	US 12,173,290 B2
	Materials and methods for controlling gene editing
Ryo Takeuchi, Cambridge, MA (US); and Abraham Scaria, Cambridge, MA (US)
Assigned to CRISPR THERAPEUTICS AG, Zug (CH); and BAYER HEALTHCARE LLC, Whippany, NJ (US)
Filed by CRISPR THERAPEUTICS AG, Zug (CH); and BAYER HEALTHCARE LLC, Whippany, NJ (US)
Filed on Jun. 26, 2020, as Appl. No. 16/912,999.
Claims priority of provisional application 62/868,209, filed on Jun. 28, 2019.
Prior Publication US 2020/0407729 A1, Dec. 31, 2020
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/11 (2006.01); C12N 9/22 (2006.01); C12N 15/64 (2006.01); C12N 15/86 (2006.01)

CPC C12N 15/64 (2013.01) [C12N 9/22 (2013.01); C12N 15/86 (2013.01); C12N 2310/122 (2013.01); C12N 2310/20 (2017.05); C12N 2750/14143 (2013.01); C12N 2750/14151 (2013.01); C12N 2800/22 (2013.01)]

28 Claims

1. A CRISPR/Cas system comprising:

a nuclease segment comprising a codon optimized nucleotide sequence that encodes a Cas9 nuclease or variant thereof, wherein the Cas9 nuclease comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 60;

a guide RNA (gRNA) segment comprising a nucleotide sequence that encodes a gRNA or sgRNA;

a promoter segment comprising a nucleotide sequence that encodes a promoter comprising one or more tetracycline operator sequence, wherein the gRNA segment is operably linked to the promoter segment; and/or

a short-hairpin RNA (shRNA) segment comprising a nucleotide sequence that encodes a shRNA that comprises sequence that is complementary to a transcript from the nuclease segment;

wherein the Cas9 nuclease comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 60 introduces a double stranded break at a site in a nucleic acid complementary to the gRNA segment.