Genome-Wide CRISPR Screening Identifies Cellular Factors Controlling Nonviral Genome Editing Efficiency

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Genome editing efficiency is hindered by cellular uptake, trafficking, and nuclear import barriers.
• These barriers vary significantly based on cell type and vector characteristics, such as lipid nanoparticles.

Purpose of the Study:

• To develop a genome-wide CRISPR screening strategy to identify genes that modulate cellular uptake, payload delivery, and gene editing efficiency in human cells.
• To uncover novel targets for enhancing nonviral genome editing delivery methods.

Main Methods:

• A genome-wide CRISPR screen targeting 19,114 genes in HEK293 cells was performed.
• Cas-based nuclease and base editing strategies were employed to interrogate cellular processes controlling genome editing.
• Short-read sequencing was used to track guide RNA identity and editing outcomes, enabling computational sorting of edited and unedited cells.

Main Results:

• Six genes were identified whose knockout increased nonviral editing efficiency by up to five-fold in human cells.
• Arrayed knockouts of top hits enhanced Cas9 editing efficiency from 5% to 50% using lipid-based nanoparticles.
• Knockouts of BET1L, GJB2, and MS4A13 genes increased targeted genome editing by over five-fold in patient-derived retinal pigment epithelium cells.

Conclusions:

• The high-throughput screening approach successfully identified key genes modulating nonviral genome editing efficiency.
• The identified genes can be leveraged to engineer improved nonviral delivery methods for gene editing.
• This strategy holds potential for enhancing genome editing efficiency in various therapeutically relevant cell types.