An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize

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). T₀ 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-II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T₁ progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target-specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize.

Keywords: Argonaute; anthocyaninless; CRISPR/Cas9; gene editing; maize; targeted mutagenesis.

Figure 1

Schematic diagram of Cas9/gRNA construction. Cloning vectors pENTR‐gRNA1 (with two HindIII sites) or pENTR‐gRNA2 (with one HindIII site) were sequentially digested with Btg ZI and BsaI restriction enzymes for the insertions of two double‐stranded oligonucleotides. The subcloning resulted in two intermediate constructs, pgRNA‐IM1 and pgRNA‐IM2, each carrying two gRNA expression cassettes. The cassettes flanked by the Gateway recombination sequences attL1 and attL2 were mobilized to the binary vector pGW‐Cas9 through Gateway recombination, resulting in a single plasmid Cas9/gRNA binary construct for Agrobacterium‐mediated gene transfer.

Figure 2

A flow chart of targeted mutagenesis in maize using Agrobacterium‐mediated transformation illustrates the main steps in CRISPR‐based mutagenesis. A minimum of 7 months is required from embryo transformation to production of mutant seeds.

Figure 3

Cas9/gRNA‐induced mutations in ZmAgo18a and ZmAgo18b. (a) Structure of the paralogous Ago18 genes present on chromosomes 1 and 2 and the gRNAs designed to generate DSBs in exons (blank bars). gRNAs, gAgo18a‐1 and gAgo18a‐2 (above the double‐strand box for ZmAgo18a), and gAgo18b‐1 and gAgo18b‐2 (below the double‐strand box for ZmAgo18b). Nucleotides in red represent target sites, and green underlined nucleotides indicate PAM sequences for the gRNAs. 10 and 76 nt represent the numbers of nucleotides between the two target sites in each gene. (b–e) Sequences from selected T₀ plants with site‐specific mutations accompanied by corresponding regions of the sequencing chromatograms. The nucleotide changes (dashes for deletion, lowercase letter for insertion and WT for unaltered) are also indicated to the right side of each sequence. Dots in Ago18a #23 and Ago18b #15 represent nucleotides not shown.

Figure 4

Cas9/gRNA‐induced mutations at the a1 (chromosome 3) and a4 (chromosome 8) target sites. (a) Gene structures of a1 and a4 loci with gRNAs designed for DSBs in exons (blank bars). Nucleotides in red represent target sites, and green underlined nucleotides indicate PAM sequences for the gRNAs. gRNAs, gA1/A4‐1 and gA1/A4‐2 are between a1 and a4 gene boxes. 165 nt and 137 nt represent the numbers of nucleotides between the two target sites in each gene. (b) and (c) Sequences from selected T₀ plants containing the site‐specific mutations. MT, mutant types; the nucleotide changes (dashes for deletion and lowercase letter in blue for insertion) are also indicated to the right side of each sequence, suffixed with a letter, if needed, to distinguish different alleles. Line, mutant line.

Figure 5

Characterization of sexually heritable new alleles after targeted mutagenesis by Cas9/gRNA in maize cells. (a) Schematic diagram showing the inheritance and segregation of original edited alleles as well as the generation of new mutations from a1//a4 CRISPR event 20. The T₀ has DA mutations for both a1 and a4. The T₁ and T₂ progeny were derived by crossing mutants to recipient lines with wild‐type a1 and a4 loci. The wild‐type allele is represented as ‘A’, the T₀ mutated alleles as ‘a’ and ‘a′’. ‘a″’ indicates alleles that potentially contain novel mutations. For the development of the T₂ generation, T₁ plant 20‐13 was used as a female (cross I), or male (cross II), and T₁ plant 20‐20 was used as a female (cross III). (b) The top panel shows the presence of Cas9 in genomic DNA in the T₂ progeny plants of event 20 as assayed by PCR. The control lane represents a plasmid‐positive control with cloned Cas9. The bottom panel shows Cas9 transcript levels by RT‐PCR. The control lane represents ‐RT (negative control), and ubiquitin 1 gene expression (Ubi) serves as the positive control. ‘+’ stands for the presence of novel mutation in T₂, and ‘−’ stands for its absence. (c) Sequence information at the a1 and a4 targeted loci for the T₀, T₁ and T₂ plants from event 20. Nucleotides in red represent target sites and green underlined nucleotides indicate PAM sequences for the gRNAs. The nucleotide variations (dashes for deletion and lowercase letter in blue for insertion) are marked on the right side of each sequence with a number, suffixed with a letter, if needed, to distinguish different alleles. Line names are listed in the middle column.