US 11,939,605 B2
Thermostable CAS9 nucleases
John Van Der Oost, Renkum (NL); Richard Van Kranenburg, Gorinchem (NL); Elleke Fenna Bosma, Denmark (NL); Ioannis Mougiakos, Wageningen (NL); and Prarthana Mohanraju, Wageningen (NL)
Assigned to Wageningen Universiteit, Wageningen (NL); and Stichting Voor De Technische Wetenschappen, Utrecht (NL)
Filed by Wageningen Universiteit, Wageningen (NL); and Stichting Voor De Technische Wetenschappen, Utrecht (NL)
Filed on Jan. 14, 2022, as Appl. No. 17/575,952.
Application 17/575,952 is a continuation of application No. 16/469,677, granted, now 11,326,162, previously published as PCT/EP2017/070796, filed on Aug. 16, 2017.
Claims priority of application No. PCT/EP2017/081077 (WO), filed on Dec. 14, 2016.
Prior Publication US 2022/0213455 A1, Jul. 7, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/22 (2006.01); C12N 15/10 (2006.01); C12N 15/11 (2006.01); C12N 15/113 (2010.01); C12N 15/63 (2006.01); C12N 15/90 (2006.01)
CPC C12N 9/22 (2013.01) [C12N 15/102 (2013.01); C12N 15/11 (2013.01); C12N 15/113 (2013.01); C12N 15/902 (2013.01); C12N 15/907 (2013.01); C12N 2310/20 (2017.05); C12N 2330/51 (2013.01)] 19 Claims
 
1. A method of producing a genetically engineered cell resulting from a host cell comprising a double stranded target polynucleotide, wherein the double stranded target polynucleotide comprises a target nucleic acid strand comprising a target nucleic acid sequence, and a non-target nucleic acid strand comprising a protospacer nucleic acid sequence complementary to the target nucleic acid sequence, said method comprising:
a. designing at least one targeting RNA molecule, wherein the targeting RNA molecule recognizes the target sequence in the target strand, and the non-target strand further comprises a protospacer adjacent motif (PAM) sequence directly adjacent the 3′ end of the protospacer sequence, wherein the PAM sequence comprises 5′-NNNNCNN-3′;
b. providing a host cell and forming a ribonucleoprotein complex within the host cell comprising the targeting RNA molecule and a Cas protein, wherein the Cas protein has the amino acid sequence of SEQ ID NO: 1, or a sequence of at least 89% identity therewith; and
c. the ribonucleoprotein complex binding, cleaving, marking or modifying the target polynucleotide within the cell, thereby producing a genetically engineered cell; and wherein said cell is not a human cell.