| Autor: |
Qian Chen, Minhui Pang, Peng Chen, Zihao Zhou, Jun Lei, Boxiao He, Zaiqiao Sun, Chonil Paek, Baowei Jing, Yankang Wu, Shiqi Liu, Yongshun Chen, Lei Yin |
| Jazyk: |
angličtina |
| Rok vydání: |
2024 |
| Předmět: |
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| Zdroj: |
Clinical and Translational Medicine, Vol 14, Iss 7, Pp n/a-n/a (2024) |
| Druh dokumentu: |
article |
| ISSN: |
2001-1326 |
| DOI: |
10.1002/ctm2.1758 |
| Popis: |
Abstract CRISPR/Cas12a‐based combinational screening has shown remarkable potential for identifying genetic interactions. Here, we describe an innovative method for combinational genetic screening with rapid construction of a dual‐CRISPR RNA (crRNA) library using gene splicing through overlap extension PCR (SOE PCR) and the adoption of CeCas12a, which we previously identified with strict PAM recognition and low off‐targeting to guarantee fidelity and efficiency. The custom‐pooled SOE crRNA array (SOCA) library for double‐knockout screening could be conveniently constructed in the laboratory for widespread use, and the CeCas12a‐mediated high‐fidelity screen displayed good performance even under a negative selection screen. By designing a SOCA dual‐crRNA library that covered most of the kinase and metabolism‐associated gene targets of FDA‐approved drugs implicated in hepatocellular carcinoma (HCC) tumourigenesis, novel cross‐talk between the two gene sets was negatively selected to inhibit HCC cell growth in vitro and in vivo and was validated using virtual double‐knockdown screening based on TCGA databases. Thus, this rapid, efficient and high‐fidelity double‐knockout screening system is promising for systemically identifying potential genetic interactions between multiple gene sets or combinations of FDA‐ approved drugs for clinical translational medicine in the future. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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