US 12,460,217 B2
Enhanced recombination of genomic loci
David G. Caldwell, St. Louis, MO (US); and Ervin D. Nagy, Lake Saint Louis, MO (US)
Assigned to Monsanto Technology LLC, St. Louis, MO (US)
Appl. No. 15/753,928
Filed by MONSANTO TECHNOLOGY LLC, St. Louis, MO (US)
PCT Filed Aug. 19, 2016, PCT No. PCT/US2016/047748
§ 371(c)(1), (2) Date Feb. 20, 2018,
PCT Pub. No. WO2017/034971, PCT Pub. Date Mar. 2, 2017.
Claims priority of provisional application 62/208,405, filed on Aug. 21, 2015.
Prior Publication US 2018/0245091 A1, Aug. 30, 2018
Int. Cl. C12N 15/82 (2006.01)
CPC C12N 15/8213 (2013.01) [C12N 15/8271 (2013.01)] 9 Claims
 
1. A method of generating a new gene array of tandemly duplicated genes, comprising
contacting a plant cell with
a first RNA-guided endonuclease of a type II guided CRISPR-Cas system and a first guide RNA that introduce a genome modification in at least one target sequence in a first gene array of tandemly duplicated genes and
a second RNA-guided endonuclease of a type II guided CRISPR-Cas system and a second guide RNA that introduce a genome modification in at least one target sequence in a second gene array of tandemly duplicated genes,
thereby inducing asymmetric recombination between the first gene array and the second gene array of tandemly duplicated genes, and
selecting at least one progeny comprising a new array of tandemly duplicated genes,
wherein the first gene array of tandemly duplicated genes, the second gene array of tandemly duplicated genes and the new gene array of tandemly duplicated genes encode nucleotide-binding site leucine-rich (NBS-LRR) disease resistance proteins,
wherein a genomic locus comprising the target sequence for the genome modification in the first gene array is homologous to at least 100 bp of the second gene array and the regions of homology are in different positions in the first and second gene array,
wherein the cell is a plant cell, wherein the genome modification is a double-strand break (DSB), wherein the duplicated genes are not supplied exogenously, wherein inducing said asymmetric recombination occurs at a frequency that is at least 10-fold higher than the rate of asymmetric recombination in comparator cells, wherein the comparator cells have not been contacted with said first and second RNA-guided endonuclease of a type II guided CRISPR-Cas system and guide RNA, and
wherein the plant is corn and the disease resistance locus is Rp1; or wherein the plant is soy and the disease resistance locus is Rpp1; or wherein the plant is soy and the disease resistance locus is Rps1; or wherein the plant is soy and the disease resistance locus is Rhg1.