A multiplex, prime editing framework for identifying drug resistance variants at scale.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• CRISPR-based genome editing advanced functional genomics, allowing simultaneous assaying of thousands of perturbations.
• Saturation genome editing (SGE) methods typically focus on one genomic region per experiment.

Purpose of the Study:

• To develop a novel CRISPR-based framework, prime-SGE, for installing and assaying libraries of specific point mutations genome-wide.
• To enable functional assessment of numerous point mutations by sequencing prime editing guide RNAs (pegRNAs).

Main Methods:

• Developed prime-SGE, a prime editing-based system for generating and assaying libraries of point mutations.
• Applied prime-SGE in two cell lines to screen thousands of point mutations in eight oncogenes.
• Assessed the ability of point mutations to confer drug resistance to tyrosine kinase inhibitors.

Main Results:

• Successfully applied prime-SGE to assay thousands of point mutations across eight oncogenes.
• Demonstrated the utility of prime-SGE in identifying mutations conferring resistance to tyrosine kinase inhibitors.
• Showcased the feasibility of pegRNA sequencing for functional assessment of point mutations.

Conclusions:

• Prime-SGE offers an efficient strategy for large-scale positive selection screens of point mutations throughout the genome.
• This method, coupled with prime editing advancements, expands the scope of functional genomics screens.
• Opens new avenues for discovering genotype-phenotype relationships related to drug resistance.