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

<jats:title>Summary</jats:title><jats:p><jats:styled-content style="fixed-case">CRISPR</jats:styled-content>/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of <jats:styled-content style="fixed-case">CRISPR</jats:styled-content>/Cas9 is simpler compared to other nuclease‐based genome editing tools, optimization requires the consideration of the <jats:styled-content style="fixed-case">DNA</jats:styled-content> delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, <jats:styled-content style="fixed-case">ISU</jats:styled-content> Maize <jats:styled-content style="fixed-case">CRISPR</jats:styled-content>, utilizing <jats:italic>Agrobacterium</jats:italic>‐delivered <jats:styled-content style="fixed-case">CRISPR</jats:styled-content>/Cas9 for high‐frequency targeted mutagenesis in maize. This system consists of an <jats:italic>Escherichia coli</jats:italic> cloning vector and an <jats:italic>Agrobacterium</jats:italic> binary vector. It can be used to clone up to four guide <jats:styled-content style="fixed-case">RNA</jats:styled-content>s for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: <jats:italic>Argonaute 18</jats:italic> (<jats:italic>ZmAgo18a</jats:italic> and <jats:italic>ZmAgo18b</jats:italic>) and <jats:italic>dihydroflavonol 4‐reductase</jats:italic> or <jats:italic>anthocyaninless</jats:italic> genes (<jats:italic>a1</jats:italic> and <jats:italic>a4</jats:italic>). T<jats:sub>0</jats:sub> transgenic events carrying mono‐ or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi‐<jats:styled-content style="fixed-case">II</jats:styled-content> and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the <jats:styled-content style="fixed-case">CRISPR</jats:styled-content> transgene could be generated in T<jats:sub>1</jats:sub> progeny. Inheritance of an active <jats:styled-content style="fixed-case">CRISPR</jats:styled-content>/Cas9 transgene leads to additional target‐specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual <jats:italic>Agrobacterium</jats:italic> strains harbouring different Cas9/<jats:styled-content style="fixed-case">gRNA</jats:styled-content> modules can be performed for improved cost efficiency. Together, the findings demonstrate that the <jats:styled-content style="fixed-case">ISU</jats:styled-content> Maize <jats:styled-content style="fixed-case">CRISPR</jats:styled-content> platform is an effective and robust tool to targeted mutagenesis in maize.</jats:p>