Patent ID: 11957114
Assignee: MIRIMUS, INC.
Field: Biotechnology (Chemistry)
Classification: CPC A  C | IPC A  C

Claim 0:
1. A method of using founder knock-in strains comprising:
a. creating a founder strain with a nucleotide sequence comprising a promoter and a miRNA backbone by
i. using a CRISPR/Cas9 system to insert a nucleotide sequence comprising promoter sequence operably linked to a miRNA backbone sequence and a reporter sequence downstream of the Col1a1 gene on chromosome 10 of rat embryo and producing a founder strain rat whose genome comprises said promoter sequence operably linked to a miRNA backbone sequence and a reporter sequence downstream of the Col1a1 gene on chromosome 10; and
ii. obtaining an embryo or a cell that is produced using the founder strain rat in step (i) and transducing the embryo or cell with a CRISPR/Cas9 system that comprises a Cas9 protein, a gRNA that is targeted to the miRNA backbone sequence, and a donor template that includes a shRNA sequence, thereby inserting said shRNA sequence into said miRNA backbone sequence of the embryo or cell and producing a subsequent founder knock-in strain rat or cell wherein the miRNA backbone sequence comprises the integrated shRNA sequence; and

b. using the founder strain to knockin a variable shRNA sequence for a subsequent strain to be produced.