Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Current CRISPR/Cas technologies for generating gene perturbation reagents are time-consuming, involving multiple ligation and cloning steps.
• Increasing guide RNA (gRNA) sequence diversity often leads to poor distribution and heterogeneous library quality.

Purpose of the Study:

• To introduce a rapid, cloning-free mutagenesis technology for generating covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents.
• To decouple sequence diversity from sequence distribution, enabling high-quality, bias-free gene perturbation libraries.
• To demonstrate the utility of 3Cs reagents for large-scale screening, including identifying factors related to doxorubicin resistance.

Main Methods:

• Development of a novel cloning-free mutagenesis technology to produce 3Cs CRISPR/Cas gRNA reagents.
• Application of 3Cs technology to target all human deubiquitinating enzymes (DUBs).
• Construction of a comprehensive gRNA library for interrogating the human genome and identifying doxorubicin resistance-related elements.

Main Results:

• The 3Cs technology efficiently generates CRISPR/Cas gRNA reagents with uncoupled sequence diversity and distribution.
• Demonstrated the fidelity and performance of 3Cs reagents by targeting human DUBs and confirming their essentiality for cell fitness.
• Successfully generated a large-scale, bias-free gRNA library for high-content screening.

Conclusions:

• The 3Cs technology offers a rapid and robust method for generating diverse, bias-free gene perturbation libraries.
• This novel approach overcomes limitations of existing CRISPR/Cas reagent generation methods.
• 3Cs technology represents a significant advancement for high-content screening and genomic research.