggests that efficiency bottlenecks are not limited to donor targeted integration; attrition in efficiency also results from partial or additional donor insertion, chimerism, and close linkage with undesired sequence insertions such as those encoding the editing machinery. This work represents a major step forward in realizing the potential of precise genome engineering in maize for basic research and biotech trait development applications.