Using Single-Cell CRISPR Screen to Decipher the Function of Core Transcription Factors in Driving NF1-Deficient MPNST
Abstract
In Aim 1, I will use the CRISPR-KO system that was previously established by transducing Cas9 vector into MPNST cell lines. Next, a pooled dual-gRNA library that targets the DNA binding domains of 85candidate CRC TFs (plus 5 non-targeting controls) will be designed, synthesized and cloned into lentiviral vectors and used to transduce the Cas9- expressing MPNST cells. The transcriptional phenotypes of these cells will be sequenced on a single-cell level and compared to the known oncogenic signature of MPNST cells. In Aim 2, I will establish the CRISPR a system by transducing the dCas9- VPR vector into the NF1-/- Schwann cells, which will be transduced with a pooled dual-gRNA library targeting the promoters of the 85 MPNST-specific TFs. Next, these Schwann cells will be cultured in soft agar with or without the selecting pressure of single-agent topotecan, etoposide, or doxorubicin. Any colonies formed will be subjected to nuclei isolation and transcriptomic profiling.
Document Details
- Document Type
- Technical Report
- Publication Date
- Aug 01, 2023
- Accession Number
- AD1213453
Entities
People
- Xiyuan Zhang
Organizations
- Geneva Foundation