Optimal LentiCRISPR-Based System for Sequential CRISPR/Cas9 Screens.

Area of Science:

• Molecular Biology
• Genomics
• Genetic Engineering

Background:

• Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening significantly advances cellular molecular systems understanding.
• Existing CRISPR screening methods often require extensive library coverage, increasing processing and sequencing demands.

Purpose of the Study:

• To present a novel sequential CRISPR knockout screening strategy named "Many vs One" CRISPR screening.
• To demonstrate the utility of this approach for exploring biological questions across the human genome efficiently.
• To introduce a modified 7SK promoter system for improved CRISPR screening.

Main Methods:

• Development of a modified lentiCRISPRv2 vector utilizing a 7SK promoter instead of the U6 promoter for single-guide RNA expression.
• Implementation of sequential CRISPR knockout screens, where initial genome-wide screens are followed by focused screens of gene candidates against a common gene.
• Comparison of the 7SK vector's performance against the standard U6 promoter in terms of lentiviral titer, knockout efficiency, and usability.

Main Results:

• The novel 7SK promoter enables "Many vs One" CRISPR screening, reducing the need for increased CRISPR library coverage.
• This method allows for sequential analysis, using remaining cells to test multiple gene candidates against a single gene of interest.
• The 7SK vector demonstrates comparable lentiviral titer, knockout efficiency, and ease of use to the original lentiCRISPRv2 vector.

Conclusions:

• The "Many vs One" CRISPR screening approach, powered by the 7SK promoter, offers a more efficient and versatile tool for genetic screening.
• This methodology streamlines screening processes, sample handling, and next-generation sequencing requirements.
• The 7SK promoter integration into the lentiCRISPRv2 backbone provides a valuable advancement for CRISPR-based biological discovery.