US 12,404,514 B2
CRISPR-systems for modifying a trait of interest in a plant
Omar Abudayyeh, Cambridge, MA (US); Joseph John Belanto, Minneapolis, MN (US); Jonathan Gootenberg, Cambridge, MA (US); Colby Garret Starker, Minneapolis, MN (US); Daniel Francis Voytas, Minneapolis, MN (US); and Feng Zhang, Cambridge, MA (US)
Assigned to THE BROAD INSTITUTE, INC., Cambridge, MA (US); MASSACHUSETTS INSTITUTE OF TECHNOLOGY, Cambridge, MA (US); and PRESIDENT AND FELLOWS OF HARVARD COLLEGE, Cambridge, MA (US)
Appl. No. 16/468,234
Filed by THE BROAD INSTITUTE, INC., Cambridge, MA (US); MASSACHUSETTS INSTITUTE OF TECHNOLOGY, Cambridge, MA (US); REGENTS OF THE UNIVERSITY OF MINNESOTA, Minneapolis, MN (US); and PRESIDENT AND FELLOWS OF HARVARD COLLEGE, Cambridge, MA (US)
PCT Filed Dec. 8, 2017, PCT No. PCT/US2017/065438
§ 371(c)(1), (2) Date Jun. 10, 2019,
PCT Pub. No. WO2018/107103, PCT Pub. Date Jun. 14, 2018.
Claims priority of provisional application 62/432,543, filed on Dec. 9, 2016.
Claims priority of provisional application 62/567,959, filed on Oct. 4, 2017.
Prior Publication US 2019/0352652 A1, Nov. 21, 2019
Int. Cl. C12N 15/82 (2006.01); C12N 9/22 (2006.01)
CPC C12N 15/8213 (2013.01) [C12N 9/22 (2013.01); C12N 15/8283 (2013.01); C12N 2310/20 (2017.05)] 19 Claims
 
1. A plant modified to express
(a) a Type VI Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) effector protein, and
(b) one or more guide RNAs (gRNAs), comprising a first and a second guide RNA, each having complementarity with a target ribonucleotide sequence of one or more plant virus RNA molecules, and each capable of forming a complex with the Type VI CRISPR effector protein and hybridizing to the target ribonucleotide sequence in said plant,
wherein the ribonucleotide sequence of the first guide RNAs does not comprise a ribonucleotide sequence of a first plant virus chosen from Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), Cauliflower mosaic virus (CaMV) (RT virus), Plum pox virus (PPV), Brome mosaic virus (BMV) and Potato virus X (PVX),
wherein the ribonucleotide sequence of the second guide RNA comprises a ribonucleotide sequence of a second plant virus chosen from Citrus tristeza virus (CTV), Barley yellow dwarf virus (BYDV), Potato leafroll virus (PLRV), Tomato bushy stunt virus (TBSV), rice tungro spherical virus (RTSV), rice yellow mottle virus (RYMV), rice hoja blanca virus (RHBV), maize rayado fino virus (MRFV), maize dwarf mosaic virus (MDMV), sugarcane mosaic virus (SCMV), Sweet potato feathery mottle virus (SPFMV), sweet potato sunken vein closterovirus (SPSVV), Arabis mosaic virus (ArMV), and Rupestris stem pitting-associated virus (RSPaV),
wherein the polynucleotide encoding the Type VI CRISPR effector protein or gRNA, or both, is stably integrated into the plant's genome,
wherein the Type VI CRISPR effector protein is chosen from Leptotrichia wadei F0279, Lachnospiraceae bacterium MA2020, or Listeriaceae bacterium FSL M6-0635, and
wherein the first and second plant viruses are different.