US 11,926,835 B1
Methods for efficient tomato genome editing
Tomás Cermák, Brookline, MA (US); Richard Bailey Flavell, Malibu, CA (US); Eva Konecna, Brookline, MA (US); Yajie Niu, Lexington, MA (US); Michael Lee Nuccio, Salem, NH (US); Jennifer A. Raji, Waltham, MA (US); Randall William Shultz, Acton, MA (US); Davide Sosso, Cambridge, MA (US); Maria Margarita D. Unson, Pawcatuck, CT (US); John Patrick Casey, Jr., Boston, MA (US); and Michael Andreas Kock, Rheinfelden (DE)
Assigned to INARI AGRICULTURE TECHNOLOGY, INC., Cambridge, MA (US)
Filed by INARI AGRICULTURE TECHNOLOGY, INC., Cambridge, MA (US)
Filed on Jan. 29, 2019, as Appl. No. 16/261,233.
Claims priority of provisional application 62/623,481, filed on Jan. 29, 2018.
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/82 (2006.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01)
CPC C12N 15/8213 (2013.01) [C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01)] 6 Claims
 
1. A method of providing a modified tomato cell having a modified genome, comprising effecting in the genome of the tomato cell targeted modifications in at least two genes, wherein the targeted modifications comprise an insertion of a predetermined sequence encoded by a single-stranded DNA donor molecule, wherein the predetermined sequence comprises an enhancer element, wherein each targeted modification is achieved by integrating the predetermined sequence comprising the enhancer element by non-homologous end joining (NHEJ) at one or more site-specific double-strand breaks (DSBs) introduced at about 50 to about 500 nucleotides upstream of the start codon of the coding sequence of the two genes; and
wherein the targeted modifications are effected in a tomato cell by site-specific effector molecules comprising at least one CRISPR nuclease, at least one guide RNA, and at least one single-stranded DNA donor molecule encoding the predetermined sequence, wherein the single-stranded DNA donor molecule has a length of 18 to 300 nucleotides and does not have homology arms; and
wherein the targeted modifications result in an increase in expression of the first gene and an increase in expression of the second gene relative to a tomato cell lacking the modifications, wherein the at least two genes are associated with the same trait, wherein the trait is selected from the group consisting of abiotic stress, architecture, biotic stress, flowering time, nutrient use efficiency, photosynthesis, resource partitioning, and senescence; wherein, after the targeted modifications are effected, the modified genome is more than 99.9% identical to an unmodified genome of the tomato cell; and wherein the targeted modifications improve the trait in a tomato cell comprising the targeted modifications relative to a tomato cell having an unmodified genome, or in a tomato plant grown from or comprising a tomato cell comprising the modifications relative to a tomato plant lacking the modifications.