Assembly and validation of versatile transcription activator-like effector libraries.

ABSTRACT: The ability to perturb individual genes in genome-wide experiments has been instrumental in unraveling cellular and disease properties. Here we introduce, describe the assembly, and demonstrate the use of comprehensive and versatile transcription activator-like effector (TALE) libraries. As a proof of principle, we built an 11-mer library that covers all possible combinations of the nucleotides that determine the TALE-DNA binding specificity. We demonstrate the versatility of the methodology by constructing a constraint library, customized to bind to a known p53 motif. To verify the functionality in assays, we applied the 11-mer library in yeast-one-hybrid screens to discover TALEs that activate human SCN9A and miR-34b respectively. Additionally, we performed a genome-wide screen using the complete 11-mer library to confirm known genes that confer cycloheximide resistance in yeast. Considering the highly modular nature of TALEs and the versatility and ease of constructing these libraries we envision broad implications for high-throughput genomic assays.

SUBMITTER: Li Y 

PROVIDER: S-EPMC4010924 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature