	US 12,188,015 B2
	Type I CRISPR system as a tool for genome editing
Ailong Ke, Ithaca, NY (US); Adam Dolan, Ithaca, NY (US); Yan Zhang, Ann Arbor, MI (US); and Zhonggang Hou, Ann Arbor, MI (US)
Assigned to Cornell University, Ithaca, NY (US); and The Regents of the University of Michigan, Ann Arbor, MI (US)
Appl. No. 17/254,169
Filed by Cornell University, Ithaca, NY (US); and The Regents of the University of Michigan, Ann Arbor, MI (US)
PCT Filed Jun. 21, 2019, PCT No. PCT/US2019/038529 § 371(c)(1), (2) Date Dec. 18, 2020, PCT Pub. No. WO2019/246555, PCT Pub. Date Dec. 26, 2019.
Claims priority of provisional application 62/829,091, filed on Apr. 4, 2019.
Claims priority of provisional application 62/688,202, filed on Jun. 21, 2018.
Prior Publication US 2022/0315920 A1, Oct. 6, 2022
Int. Cl. C12N 15/11 (2006.01); C12N 9/22 (2006.01); C12N 15/90 (2006.01)

CPC C12N 15/11 (2013.01) [C12N 9/22 (2013.01); C12N 15/907 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01)]

19 Claims

1. A method of modifying DNA in eukaryotic cells, the method comprising introducing into the eukaryotic cells:

(i) a combination of proteins comprising Cas3, Cse1/CasA, Cse2/CasB, Cas7/CasC, Cas5e/CasD and Cas6e/CasE, each comprising an amino acid sequence that is at least 85% homologous across its entire length to a Thermobifida Fusca (T. fusca) protein, wherein the Cas3 protein comprises the sequence of SEQ ID NO: 1 or a sequence that is at least 85% homologous across its entire length to the sequence of SEQ ID NO: 1, wherein the sequence of the Cse2/CasB protein comprises or a sequence that is at least 85-99% homologous across the entire length sequence of SEQ ID NO: 2, wherein the sequence that is at least 85-99% homologous to the sequence of SEO ID NO: 2 comprises an amino acid at position 23 of SEO ID NO: 2 that is not an N, and wherein the modification of the DNA is performed at a temperature of about 37° C.;

(ii) a guide RNA (a targeting RNA) comprising a sequence that is complementary to a targeted site in a segment of the DNA, the targeted site comprising a spacer sequence; and

(iii) allowing the combination of the proteins and the guide RNA to modify the DNA by nicking, causing a double stranded break (DSB), and/or unidirectional deleting of a single strand of the DNA, wherein the targeted site is not modified, and wherein the DNA is comprised by a chromosome or an extrachromosomal element.