A CRISPR-del-based pipeline for complete gene knockout in human diploid cells

Area of Science:

• Molecular Biology
• Genetics Engineering

Background:

• CRISPR/Cas9 technology facilitates gene knockout via insertion-deletion mutations (indels).
• Indels can cause frameshifts and premature stop codons, but often fail to achieve complete gene disruption.
• Limitations exist in reliably knocking out genes using conventional CRISPR/Cas9 indel methods.

Purpose of the Study:

• To establish an efficient pipeline for 'complete' gene knockout in human diploid cells.
• To develop a method that induces large chromosomal deletions for guaranteed gene inactivation.
• To create a practical approach for generating gene knockout cell lines.

Main Methods:

• Development of the CRISPR-del pipeline, utilizing dual guide RNAs to induce double-strand breaks at two distinct sites.
• Induction of large chromosomal deletions via the non-homologous end joining (NHEJ) repair pathway.
• Quantitative analysis of deletion frequency and genomic region lengths.

Main Results:

• The CRISPR-del pipeline demonstrated a significantly higher frequency of gene deletion compared to conventional CRISPR-del methods.
• Generated deletions were larger than 95% of human protein-coding genes, ensuring comprehensive gene inactivation.
• Successfully generated a model cell line with a bi-allelic cancer-associated chromosomal deletion.

Conclusions:

• The CRISPR-del pipeline offers an efficient and practical strategy for achieving 'complete' gene knockout in human diploid cells.
• This method overcomes the limitations of indel-based gene disruption for reliable gene function studies.
• Enables the creation of advanced cell models for disease research, including cancer.