A simple and highly efficient method for multi-allelic CRISPR-Cas9 editing in primary cell cultures

Area of Science:

• Molecular Biology
• Genomics
• Gene Editing Technologies

Background:

• CRISPR-Cas9 technology has transformed mammalian genome manipulation.
• Limitations in delivery and efficacy hinder its application in primary cells.

Purpose of the Study:

• To develop an optimized protocol for efficient, reproducible, and rapid genome editing in primary cell cultures.
• To demonstrate proof-of-concept using patient-derived glioblastoma stem-like cells (GSCs) and human neural stem/progenitor cells (NSCs).

Main Methods:

• Utilized transient nucleofection of Cas9:sgRNA ribonucleoprotein complexes with chemically synthesized sgRNAs.
• Measured insertion-deletion mutation (indel) frequency and size via Sanger sequencing deconvolution.
• Employed Western blotting and RNA-sequencing for assessing gene knockout and expression changes.

Main Results:

• Achieved >90% indel formation within 3 days in primary cells.
• Demonstrated near-complete protein loss for target genes in cell pools.
• Successfully created targeted genomic deletions and assessed gene expression changes in edited NSCs.

Conclusions:

• Developed a simple, highly efficient, and rapid gene knockout and genomic deletion method applicable to diverse cell types, including GSCs.
• Provides a valuable tool for cancer research to inactivate coding genes, non-coding RNAs, UTRs, enhancers, and promoters.
• Facilitates rapid functional studies of oncogenic activities through quick gene knockout creation.