CRISPR-based screening methods using single-cell RNA sequencing (scRNA-seq) technology enable comprehensive profiling of gene perturbations from knock-out mutations. However, evaluating substitution mutations using scRNA-seq is currently limited. We combined CRISPR RNA-guided deaminase and scRNA-seq technology to develop a platform for introducing mutations in multiple genes and assessing the mutation-associated signatures. Using this platform, we generated a library consisting of 420 sgRNAs, performed sgRNA tracking analysis, and assessed the effect size of the response to vemurafenib in the human melanoma cell line, which has been well-studied via knockout-based drop-out screens. However, a substitution mutation library screen has not been applied and transcriptional information for mechanisms of action was not assessed. Our platform permits discrimination of several candidate mutations that function differently from other mutations by integrating sgRNA candidates and gene expression readout. We anticipate that our platform will enable high-throughput analyses of the mechanisms related to a variety of biological events.
Bibliographical noteFunding Information:
This work was supported by: (i) the Mid-career Researcher Program (NRF-2018R1A2A1A05079172), (ii) the Bio & Medical Technology Development Program (NRF-2016M3A9B6948494), (iii) the Bio & Medical Technology Development Program (NRF-2018M3A9H3024850), (iv) the Bio & Medical Technology Development Program (NRF-2018M3A9D7079485), (v) Basic Science Research Program (NRF-2018R1A2B2001322) of the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Planning, and (vi) Korea Health Technology R&D Project (HI18C2282) through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare.
© 2020, The Author(s).
All Science Journal Classification (ASJC) codes
- Biochemistry, Genetics and Molecular Biology(all)
- Agricultural and Biological Sciences(all)
- Medicine (miscellaneous)