IP Library › Granted Patent US 12,404,514
Granted Patent B2
US 12,404,514 · App. 16/468,234 · Granted Sep 2, 2025

CRISPR-systems for modifying a trait of interest in a plant

Inventors: Omar Abudayyeh (Cambridge, MA); Joseph John Belanto (Minneapolis, MN); Jonathan Gootenberg (Cambridge, MA); Colby Garret Starker (Minneapolis, MN); Daniel Francis Voytas (Minneapolis, MN); Feng Zhang (Cambridge, MA)
Assignees: THE BROAD INSTITUTE, INC.; MASSACHUSETTS INSTITUTE OF TECHNOLOGY; PRESIDENT AND FELLOWS OF HARVARD COLLEGE
C12N15/8213C12N9/22C12N15/8283C12N2310/20
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Quick Facts
Patent No.
US 12,404,514
App. No.
16/468,234
Granted
Sep 2, 2025
Kind
B2
Abstract

The present invention generally relates to plants comprising a CRISPR system or parts of a CRISPR system, compositions, containers, polynucleotide, vectors, delivery systems, parts of plants, methods for production, CRISPR systems, and components thereof. Further aspects of the invention include a method for identifying a CRISPR system which is functional in a plant cell and a method for improving a CRISPR system in a plant.

Claims (33)

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.

2. The plant of claim 1 , wherein the one or more guide RNAs further comprise a third guide RNA, and wherein the ribonucleotide sequence of the third guide RNA comprises a ribonucleotide sequence of a third plant virus.

3. The plant of claim 1 , wherein the one or more guide RNAs (gRNAs) in a complex with the Type VI CRISPR can hybridize to the target ribonucleotide sequence of a plant virus RNA molecule which triggers:

(i) cleavage of the target ribonucleotide sequence,

(ii) an increase in translation of the target RNA molecule,

(iii) a reduction in translation of the target RNA molecule, or

(iv) a change in splicing of the target RNA molecule.

4. The plant of claim 1 , wherein the target ribonucleotide sequence of the one or more guide RNAs comprises one or more target ribonucleotide sequences of:

(i) an mRNA molecule of a pathogen susceptibility gene;

(ii) an mRNA of a herbicide resistance gene; or

(iii) an mRNA of an enzyme required for lignin or volatile organic compound (VOC) biosynthesis.

5. The plant of claim 1 , wherein said Type VI CRISPR effector protein cleaves the target ribonucleotide sequence of the first and second plant viruses if the viruses infect or have infected said plant.

6. The plant of claim 1 , wherein said Type VI CRISPR effector protein is expressed in said plant from an inducible promoter.

7. The plant of claim 1 , wherein the one or more guide RNAs are not expressed from a DNA sequence in said plant.

8. The plant of claim 4 , wherein the pathogen susceptibility gene is chosen from translation initiation like factors elF4E and elF(iso)4E, Mildew-resistance locus (MLO) proteins, ERF transcription factor gene OSERF922, alcohol dehydrogenase and polyphenol oxidase (PPO).

9. The plant of claim 1 , wherein the plant is selected from the group consisting of Oryza sativa, Solanum tuberosum, Solanum lycopersicum, Zea mays, Triticum spp., Triticum aestivum, Sorghum bicolor, Dioscorea spp., Musa spp., Manihot esculenta, Glycine max, Gossypium hirsutum, Hordeum vulgare, Avena sativa, Secale cereale, Brassica rapa and Brassica napus.

10. The plant of claim 1 , wherein said plant is a cereal plant, a pseudocereal plant, or a vegetable plant.

11. The plant of claim 4 , wherein the enzyme required for lignin biosynthesis is chosen from 4-coumarate 3-hydroxylase (C3H), phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), hydroxycinnamoyl transferase (HCT), caffeic acid O-methyltransferase (COMT), caffeoyl COA 3-O-methyltransferase (CCOAOMT), ferulate 5-hydroxylase (F5H), cinnamyl alcohol dehydrogenase (CAD), cinnamoyl CoA-reductase (CCR), 4-coumarate-CoA ligase (4CL), monolignol-lignin-specific glycosyl-transferase, and aldehyde dehydrogenase (ALDH), and the enzyme required for herbicide resistance is chosen from Resistance to Phytophora infestans (RPi) genes, 5-enolpyruvylshikimate-3-phosphate synthase, acetolactate synthase (ALS) and 15-cis-phytoene desaturase chloroplastic/chromoplastic, and wherein the gene required for volatile organic compound (VOC) biosynthesis is chosen from patchoulol synthase, linalool/nerolidol synthase and E-(b) caryophyllene synthase.

12. A plant part of the plant of claim 1 , wherein the plant part comprises the Type VI CRISPR effector protein and a nucleotide sequence encoding the Type VI CRISPR effector protein.

13. The plant part of claim 12 , wherein said plant part is chosen from the group consisting of a plant cell, a somatic embryo, a pollen, gametophyte, ovule, a leaveleaf, a seedling, a stem, a callus, a stolon, a microtuber, a shoot, a seed, a fruit and a spore.

14. A composition comprising at least two plant parts of claim 13 .

15. A packaging comprising the plant of claim 1 .

16. The plant of claim 4 , wherein the pathogen is chosen from Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), the RT virus Cauliflower mosaic virus (CaMV), Plum pox virus (PPV), Brome mosaic virus (BMV), Potato virus X (PVX), 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), Grapevine fanleaf virus (GFLV), Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine fleck virus (GFkV), Grapevine leafroll-associated virus-1, -2, and -3, (GLRaV-1, -2, and -3), Arabis mosaic virus (ArMV), or Rupestris stem pitting-associated virus (RSPaV).

17. The plant of claim 4 , wherein the one or more guide RNAs are capable of binding to a same or different target RNA molecule.

18. The plant of claim 10 , wherein said plant is a rice plant.

19. The plant of claim 18 , wherein said rice plant is Oryza sativa.

Assignments (4)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 25, 2019
From: BELANTO, JOSEPH JOHN
To: REGENTS OF THE UNIVERSITY OF MINNESOTA
Reel/Frame 049582/0503 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 25, 2019
From: ZHANG, FENG
To: THE BROAD INSTITUTE, INC.; MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 049583/0220 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 25, 2019
From: ABUDAYYEH, OMAR
To: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 049583/0477 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 25, 2019
From: GOOTENBERG, JONATHAN
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 049583/0530 →
Continuity (3)
Provisional Application 62432543 · Dec 9, 2016
Provisional Application 62567959 · Oct 4, 2017
Related Publication 20190352652A1 · Nov 21, 2019
References Cited (211)
US 4186183A · Steck et al. · 1980 [cited by applicant]
US 4217344A · Vanlerberghe et al. · 1980 [cited by applicant]
US 4235871A · Papahadjopoulos et al. · 1980 [cited by applicant]
US 4261975A · Fullerton et al. · 1981 [cited by applicant]
US 4485054A · Mezei et al. · 1984 [cited by applicant]
US 4501728A · Geho et al. · 1985 [cited by applicant]
US 4774085A · Fidler · 1988 [cited by applicant]
US 4837028A · Allen · 1989 [cited by applicant]
US 4897355A · Eppstein et al. · 1990 [cited by applicant]
US 4946787A · Eppstein et al. · 1990 [cited by applicant]
US 5049386A · Eppstein et al. · 1991 [cited by applicant]
US 5210015A · Gelfand et al. · 1993 [cited by applicant]
US 5445934A · Fodor et al. · 1995 [cited by applicant]
US 5543158A · Gref et al. · 1996 [cited by applicant]
US 5563055A · Townsend et al. · 1996 [cited by applicant]
US 5855913A · Hanes et al. · 1999 [cited by applicant]
US 5985309A · Edwards et al. · 1999 [cited by applicant]
US 6007845A · Domb et al. · 1999 [cited by applicant]
US 6603061B1 · Armstrong et al. · 2003 [cited by applicant]
US 7776321B2 · Cascalho et al. · 2010 [cited by applicant]
US 7868149B2 · Boukharov et al. · 2011 [cited by applicant]
US 8044019B2 · Uno et al. · 2011 [cited by applicant]
US 8372951B2 · Chang et al. · 2013 [cited by applicant]
US 8575305B2 · Gait et al. · 2013 [cited by applicant]
US 8614194B1 · Chen et al. · 2013 [cited by applicant]
US 8697359B1 · Zhang · 2014 [cited by applicant]
US 8709843B2 · Shakuda · 2014 [cited by applicant]
US 8771945B1 · Zhang · 2014 [cited by applicant]
US 8795965B2 · Zhang · 2014 [cited by applicant]
US 8865406B2 · Zhang et al. · 2014 [cited by applicant]
US 8871445B2 · Cong et al. · 2014 [cited by applicant]
US 8889356B2 · Zhang · 2014 [cited by applicant]
US 8889418B2 · Zhang et al. · 2014 [cited by applicant]
US 8895308B1 · Zhang et al. · 2014 [cited by applicant]
US 8906616B2 · Zhang et al. · 2014 [cited by applicant]
US 8932814B2 · Cong et al. · 2015 [cited by applicant]
US 8945839B2 · Zhang · 2015 [cited by applicant]
US 8993233B2 · Zhang et al. · 2015 [cited by applicant]
US 8999641B2 · Zhang et al. · 2015 [cited by applicant]
US 20040142476A1 · Evans et al. · 2004 [cited by applicant]
US 20040171156A1 · Hartley et al. · 2004 [cited by applicant]
US 20090100536A1 · Adams et al. · 2009 [cited by applicant]
US 20110027239A1 · Paek et al. · 2011 [cited by applicant]
US 20110265198A1 · Gregory et al. · 2011 [cited by applicant]
US 20120017290A1 · Cui et al. · 2012 [cited by applicant]
US 20130185823A1 · Kuang et al. · 2013 [cited by applicant]
US 20130236946A1 · Gouble · 2013 [cited by applicant]
US 20140170753A1 · Zhang · 2014 [cited by applicant]
US 20140179006A1 · Zhang · 2014 [cited by applicant]
US 20140179770A1 · Zhang et al. · 2014 [cited by applicant]
US 20140186843A1 · Zhang et al. · 2014 [cited by applicant]
US 20140186919A1 · Zhang et al. · 2014 [cited by applicant]
US 20140186958A1 · Zhang et al. · 2014 [cited by applicant]
US 20140189896A1 · Zhang et al. · 2014 [cited by applicant]
US 20140227787A1 · Zhang · 2014 [cited by applicant]
US 20140234972A1 · Zhang · 2014 [cited by applicant]
US 20140242664A1 · Zhang et al. · 2014 [cited by applicant]
US 20140242699A1 · Zhang · 2014 [cited by applicant]
US 20140242700A1 · Zhang et al. · 2014 [cited by applicant]
US 20140248702A1 · Zhang et al. · 2014 [cited by applicant]
US 20140256046A1 · Zhang et al. · 2014 [cited by applicant]
US 20140273231A1 · Zhang et al. · 2014 [cited by applicant]
US 20140273232A1 · Zhang et al. · 2014 [cited by applicant]
US 20140273234A1 · Zhang et al. · 2014 [cited by applicant]
US 20140273235A1 · Voytas et al. · 2014 [cited by applicant]
US 20140287938A1 · Zhang et al. · 2014 [cited by applicant]
US 20140310830A1 · Zhang et al. · 2014 [cited by applicant]
US 20140335620A1 · Zhang et al. · 2014 [cited by applicant]
US 20150291966A1 · Zhang et al. · 2015 [cited by applicant]
US 20160208243A1 · Zhang · 2016 [cited by examiner]
US 20170211142A1 · Smargon et al. · 2017 [cited by applicant]
EP 2764103B1 · 2014 [cited by applicant]
EP 2771468B1 · 2014 [cited by applicant]
EP 2784162A1 · 2014 [cited by applicant]
WO 9116024A1 · 1991 [cited by applicant]
WO 9117424A1 · 1991 [cited by applicant]
WO 2008042156A1 · 2008 [cited by applicant]
WO 2011028929A3 · 2011 [cited by applicant]
WO 2014018423A2 · 2014 [cited by applicant]
WO 2014093595A1 · 2014 [cited by applicant]
WO 2014093622A2 · 2014 [cited by applicant]
WO 2014093635A1 · 2014 [cited by applicant]
WO 2014093655A2 · 2014 [cited by applicant]
WO 2014093661A2 · 2014 [cited by applicant]
WO 2014093694A1 · 2014 [cited by applicant]
WO 2014093701A1 · 2014 [cited by applicant]
WO 2014093709A1 · 2014 [cited by applicant]
WO 2014093712A1 · 2014 [cited by applicant]
WO 2014093718A1 · 2014 [cited by applicant]
WO 2014204723A1 · 2014 [cited by applicant]
WO 2014204724A1 · 2014 [cited by applicant]
WO 2014204725A1 · 2014 [cited by applicant]
WO 2014204726A1 · 2014 [cited by applicant]
WO 2014204727A1 · 2014 [cited by applicant]
WO 2014204728A1 · 2014 [cited by applicant]
WO 2014204729A1 · 2014 [cited by applicant]
WO 2015065964A1 · 2015 [cited by applicant]
WO 2015089419A2 · 2015 [cited by applicant]
WO 2015189693A1 · 2015 [cited by applicant]
WO WO2016205764A1 · 2016 [cited by examiner]
WO 2017070605A1 · 2017 [cited by applicant]
WO 2017219027A1 · 2017 [cited by applicant]
WO 2018035250A1 · 2018 [cited by applicant]
WO WO2018107103A1 · 2018 [cited by examiner]
WO 2019005866A1 · 2019 [cited by applicant]
Xie et al (RNA-Guided Genome Editing in Plants Using a CRISPR-Cas System. Molecular Plant 6:1975-1983, 2013) (Year: 2013). [cited by examiner]
Yamano et al (Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA. Cell. 165: 949-962, May 2016). (Year: 2016). [cited by examiner]
Zetsche et al (Cpf1 is a single RNA-guided endonuclease of a Class 2 CRISPR-Cas system. Cell. 163(3): 759-771, published Oct. 2015). (Year: 2015). [cited by examiner]
Endo et al (Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida. Nature Scientific Report. Published Dec. 1, 2016). (Year: 2016). [cited by examiner]
Li et al (Cas9-Guide RNA Directed Genome Editing in Soybean. Plant Physiology, 169, pp. 960-970, 2015) (Year: 2015). [cited by examiner]
Abudayyeh et al (C2c2 is a single-component programmable RNA-guided RNA targeting CRISPR effector. Science. 535: 1-23, published Aug. 2016) (Year: 2016). [cited by examiner]
East-Seletsky et al (Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature. 538: 270-275, Oct. 2016). (Year: 2016). [cited by examiner]
Sanfacon (Plant Translation Factors and Virus Resistance. Viruses, 7, 3392-3419, 2015) (Year: 2015). [cited by examiner]
Collins et al (Self-Cleaving Circular RNA Associated with Rice Yellow Mottle Virus Is the Smallest Viroid-like RNA. Virology 241, 269-275, 1998) (Year: 1998). [cited by examiner]
Abudayyeh (C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science. 1-23, 2016) (Year: 2016). [cited by examiner]
International Preliminary Report on Patentability issued in International Application No. PCT/US2017/065438, mailed on Jun. 20, 2019, 10 pages. [cited by applicant]
Abil, et al., “Engineering Reprogrammable RNA-Binding Proteins for Study and Manipulation of the Transcriptome”, Molecular BioSystems, The Royal Society of Chemistry, vol. 11, No. 10, Jul. 6, 2015, 2658-2665. [cited by applicant]
Abudayyeh, et al., “C2c2 is a Single-Component Programmable RNA-Guided RNA-Targeting CRISPR Effector”, Science, vol. 353, No. 6299, Aug. 5, 2016, 11 pages. [cited by applicant]
Belhaj, et al., “Plant Genome Editing Made Easy: Targeted Mutagenesis in Model and Crop Plants Using the CRISPR/Cas System”, Plant Methods, vol. 9, No. 39, Oct. 11, 2013, 1859-1872. [cited by applicant]
Bocobza, et al., “Small Molecules that Interact with RNA: Riboswitch-Based Gene Control and its Involvement in Metabolic Regulation in Plants and Algae”, The Plant Journal, vol. 79, No. 4, 2014, 693-703. [cited by applicant]
Brooks, et al., “Efficient Gene Editing in Tomato in the First Generation Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated9 System”, Plant Physiology, vol. 166, No. 3, Nov. 2014, 129… [cited by applicant]
Caliando, et al., “Targeted DNA Degradation using a CRISPR Device Stably Carried in the Host Genome”, Nature Communications, vol. 6, No. 6989, May 19, 2015, 10 pages. [cited by applicant]
Canver, et al., “BCL11A Enhancer Dissection by Cas9-Mediated In Situ Saturating Mutagenesis”, Nature, vol. 527, Nov. 12, 2015, 192-197. [cited by applicant]
Cheah, et al., “Control of Alternative RNA Splicing and Gene Expression by Eukaryotic Riboswitches”, Nature, vol. 447, No. 7143, May 24, 2007, 497-500. [cited by applicant]
Chen, et al., “A Highly Efficient Transient Protoplast System for Analyzing Defence Gene Expression and Protein-Protein Interactions in Rice”, Molecular Plant Pathology, vol. 7, No. 5, Sep. 2006, 417-427. [cited by applicant]
Chen, et al., “Genome-Wide CRISPR Screen in a Mouse Model of Tumor Growth and Metastasis”, Cell, vol. 160, No. 6, Mar. 12, 2015, 1246-1260. [cited by applicant]
Chen, et al., “Predicting Peptide-Mediated Interactions on a Genome-Wide Scale”, PLOS Computational Biology, vol. 11, No. 5, May 4, 2015, 13 pages. [cited by applicant]
Chen, et al., “RNA Imaging. Spatially Resolved, Highly Multiplexed RNA Profiling in Single Cells”, Science, vol. 348, No. 6233, Apr. 24, 2015, 14 pages. [cited by applicant]
Cong, et al., “Multiplex Genome Engineering Using CRISPR/Cas Systems”, Science, vol. 339, No. 6121, Feb. 15, 2013, 819-823. [cited by applicant]
CRISPR-Plant, “A Portal of CRISPR-Cas9 Mediated Genome Editing”, CRISPR-Plant is supported by Penn State and AGI, Oct. 15, 2010, 2 pages. [cited by applicant]
Curtin, et al., “A Genome Engineering Toolbox for Legume Functional Genomics”, International Plant and Animal Genome Conference XXII 2014, 2014. [cited by applicant]
Dey, et al., “Toward a “Structural BLAST”: Using Structural Relationships to Infer Function”, Protein Science, vol. 22, No. 4, Apr. 2013, 359-366. [cited by applicant]
Doench, et al., “Rational Design of Highly Active SgRNAs for CRISPR-Cas9-Mediated Gene Inactivation”, Nature Biotechnology, vol. 32, No. 12, Dec. 2014, 1262-1267. [cited by applicant]
Dudareva, et al., “Biosynthesis, Function and Metabolic Engineering of Plant Volatile Organic Compounds”, New Phytologist, vol. 198, No. 1, Apr. 2013, 16-32. [cited by applicant]
Feng, et al., “Efficient Genome Editing in Plants using a CRISPR/Cas System”, Cell Research, vol. 23, Aug. 20, 2013, 1229-1232. [cited by applicant]
Fu, et al., “Gene Expression Regulation Mediated Through Reversible M6A RNA Methylation”, Nature Reviews Genetics, vol. 15, No. 5, May 2014, 293-306. [cited by applicant]
Gao, et al., “Engineered Cpf1 Enzymes with Altered PAM Specificities”, BioRxiv, Dec. 4, 2016, 17 pages. [cited by applicant]
Gerbasch, et al., “Pulling the Strings of Our Genetic Puppetmasters: Engineers Gain Control of Gene Activity”, Nature Biotechnology, Apr. 6, 2015. [cited by applicant]
Goldfless, et al., “Direct and Specific Chemical Control of Eukaryotic Translation with a Synthetic RNA-Protein Interaction”, Nucleic Acids Research, vol. 40, No. 9, 2012, 1-12. [cited by applicant]
Green, “Current State of Herbicides in Herbicide-Resistant Crops”, Society of Chemical Industry, Pest Management Science, vol. 70, No. 9, Jan. 20, 2014, 1351-1357. [cited by applicant]
Grennan, “To Thy Proteins Be True: RNA Editing in Plants”, Plant Physiology, vol. 156, No. 2, Jun. 2011, 2 pages. [cited by applicant]
Guidi, et al., “Application of RNAi to Genomic Drug Target Validation in Schistosomes”, PLOS Neglected Tropical Diseases, University of Cambridge, United Kingdom, vol. 9, No. 5, May 20, 2015, 22 pages. [cited by applicant]
Hebelstrup, et al., “The Future of Starch Bioengineering: GM Microorganisms or GM Plants?”, Frontiers in Plant Science, vol. 6, Article 247, Apr. 23, 2015, 6 pages. [cited by applicant]
Hlavova, et al., “Improving Microalgae for Biotechnology—From Genetics to Synthetic Biology”, Biotechnology Advances, vol. 33, Issue 6, Part 2, Nov. 2015, 1194-1203. [cited by applicant]
Hsu, et al., “Development and Applications of CRISPR-Cas9 for Genome Engineering”, Cell, vol. 157, No. 6, Jun. 5, 2014, 1262-1278. [cited by applicant]
Hsu, et al., “DNA Targeting Specificity of RNA-Guided Cas9 Nucleases”, Nature Biotechnology, vol. 31, No. 9, Sep. 2013, 827-832. [cited by applicant]
Jain, et al., “Validation of Housekeeping Genes as Internal Control for Studying Gene Expression in Rice by Quantitative Real-Time PCR”, Biochemical and Biophysical Research Communications, vol. 345, No. 2, Jun. 30, 200… [cited by applicant]
Jiang, et al., “RNA-Guided Editing of Bacterial Genomes Using CRISPR-Cas Systems”, Nature Biotechnology, vol. 31, No. 3, Mar. 2013, 233-239. [cited by applicant]
Joung, et al., “Genome-Scale Crispr-Cas9 Knockout and Transcriptional Activation Screening”, Nature Protocols, vol. 12, No. 4, Apr. 2017, 828-863. [cited by applicant]
Kabadi, et al., “Multiplex CRISPR/Cas9-based Genome Engineering from a Single Lenttiviral Vector”, Nucleic Acids Research, vol. 42, No. 19, Aug. 13, 2014, 11 pages. [cited by applicant]
Kawai, et al., “Transformation of [cited by applicant]
Kim, et al., “RNA Interference: Applications and Advances in Insect Toxicology and Insect Pest Management”, Pesticide Biochemistry and Physiology, vol. 120, 2015, 109-117. [cited by applicant]
Konermann, et al., “Genome-Scale Transcriptional Activation by an Engineered CRISPR-Cas9 Complex”, Nature, vol. 517, No. 7536, Jan. 29, 2015, 583-588. [cited by applicant]
Konermann, et al., “Optical Control of Mammalian Endogenous Transcription and Epigenetic States”, Nature, vol. 500, No. 7463, Aug. 22, 2013, 472-476. [cited by applicant]
Kurth, et al., “Virus-Derived Gene Expression and RNA Interference Vector for Grapevine”, Journal of Virology, vol. 86, No. 11, Jun. 2012, 6002-6009. [cited by applicant]
Lowder, et al., “A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation”, Plant Physiology, vol. 169., Oct. 2015, 15 pages. [cited by applicant]
Ma, et al., “A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants”, Molecular Plant, vol. 8, No. 8, Aug. 3, 2015, 1274-1284. [cited by applicant]
MacKay, et al., “The Prospects for Designer Single-Stranded RNA-Binding Proteins”, Nature Structural & Molecular Biology, vol. 18, No. 3, Mar. 2011, 256-261. [cited by applicant]
Makkonen, et al., “Baculovirus-Mediated Gene Delivery and RNAi Applications”, Viruses, vol. 7, No. 4, 2015, 2099-2125. [cited by applicant]
Miyamura, et al., “Mutations of the RNA-Specific Adenosine Deaminase Gene (DSRAD) are Involved in Dyschromatosis Symmetrica Hereditaria”, American Journal of Human Genetics, vol. 73, 2003, 693-699. [cited by applicant]
Morrell, et al., “Crop Genomics: Advances and Applications”, Nature Reviews Genetics, vol. 13, Feb. 2012, 85-96. [cited by applicant]
Murray, et al., “Suppressors of RNAi from Plant Viruses are Subject to Episodic Positive Selection”, Proceedings of the Royal Society B, vol. 280, No. 1765, Jun. 2013, 1-9. [cited by applicant]
Nelles, et al., “Applications of Cas9 as an RNA-Programmed RNA-Binding Protein”, Bioessays, vol. 37, 2015, 1-8. [cited by applicant]
Nishimasu, et al., “Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA”, Cell, vol. 156, No. 5, Feb. 27, 2014, 935-949. [cited by applicant]
Platt, et al., “CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modelling”, Cell, vol. 159, No. 2, Oct. 9, 2014, 440-455. [cited by applicant]
Zhou, et al., “RNase Z(S1) Processes UbL40 mRNAs and Controls Thermosensitive Genic Male Sterility in Rice”, Nature Communications, vol. 5, No. 4884, Sep. 11, 2014, 9 pages. [cited by applicant]
Nishimasu, et al., “Crystal Structure of [cited by applicant]
Osakabe, et al., “Optimization of CRISPR/Cas9 Genome Editing to Modify Abiotic Stress Responses in Plants”, Scientific Reports, vol. 6, No. 26685, May 26, 2016, 10 pages. [cited by applicant]
Parnas, et al., “A Genome-Wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks”, Cell, vol. 162, No. 3, Jul. 30, 2015, 675-686. [cited by applicant]
Petersen, et al., “Towards precisely glyco engineered plants”, Plant Biotech Denmark Annual Meeting, Jan. 28-29, 2015, 6 pages. [cited by applicant]
Price, et al., “Cas9-Mediated Targeting of Viral RNA in Eukaryotic Cells”, Proceedings of the National Academy of Sciences , vol. 112, No. 19, May 12, 2015, 6164-6169. [cited by applicant]
Ramakrishna, et al., “Gene Disruption by Cell-Penetrating Peptide-Mediated Delivery of Cas9 Protein and Guide RNA”, Genome Research, vol. 24, No. 6, Jun. 2014, 1020-1027. [cited by applicant]
Ramanan, et al., “CRISPR/Cas9 Cleavage of Viral DNA Efficiently Suppresses Hepatitis B Virus”, Scientific Reports, vol. 5, No. 10833, Jun. 2, 2015, 9 pages. [cited by applicant]
Ran, et al., “Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity”, Cell, vol. 154, No. 6, Sep. 12, 2013, 1380-1389. [cited by applicant]
Ran, et al., “Genome Engineering Using the CRISPR-Cas9 System”, Nature Protocols, vol. 8, No. 11, Nov. 2013, 2281-2308. [cited by applicant]
Ran, et al., “In Vivo Genome Editing using [cited by applicant]
Sainsbury, et al., “pEAQ: Versatile Expression Vectors for Easy and Quick Transient Expression of Heterologous Proteins in Plants”, Plant Biotechnology Journal, vol. 7, No. 7, 2009, 682-693. [cited by applicant]
Samai, et al., “Co-Transcriptional DNA and RNA Cleavage During Type III CRISPR-Cas Immunity”, Cell, vol. 161, No. 5, May 21, 2015, 1164-1174. [cited by applicant]
Sapranauskas, et al., “The [cited by applicant]
Shalem, et al., “Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells”, Science, vol. 343, No. 6166, Jan. 3, 2014, 84-87. [cited by applicant]
Shalem, et al., “High-Throughput Functional Genomics Using CRISPR-Cas9”, Nature Reviews Genetics, vol. 16, No. 5, May 2015, 299-311. [cited by applicant]
Shan, et al., “Targeted Genome Modification of Crop Plants Using a CRISPR-Cas System”, Nature biotechnology, vol. 31, No. 8, Aug. 2013, 686-688. [cited by applicant]
Sharma, et al., “RNA Interference: A Novel Tool for Plant Disease Management”, African Journal of Biotechnology, Academic Journals, vol. 12, No. 18, May 1, 2013, 2303-2312. [cited by applicant]
Shmakov, et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems”, Molecular Cell, vol. 60, No. 3, Nov. 5, 2015, 385-397. [cited by applicant]
Smargon, et al., “Casl3B is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28”, Molecular Cell, vol. 65, No. 4, Feb. 16, 2017, 618-630. [cited by applicant]
Stovicek, et al., “CRISPR-Cas System Enables Fast and Simple Genome Editing of Industrial [cited by applicant]
Sugano, et al., “CRISPR/Cas9-Mediated Targeted Mutagenesis in the [cited by applicant]
Swiech, et al., “In Vivo Interrogation of Gene Function in the Mammalian Brain Using CRISPR-Cas9”, Nature Biotechnology, vol. 33, No. 1, Jan. 2015, 102-106. [cited by applicant]
Tsai, et al., “Dimeric CRISPR RNA-Guided Fokl Nucleases for Highly Specific Genome Editing”, Nature Biotechnology, vol. 32, No. 6, Jun. 2014, 569-577. [cited by applicant]
Wang, et al., “Enhanced Rice Blast Resistance by CRISPR/Cas9-Targeted Mutagenesis of the ERF Transcription Factor Gene OsERF922”, PLoS One, vol. 11, No. 4, Apr. 26, 2016, 18 pages. [cited by applicant]
Wang, et al., “Genetic Screens in Human Cells Using the CRISPR/Cas9 System”, Science, vol. 343, No. 6166, Jan. 3, 2014, 80-84. [cited by applicant]
Wang, et al., “One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering”, Cell, vol. 153, No. 4, May 9, 2013, 910-918. [cited by applicant]
Witek, et al., “Accelerated Cloning of a Potato Late Blight-Resistance Gene Using RenSeq and SMRT Sequencing”, Nature Biotechnology, vol. 34, No. 6, Jun. 2016, 656-660. [cited by applicant]
Woo, et al., “DNA-Free Genome Editing in Plants with Preassembled CRISPR-Cas9 Ribonucleoproteins”, Nature Biotechnology, vol. 33, No. 11, Nov. 2015, 1162-1164. [cited by applicant]
Wroblewska, et al., “Mammalian Synthetic Circuits with RNA Binding Proteins for RNA-Only Delivery”, Nature Biotechnology, vol. 33, No. 8, Aug. 2015, 839-841. [cited by applicant]
Wu, et al., “Genome-Wide Binding of the CRISPR Endonuclease Cas9 in Mammalian Cells”, Nature Biotechnology, vol. 32, No. 7, Jul. 2014, 670-676. [cited by applicant]
Xie, et al., “RNA-Guided Genome Editing in Plants Using a CRISPR-Cas System”, Molecular Plant, vol. 6, No. 6, Nov. 2013, 1975-1983. [cited by applicant]
Xing, et al., “A CRISPR/Cas9 Toolkit for Multiplex Genome Editing in Plants”, BMC Plant Biology, vol. 14, No. 327, 2014, 12 pages. [cited by applicant]
Xu, et al., “Gene Targeting Using the Agrobacterium Tumefaciens-Mediated CRISPR-Cas System in Rice”, Rice, vol. 7, No. 5, 2014, 4 pages. [cited by applicant]
Xu, et al., “Sequence Determinants of Improved CRISPR SgRNA Design”, Genome Research, vol. 25, No. 8, Aug. 2015, 1147-1157. [cited by applicant]
Zahir, et al., “CRISPR/Cas9-Mediated Viral Interference in Plants”, Genome Biology, vol. 16, No. 238, Nov. 11, 2015, 11 pages. [cited by applicant]
Zetsche, et al., “A Split-Cas9 Architecture for Inducible Genome Editing and Transcription Modulation”, Nature Biotechnology, vol. 33, No. 2, Feb. 2015, 139-142. [cited by applicant]
Zetsche, et al., “Cpf1 is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System”, Cell, vol. 163, No. 3, Oct. 22, 2015, 759-771. [cited by applicant]
Zhang, et al., “A Highly Efficient Rice Green Tissue Protoplast System for Transient Gene Expression and Studying Light/Chloroplast-Related Processes”, Plant Methods, vol. 7, No. 30, 2011. [cited by applicant]
Zhang, et al., “Structure-Based Prediction of Protein-Protein Interactions on a Genome-Wide Scale”, Nature, vol. 490, Oct. 25, 2012, 556-560. [cited by applicant]
Zhou, et al., “Exploiting SNPs for Biallelic CRISPR Mutations in the Outcrossing Woody Perennial Populus Reveals 4-coumarate:CoA ligase Specificity and Redundancy”, New Phytologist, vol. 208, Oct. 2015, 298-301. [cited by applicant]
Crotty, et al., “In Vivo RNAi Screens: Concepts and Applications”. Trends in Immunology, vol. 36, No. 5, May 2015, 315-322. [cited by applicant]
“PCT International Search Report and Written Opinion issued in PCT/US2017/065438 mailed Feb. 12, 2018”, Feb. 12, 2018, 1-13. [cited by applicant]
Abudayyeh, et al. “RNA targeting with CRISPR-Cas13a,” Nature. Oct. 12, 2017; 550(7675): 280-284. [cited by applicant]
Aman, et al., “RNA virus interference via CRISPR/Cas13a system in plants,” Genome Biology (2018) 19:1, all enclosed pages cited. [cited by applicant]
Zaidi, et al., “Engineering Plant Immunity: Using CRISPR/Cas9 to Generate Virus Resistance,” Frontiers in Plant Science, Nov. 2016, vol. 7, Article 1673, all enclosed pages cited. [cited by applicant]