Demonstration of targeted crossovers in hybrid maize using CRISPR technology

Abstract

Naturally occurring chromosomal crossovers (CO) during meiosis are a key driver of genetic diversity. The ability to target CO at specific allelic loci in hybrid plants would provide an advantage to the plant breeding process by facilitating trait introgression, and potentially increasing the rate of genetic gain. We present the first demonstration of targeted CO in hybrid maize utilizing the CRISPR Cas12a system. Our experiments showed that stable and heritable targeted CO can be produced in F1 somatic cells using Cas12a at a significantly higher rate than the natural CO in the same interval. Molecular characterization of the recombinant plants demonstrated that the targeted CO were driven by the non-homologous end joining (NHEJ) or HDR repair pathways, presumably during the mitotic cell cycle. These results are a step towards the use of RNA-guided nuclease technology to simplify the creation of targeted genome combinations in progeny and accelerate breeding.

Fig. 1. Schematic diagram of the two target regions (gRNA1 and gRNA2) and the locations of the polymorphic SNP markers (M1-M34) used in the genotyping assays.

Schematic SNP marker positions are shown as black, brown, and blue triangles. The blue triangles represent SNP markers that were shared in the two different genome editing experiments. The physical genome coordinates of the gRNA target sites are based on the B73 genome reference public assembly: Zm-B73-REFERENCE-NAM-5.0. (https://www.maizegdb.org). The physical position of each SNP marker can be found in Supplementary Data 3.

Fig. 2. Experimental workflow to demonstrate guided homologous chromosome recombination in maize.

a Two maize inbred lines were crossed to produce F1 hybrid material. b Isolated F1 embryo explants were separated into two groups for Agrobacterium -mediated transformation: (I) editing treatment; (II) control treatment. c, d The regenerated F1-T0 plants were reciprocally backcrossed to Parent B (the first experiment: gRNA1) or Parent A (the second experiment: gRNA2) for the identification of CO by genotyping. e The harvested BC1-F1 seeds were chipped to isolate a small amount of endosperm tissue for genotyping analysis using SNP TaqMan assays. f The genotyping results were examined for the presence of the expected reciprocal chromosomal recombination between the parental chromosomes.

Fig. 3. Analysis of DNA editing patterns at the sites of targeted CO in the BC1-F1 plants identified in the genome editing experiments with gRNA1 and gRNA2 using Illumina sequencing.

DNA was isolated from a single leaf disc sampled from each BC1-F1 plant for library construction and sequencing. Each bar on the graph represents a result derived from a single BC-F1 plant (n = 1). a The gRNA1 target site showing LbCas12a cutting pattern: the PAM sequence (red); gRNA target sequence (italic blue); a fragment of the downstream DNA sequence (black). b The graph shows the proportion of edited (blue) and non-edited (red) reads in 13 LbCas12a negative BC1-F1 plants with the targeted CO and two control plants. BC1-F1 plants selected for further characterization are highlighted with red stars (*) c The gRNA2 target site showing LbCas12a cutting pattern; d The graph shows the proportion of edited (blue) and non-edited (red) reads in 14 randomly selected LbCas12a negative BC1-F1 plants with targeted CO derived from F1-T0 by Parent A backcross.

Fig. 4. Segregation analysis of BC1-F2 populations and identification of homozygous recombinant plants.

a Schematic diagram of the segregation analysis of the BC1-F2 populations; b The result of the genotyping analysis of the BC1-F2 segregating population. An example of the segregation analysis of the SNP markers in 21 BC1-F2 plants from Event-2.11 is shown; c Confirmation of DNA editing pattern at the site of the targeted CO in homozygous BC1-F2 plants with Illumina sequencing. DNA was isolated from a single leaf disc sampled from each BC1-F2 plant for library construction and sequencing. Each bar on the graph represents a result derived from a single BC-F2 plant (n = 1). The proportion of edited (blue) and non-edited (red) amplicons in homozygous recombinant plants from three evaluated populations is shown on the graph. The pictures under the graph represent alignments of the sequences at the site of targeted CO with the inbred reference. gRNA1 target sequence is highlighted in yellow. The gray highlights nucleotides microhomology that could contribute to the repair of DSBs. S2d23 is an abbreviated description of the DNA editing pattern. S2 indicates the nucleotide position where the deletion starts; d23 indicates the length of the deleted nucleotides.