CPC C12N 9/22 (2013.01) [C12N 15/8202 (2013.01); A01H 4/008 (2013.01); C12N 2310/20 (2017.05)] | 19 Claims |
1. A method of altering a target nucleotide sequence in a population of protoplasts of a plant, comprising delivering a preassembled ribonucleoprotein composition into the population of protoplasts, wherein the preassembled ribonucleoprotein composition comprises:
(a) an RNA-guided nuclease protein, and
(b) a guide RNA (gRNA) that has a nucleotide sequence designed to alter a target nucleotide sequence in the population of protoplasts, and is provided as:
(i) a CRISPR RNA (crRNA) that comprises the gRNA, or
(ii) a single guide RNA (sgRNA) that comprises the gRNA;
wherein the RNA-guided nuclease protein of (a) is complexed with the gRNA of (b); and
wherein the delivering comprises contacting the population of protoplasts with the preassembled ribonucleoprotein composition and concurrently incubating the population of protoplasts at 37 degrees Celsius for a period of between 10 minutes to about 2 hours, thereby altering the target nucleotide sequence.
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