LAUSR

Although homology directed repair (HDR)-mediated gene knockout / knockin is well established, it cannot necessarily be applied in some cell types and organisms with low HDR frequencies. Herein we describe an improved technology for genome-wide gene knockout / knockin using CRISPR-Cas9 targeted genome editing: CRISPR KN2.0. CRISPR KN2.0 technology is specifically designed to provide an easy-to-use and get around 50% biallelic knockout. This gene knockout / knockin technology is designed to knock out any DNA locus and knock in a functional cassette containing Green Fluorescence Protein (GFP) and puromycin resistant gene to facilitate screening process. Both of these cassettes are expressed under EF1A promoter after genomic integration. CRISPR KN 2.0 has been used to successfully knock out many genes / knocked in functional cassette in HeLa, HEK293T and MIA PaCa-2 (a human pancreatic carcinoma cell line) cells. Studies carried out in house and by some colleagues show that CRISPR KN 2.0 is highly efficient and render improved knockout/knockin rate (over 50% positively integrated colonies after puromycin selection). Citation Format: Yongqi Li, Li Min, Ellen Liu, Yiran Wang. CRISPR knockout: How to get high biallelic knockout [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5243.