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.
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