Heterozygous CENH3 monocots and methods of use thereof for haploid induction and simultaneous genome editing
View Patent ↗Monocot plants heterozygous for centromeric histone 3 (CenH3) and optionally expressing gene editing constructs, for use in inducing haploids of a monocot target plant and optionally pass-through gene editing are provided. The monocot haploid inducer plants are typically composed of diploid plant cells having only one allele encoding a functional CENH3 protein. The diploid plant cells can also include, for example, one CenH3 allele encoding non-functional CENH3 protein. In some embodiments, the allele encoding non-functional CENH3 protein is a frameshift mutation, protein null allele, an RNA null allele, or a combination thereof. The monocot haploid inducer plant can also include gene editing machinery, such as a site-directed nuclease and optionally a guide RNA stably expressed by cells of the monocot plant. Methods of inducing formation of a target haploid monocot plant while optionally simultaneously modifying the target monocot plant's genome are also provided.
1 . A diploid monocot haploid inducer plant heterozygous for centromeric histone 3 (CenH3) comprising diploid plant cells comprising only two CenH3 alleles, wherein one allele encodes wildtype CENH3 protein and the other allele is a null allele, and wherein
the diploid plant cells express an exogenous CRISPR-based site-directed nuclease and a guide RNA, and
the plant's genome further comprises a donor nucleic acid sequence to be introduced by recombination at a cleavage site induced by the nuclease.
2 . The monocot plant of claim 1 , wherein the sperm or eggs of the plant have less CENH3 than a CENH3 homozygous wildtype plant's sperm or eggs.
3 . The monocot plant of claim 1 , wherein the plant is a diploid species of maize, wheat, rice, sorghum, oats, pearl millet, finger millet, foxtail millet, banana, bamboo, Miscanthus, asparagus, onion, garlic, chives, or yam.
4 . The monocot plant of claim 1 , wherein the CRISPR-based nuclease is selected from Cas9 nuclease, Cpf1 nuclease, dCas9-FokI, dCpf1-FokI, chimeric Cas9-cytidine deaminase, chimeric Cas9-adenine deaminase, chimeric FEN1-FokI, nickase Cas9 (nCas9), chimeric dCas9 non-FokI nuclease, dCpf1 non-FokI nuclease, chimeric Cpf1-cytidine deaminase, and Cpf1-adenine deaminase.
5 . The monocot plant of claim 1 , wherein the plant's genome comprises a heterologous nucleic acid construct encoding the gRNA.
6 . An egg cell formed by the plant of claim 1 , the egg cell lacking the allele encoding wildtype CENH3 protein and comprising no more than about 12.5% wildtype CENH3 protein relative to a corresponding egg cell formed by a CenH3 homozygous plant.
7 . A sperm cell formed by the plant of claim 1 , the sperm cell lacking the allele encoding wildtype CENH3 protein and comprising no more than about 25% wildtype CENH3 protein relative to a corresponding sperm cell formed by a CenH3 homozygous plant.
8 . A method of inducing formation of a target haploid monocot plant comprising pollinating a parent monocot target plant with pollen from a diploid monocot haploid inducer plant and selecting at least one haploid progeny produced by the pollination,
wherein the diploid monocot haploid inducer plant is heterozygous for centromeric histone 3 (CenH3) comprising diploid plant cells comprising only two CenH3 alleles, wherein one allele encodes wildtype CENH3 protein and the other allele is a null allele.
9 . A method of inducing formation of a target haploid monocot plant comprising pollinating a diploid monocot haploid inducer plant with pollen from a parent monocot target plant and selecting at least one haploid progeny produced by the pollination,
wherein the diploid monocot haploid inducer plant is heterozygous for centromeric histone 3 (CenH3) comprising diploid plant cells comprising only two CenH3 alleles, wherein one allele encodes wildtype CENH3 protein and the other allele is a null allele.
10 . The method of claim 8 ,
wherein the monocot haploid inducer plant comprises an exogenous site-directed nuclease expressed by cells of the monocot haploid inducer plant, and
wherein the haploid progeny comprises the genome of the monocot target plant but not the monocot haploid inducer plant, and the genome of the haploid progeny has been modified by the site directed nuclease and optionally at least one guide RNA delivered by the monocot haploid inducer plant.
11 . The method of claim 8 , further comprising chromosome doubling of the selected haploid progeny.
12 . The method of claim 9 ,
wherein the monocot haploid inducer plant comprises an exogenous site-directed nuclease expressed by cells of the monocot haploid inducer plant, and
wherein the haploid progeny comprises the genome of the monocot target plant but not the monocot haploid inducer plant, and the genome of the haploid progeny has been modified by the site directed nuclease and optionally at least one guide RNA delivered by the monocot haploid inducer plant.
13 . The method of claim 9 , further comprising chromosome doubling of the selected haploid progeny.
14 . The method of claim 10 , wherein the monocot haploid inducer plant comprises the at least one guide RNA.
15 . The method of claim 12 , wherein the monocot haploid inducer plant comprises the at least one guide RNA.
16 . The method of claim 8 , wherein the monocot haploid inducer plant is a diploid species of maize, wheat, rice, sorghum, oats, pearl millet, finger millet, foxtail millet, banana, bamboo, Miscanthus, asparagus, onion, garlic, chives, or yam.
17 . The method of claim 8 , wherein the monocot haploid inducer plant is maize.
18 . The method of claim 9 , wherein the monocot haploid inducer plant is a diploid species of maize, wheat, rice, sorghum, oats, pearl millet, finger millet, foxtail millet, banana, bamboo, Miscanthus, asparagus, onion, garlic, chives, or yam.
19 . The method of claim 9 , wherein the monocot haploid inducer plant is maize.