Optimization of Genomewide CRISPR Screens Using AsCas12a and Multi-Guide Arrays

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Genomewide loss-of-function (LOF) screening using clustered regularly interspaced short palindromic repeats (CRISPR) is crucial for discovering gene functions.
• Conventional CRISPR-Cas9 libraries are large and resource-intensive, limiting their accessibility.
• Multiplexing guides per gene on a single construct offers a solution to streamline screening.

Purpose of the Study:

• To determine the optimal design for multiplexed CRISPR-Cas12a libraries for genomewide LOF screening.
• To assess the performance of optimized CRISPR-Cas12a libraries in robotic cell culture systems.

Main Methods:

• CRISPR-Cas12a library design with varying numbers of guides per gene.
• Genomewide LOF screening experiments.
• Adaptation and validation of screening in robotic cell culture.

Main Results:

• CRISPR-Cas12a screening performed optimally with three guides arrayed per gene construct.
• The optimized three-guide CRISPR-Cas12a library design showed comparable performance to larger CRISPR-Cas9 libraries.
• Screening could be successfully adapted to robotic cell culture without compromising performance.

Conclusions:

• A three-guide array per gene construct represents an optimal design for CRISPR-Cas12a genomewide LOF screening.
• Streamlined CRISPR-Cas12a libraries are compatible with robotic cell culture, enhancing efficiency and accessibility.
• This study provides a more efficient and accessible approach to genomewide LOF screening.