Combining multiplex gene editing and doubled haploid technology in maize

Abstract

A major advantage of using CRISPR/Cas9 for gene editing is multiplexing, that is, the simultaneous targeting of many genes. However, primary transformants typically contain hetero-allelic mutations or are genetic mosaic, while genetically stable lines that are homozygous are desired for functional analysis. Currently, a dedicated and labor-intensive effort is required to obtain such higher-order mutants through several generations of genetic crosses and genotyping. We describe the design and validation of a rapid and efficient strategy to produce lines of genetically identical plants carrying various combinations of homozygous edits, suitable for replicated analysis of phenotypical differences. This approach was achieved by combining highly multiplex gene editing in Zea mays (maize) with in vivo haploid induction and efficient in vitro generation of doubled haploid plants using embryo rescue doubling. By combining three CRISPR/Cas9 constructs that target in total 36 genes potentially involved in leaf growth, we generated an array of homozygous lines with various combinations of edits within three generations. Several genotypes show a reproducible 10% increase in leaf size, including a septuple mutant combination. We anticipate that our strategy will facilitate the study of gene families via multiplex CRISPR mutagenesis and the identification of allele combinations to improve quantitative crop traits.

Keywords: CRISPR/Cas9; doubled haploids; gene editing; gene family; haploid induction; maize; multiplex gene editing; mutation stacking.

Fig. 1. Efficient haploid induction and haploid doubling in Zea mays (maize) B104.

(a) Number of embryos obtained per cross between B104 background (female) and RWS-GFP (male) (n = 38 independent pollinations). (b) Haploid induction rate (HIR). Percentage of embryos scored as haploid based on the absence of GFP from a cross between B104 background and RWS-GFP (n = 38). (c) Percentage of haploid plants in each independent experiment surviving the embryo rescue doubling process (n = 30). (d) Haploid doubling rate. Percentage of plants in each experiment scored either as doubled haploid (DH), mixoploid, or haploid based on flow cytometry analysis of leaf 3 or 4, calculated per treated haploid embryo (n = 28). Boxplots with jittered data points; center lines show the medians; box limits indicate the 25^th and 75^th percentiles; whiskers extend 1.5 times the interquartile range from the 25^th and 75^th percentiles, and the mean is indicated as a cross.

Fig. 2. Combining multiplex gene editing and doubled haploid breeding in Zea mays (maize).

(a) Overview of the GEDH strategy. For simplicity, four GE target loci are shown to represent a genotype, stacked colored bars represent different edited alleles, and white bars indicate reference alleles, bar lengths are proportional to the allele frequencies. More than two alleles can be present in a plant due to genetic mosaicism from ongoing Cas9 activity. Multiplex edited T0 plants are crossed with wild-type (WT) B104 plants resulting in BC1 plants that are either heterozygous or wild-type at each locus and can be selected for the absence of the EDITOR T-DNA to avoid further editing. BC1 plants are pollinated using a haploid inducer line carrying a GFP transgene (RWS-GFP). Two weeks after pollination, embryos are isolated, haploid embryos are selected for the absence of GFP fluorescence (GFP-), and incubated on a medium containing colchicine. Colchicine-treated embryos are germinated in vitro and chromosome doubling is confirmed by flow cytometry. Every haploid embryo and doubled haploid plant will show a random combination of edited loci. Doubled haploids (DH0) are self-pollinated to obtain genetically identical homozygous DH1 seeds. (b) Diagrams of the EDITOR and SCRIPT T-DNAs. RB, right border; pZmUBI, maize UBIQUITIN-1 promoter; tNOS, Agrobacterium NOPALINE SYNTHASE terminator; p35S, double Cauliflower mosaic virus 35S promoter; HYG,HYGROMYCINE RESISTANCE; BAR, BIALAPHOS RESISTANCE, LB, left border. In the SCRIPT T-DNA, 12 gRNAs are alternately expressed by either an OsU3 or TaU3 promoter.

Fig. 3. Combining haploid induction and multiplex gene editing in Zea mays (maize) with SCRIPT 4.

Maize B104 immature embryos heterozygous for the EDITOR T-DNA were supertransformed with the SCRIPT 4 gRNA construct, targeting the 12 genes listed on top. Experimentally obtained genotypes for a representative subset of plants are shown for each generation. Colored, horizontally stacked bars each indicate different mutant out-of-frame alleles per target locus, white bars indicate wild-type (reference) alleles, light gray bars indicate in-frame mutant alleles and colored bar lengths are proportional to the fraction of sequence reads per locus containing the allele. Bar lengths <50% are indicative of mosaicism.

Fig. 4. Phenotypic analysis of Zea mays (maize) SCRIPT 4 DH lines.

Genotypes and corresponding phenotypes observed in DH1 SCRIPT 4 plants homozygous for various combinations of out-of-frame alleles (green squares) and in-frame mutated alleles (gray squares); the size of the indel (in bp) is indicated in the squares. White squares indicate that the wild-type reference (REF) allele was identified by genotyping. Each row represents an independent DH line. Boxplots with jittered data points on the right display measurements of pseudo leaf 3 area (PLA3) for edited plants compared with non-edited control plants (wild-type B104 and two wild-type doubled haploids (HIC01 and HIC03)). DH lines are sorted from lowest to highest mean PLA3. 24 to 29 seeds were sown for each DH line; n, number of germinated plants phenotyped. The compact letter display shows the result of the pairwise comparisons of the Wilcoxon rank sum test (significance level of 5% with Holm correction).

Fig. 5. Combining haploid induction and multiplex gene editing in Zea mays (maize) with inter-script crosses.

Experimentally obtained genotypes for a representative subset of plants of (a) S2xS4 and (b) S4xS3 are shown for each generation. Colored, horizontally stacked bars each indicate different mutant out-of-frame alleles per locus, white bars indicate wild-type (reference) alleles, light gray bars indicate in-frame mutant alleles and colored bar lengths are proportional to the fraction of sequence reads per locus containing the allele. Absent bars indicate missing data due to low-quality sequencing. Gene locus names are indicated above each column, as well as their respective SCRIPT construct.

Fig. 6. Phenotypic analysis of Zea mays (maize) DH1 and DH2 plants.

(a) Observed genotypes and corresponding phenotypes in DH1 and DH2 for four different homozygous edited genotypes and the non-edited control HIC01. Out-of-frame mutated alleles (green squares), in-frame mutated alleles (gray squares) and reference alleles (white squares), the size of the indel (in bp) is indicated in the squares. On the left, each row represents the genotype of a line (DH1 or DH2 generation), on the right, corresponding boxplots with jittered data points display measurements of pseudo leaf 3 area (PLA3). 24 to 35 seeds of each line were sown; n, number of plants phenotyped. The compact letter display shows the result of the pairwise comparisons of the Wilcoxon rank sum test (significance level of 5% with Holm correction). (b) Representative seedlings at V3 stage for HIC01 and the septuple mutant Line25, one plant of each generation (DH1 and DH2).