Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Robust gene expression perturbation is crucial for understanding gene function.
• CRISPR-Cas9 technology offers a powerful platform for genetic manipulation.

Purpose of the Study:

• To engineer a CRISPR-Cas9 complex for efficient transcriptional activation at endogenous genomic loci.
• To investigate single-guide RNA (sgRNA) targeting rules for transcriptional activation.
• To screen for genes conferring resistance to a BRAF inhibitor upon activation.

Main Methods:

• Structure-guided engineering of CRISPR-Cas9 complexes for transcriptional activation.
• Utilizing engineered Cas9 activation complexes to study sgRNA targeting rules.
• Performing multiplexed gene activation and upregulating long intergenic non-coding RNA (lincRNA) transcripts.
• Synthesizing a large library of guides for a genome-wide screen for BRAF inhibitor resistance.

Main Results:

• Demonstrated efficient transcriptional activation at endogenous loci using engineered Cas9 activators.
• Identified sgRNA targeting rules for effective transcriptional activation.
• Successfully achieved multiplexed activation of ten genes and upregulated lincRNA transcripts.
• Discovered known and novel genes conferring BRAF inhibitor resistance through a large-scale screen.
• Validated screening hits and found a gene expression signature correlating with BRAF inhibitor resistance.

Conclusions:

• Engineered Cas9 activators represent a powerful technology for genetic perturbation.
• This approach enables efficient transcriptional activation and large-scale functional genomic screens.
• The findings have implications for understanding drug resistance mechanisms and developing new therapeutic strategies.