RT Journal Article
SR Electronic
T1 Pooled AAV-CRISPR Screen with Targeted Amplicon Sequencing
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 153643
DO 10.1101/153643
A1 Guangchuan Wang
A1 Ryan D. Chow
A1 Lupeng Ye
A1 Christopher D. Guzman
A1 Xiaoyun Dai
A1 Matthew B. Dong
A1 Feng Zhang
A1 Phillip A. Sharp
A1 Randall J. Platt
A1 Sidi Chen
YR 2017
UL http://biorxiv.org/content/early/2017/06/22/153643.abstract
AB High-resolution, high-throughput direct in vivo screening of functional genetic factors in native tissues has long been challenging. Adeno-associated viruses (AAV) are powerful carriers of transgenes and have been shown to mediate efficient genome editing in various organs in mice. Here, we developed a new technological approach, Pooled AAV-CRISPR Screen with Targeted Amplicon Sequencing (PASTAS), and demonstrated its application for directly mapping functional cancer driver variants in the mouse liver in an autochthonous manner. Intravenous delivery of an AAV-CRISPR library targeting a set of the most frequently mutated tumor suppressor genes into fully immunocompetent conditional Cas9 knock-in mice consistently generated highly complex autochthonous liver tumors. The molecular landscapes of these genetically diverse tumors were mapped out by deep direct readout of Cas9-generated variants at predicted sgRNA cut sites using molecular inversion probe sequencing. Co-occurrence and correlation analyses as well as validation with lower complexity minipools further confirmed the potency of various co-mutated drivers. The PASTAS method can be applied to virtually any gene sets, any cancer types, or any type of in vivo genetic studies other than cancer.