# US 2020/0399647 A1 · Genome editing by pollen-mediated transformation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2020/0399647 A1 - Document ID: US20200399647A1 - Status: Published application - Kind: A1 - Application number: 16/769,829 - Filed: 2018-12-04 - Published: 2020-12-24 - Earliest claimed: 2017-12-04 - Assignees: University of Florida Research Foundation, Inc. - Applicants: University of Florida Research Foundation - Inventors: Marcio Fernando RESENDE, Jr.; Kelly Mayrink BALMANT; Kristen LEACH; Matias KIRST; Christopher DERVINIS - Web page: https://patentgrep.com/patent/US20200399647A1 ## CPC - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein} - C12N 15/8202: {by biological means, e.g. cell mediated or natural vector} - C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination} - C07K 2319/10: containing a tag for extracellular membrane crossing, e.g. TAT or VP22 - C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR] - C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract Disclosed herein are methods of genome-editing transformation that delivers to the cell the RNA-guided endonucleases as well as the required guide RNA, without the need of tissue culture to regenerate the plant. Thus, methods as described herein enable any plant species to have its genome edited in a process that is simple and efficient. This process utilizes genome-editing pollen cells, and delivering the required components for the editing by combining them with cell-penetrating peptides. ## Claims ### Claim 1 1) A method for modifying the genomic material in a plant, comprising: (a) providing a RNA-guided endonuclease and cell penetrating peptide (CPP) conjugate; (b) providing one or more guide RNA (gRNA) and CPP conjugates; (c) incubating mature pollen grains from a donor plant in a solution comprising the RNA-guided endonuclease and CPP conjugate and one or more guide RNA and CPP conjugates to form treated pollen grains; and (d) pollinating a maternal plant with the treated pollen grains to produce a plant with modified genomic material. ### Claim 2 (depends on 1) 2) The method of claim 1, wherein the RNA-guided endonuclease is a Cas9, a Cpf1, a C2c1, or a C2c2. ### Claim 3 (depends on 1) 3) The method of claim 1, wherein the CPP comprises one of SEQ ID NOs. 1-16 or a modified variant thereof. ### Claim 4 4) (canceled) ### Claim 5 (depends on 1) 5) The method of claim 1, wherein the gRNA comprises crRNA and a tracrRNA, or a chimeric cr/tracrRNA hybrid. ### Claim 6 (depends on 1) 6) The method of claim 1, wherein the donor plant is a monocotyledonous plant, food plant, or dicotyledonous plant. ### Claim 7 (depends on 1) 7) The method of claim 1, wherein the donor plant is a wheat, maize, rice, orchid, onion, aloe, true lily, grass, Setaria, woody shrub, tree, palm tree, bamboo, pineapple, sugar cane, tomato, cassava, soybean, tobacco, potato, Arabidopsis, rose, pansy, sunflower, grape, strawberry, squash, bean, pea, or peanut. ### Claim 8 (depends on 1) 8) The method of claim 1, wherein the donor plant is tobacco or maize. ### Claim at position 9 9-16) (canceled) ### Claim 17 (depends on 1) 17) The method of claim 1, wherein the solution further comprises a donor DNA sequence and CPP conjugate. ### Claim 18 (depends on 1) 18) The method of claim 1, further comprising, before step (a), incubating a RNA-guided endonuclease and cell penetrating peptide (CPP) for at least 30 minutes to form a RNA-guided endonuclease and CPP conjugate. ### Claim at position 12 19-20) (canceled) ### Claim 21 (depends on 18) 21) The method of claim 18, further comprising, before steps (a) or (b), incubating one or more guide RNA (gRNA) and cell penetrating peptide (CPP) for at least 30 minutes to form one or more gRNA and CPP conjugates. ### Claim at position 14 22-23) (canceled) ### Claim 24 (depends on 1) 24) The method of claim 1, further comprising electroporating, sonicating, or both, the mature pollen grains in solution during step (c), after step (c), or both. ### Claim 25 (depends on 1) 25) The method of claim 1, further comprising storing the treated pollen after step (c). ### Claim 26 (depends on 1) 26) The method of claim 1, further comprising screening the treated pollen for double strand breaks after step (c). ### Claim 27 (depends on 1) 27) The method of claim 1, further comprising screening the genotype of the plant with modified genomic material, phenotype of the plant with modified genomic material, or both, after step (d). ### Claim 28 28) A kit, comprising: one or more mature pollen grains; a RNA-guided endonuclease; one or more gRNA; and a CPP. ### Claim 29 (depends on 28) 29) The kit of claim 28 wherein the RNA-guided endonuclease is a Cas9, a Cpf1, a C2c1, or a C2c2. ### Claim 30 (depends on 28) 30) The kit of claim 28, wherein the CPP comprises one of SEQ ID NOs. 1-16 or a modified variant thereof. ### Claim 31 31) (canceled) ### Claim 32 (depends on 28) 32) The kit of any claim 28, wherein the gRNA comprises crRNA and a tracrRNA, or a chimeric cr/tracrRNA hybrid. ### Claim 33 (depends on 28) 33) The kit of claim 28, wherein one or more mature pollen grains are from a monocotyledonous plant, food plant, or dicotyledonous plant. ### Claim 34 34) (canceled) ### Claim 35 (depends on 28) 35) The kit of claim 28, wherein one or more mature pollen grains are tobacco or maize. ### Claim at position 27 36-39) (canceled)