	US 12,146,135 B2
	Method for editing filamentous fungal genome through direct introduction of genome-editing protein
Kazuhiro Iwashita, Higashihiroshima (JP); Ken Oda, Higashihiroshima (JP); Kohei Shimamoto, Higashihiroshima (JP); and Yusaku Wada, Atsugi (JP)
Assigned to NATIONAL RESEARCH INSTITUTE OF BREWING, Higashihiroshima (JP); and FASMAC CO., LTD., Atsugi (JP)
Appl. No. 16/473,492
Filed by NATIONAL RESEARCH INSTITUTE OF BREWING, Higashihiroshima (JP); and FASMAC CO., LTD., Atsugi (JP)
PCT Filed Aug. 25, 2017, PCT No. PCT/JP2017/030501 § 371(c)(1), (2) Date Oct. 4, 2019, PCT Pub. No. WO2018/123134, PCT Pub. Date Jul. 5, 2018.
Claims priority of application No. 2016-250518 (JP), filed on Dec. 26, 2016.
Prior Publication US 2020/0040330 A1, Feb. 6, 2020
Int. Cl. C12N 15/11 (2006.01); C12N 9/22 (2006.01)

CPC C12N 15/11 (2013.01) [C12N 9/22 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01)]

13 Claims

1. A method of editing a gene in a koji fungal genome, the method comprising the step of:

directly introducing a genome editing protein complex for a target gene into a cell of a koji fungus,

wherein the genome editing protein complex is a complex containing a Cas9 protein and a guide RNA; complexes containing a nickase-modified Cas9 protein and each of a pair of guide RNAs; or a complex containing a nickase-modified or null mutant Cas9 protein to which a deaminase is linked and a guide RNA, and

wherein the genome editing protein complex containing not less than 10 μg of said Cas9 protein is added with respect to 10⁶koji fungal protoplasts in the direct introduction step.