US 11,859,219 B1
Methods of altering a target nucleotide sequence with an RNA-guided nuclease and a single guide RNA
Yajie Niu, Lexington, MA (US); Kristine Yu, Cambridge, MA (US); Frank Anthony Skraly, Watertown, MA (US); Randall William Shultz, Acton, MA (US); John P. Casey, Jr., Boston, MA (US); and Brian Prescott Fiske, Cambridge, MA (US)
Assigned to Flagship Pioneering Innovations V, Inc., Cambridge, MA (US)
Filed by Flagship Pioneering Innovations V, Inc., Cambridge, MA (US)
Filed on Dec. 29, 2017, as Appl. No. 15/858,581.
Claims priority of provisional application 62/440,676, filed on Dec. 30, 2016.
Int. Cl. C12N 9/22 (2006.01); C12N 15/82 (2006.01); A01H 4/00 (2006.01)
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.