Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Canonical CRISPR-knockout (KO) screens use Cas9 to induce DNA double-strand breaks (DSBs) for gene disruption.
• DSB induction can cause cytotoxicity and confounding artifacts, particularly at high-copy sites, potentially skewing screening results.
• Existing methods may not accurately reflect gene perturbation effects due to DSB-associated cellular responses.

Purpose of the Study:

• To introduce a novel, DNA double-strand break-independent genome-wide CRISPR screening method.
• To enhance screening accuracy and efficiency by mitigating DSB-induced artifacts.
• To provide a complementary tool to existing CRISPR-KO screening methodologies.

Main Methods:

• Developed iBARed cytosine base editing-mediated gene KO (BARBEKO), a genome-wide screening method.
• Utilized CRISPR cytosine base editors to perturb gene start codons, splice sites, or introduce premature termination codons.
• Integrated BARBEKO with the iBAR strategy for improved screening quality and efficiency, using high multiplicity of infection lentiviral transduction.

Main Results:

• BARBEKO successfully generated genome-scale gene KOs without inducing DSBs.
• Screens using BARBEKO showed no DNA cleavage-induced cytotoxicity in various cell lines, including DSB-sensitive cells.
• Achieved efficient and accurate screening with reduced starting cell numbers due to high MOI lentiviral infection.

Conclusions:

• BARBEKO offers a robust, DSB-independent alternative for CRISPR genome-wide screens.
• This method overcomes limitations of Cas9-based KO screens by eliminating DSB-associated artifacts.
• BARBEKO is anticipated to be a valuable tool for complementing current CRISPR screening approaches in diverse biological research.