CRISPR-Mediated Genome Engineering for Protein Depletion

摘要

The present invention provides compositions and methods for tagging a target gene with a degron (e.g., auxin-inducible degron) in a variety of eukaryotic cells using the CRISPR genome-editing technology. Also provided are cells that have been genetically modified using such compositions and methods.

主权项

1. A method of tagging a target gene in a cell with a nucleotide sequence encoding an auxin-inducible degron (AID), comprising:
a) introducing into a cell:
1) a nucleic acid comprising a nucleotide sequence encoding a synthetic guide ribonucleic acid (sgRNA), wherein the sgRNA is complementary to a target nucleotide sequence in or near a target gene;2) a nucleic acid comprising a nucleotide sequence encoding a clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9);3) a repair template comprising a nucleotide sequence encoding an AID; and b) expressing the sgRNA and Cas9 nuclease in the presence of the repair template in the cell,
thereby tagging the target gene with the nucleotide sequence encoding the AID.