An <i>Agrobacterium</i>‐delivered <scp>CRISPR</scp>/Cas9 system for high‐frequency targeted mutagenesis in maize

TLDR

CRISPR/Cas9 is a powerful genome editing tool across many species, but achieving high accuracy and efficiency requires optimizing DNA delivery and tissue regeneration methods for each species. The study introduces the ISU Maize CRISPR system, an Agrobacterium‑delivered CRISPR/Cas9 platform for high‑frequency targeted mutagenesis in maize. The system uses an E. coli cloning vector and an Agrobacterium binary vector that can accommodate up to four guide RNAs for single or multiplex targeting, and was tested on four maize genes (ZmAgo18a, ZmAgo18b, a1, a4) to assess mutagenesis frequency and heritability.

Abstract

CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease-based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium-delivered CRISPR/Cas9 for high-frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4-reductase or anthocyaninless genes (a1 and a4). T