Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line

Area of Science:

• Human genetics
• Genome engineering
• Cell biology

Background:

• Near-haploid human cell lines are valuable for genetic screens due to simplified gene inactivation.
• The absence of a completely haploid human cell line limits genetic accessibility for certain genes.
• The HAP1 cell line is near-haploid, possessing a heterozygous 30-megabase fragment on Chromosome 15.

Purpose of the Study:

• To engineer a fully haploid human cell line.
• To excise a large heterozygous chromosomal fragment from the HAP1 cell line.
• To establish a reproducible method for deriving and validating fully haploid cells.

Main Methods:

• CRISPR/Cas9 genome engineering was employed to target and excise the 30-megabase fragment.
• Spectral karyotyping and single-nucleotide polymorphism (SNP) genotyping were used for chromosomal analysis.
• Whole-genome sequencing and transcriptome analysis were performed to assess genetic and transcriptional integrity.

Main Results:

• Engineered-HAPloid (eHAP) cells were successfully derived, exhibiting a fully haploid state.
• No gross chromosomal aberrations were detected in eHAP cells post-CRISPR/Cas9 editing.
• Transcriptional changes were confined to the excised Chromosome 15 fragment, indicating minimal off-target effects.

Conclusions:

• CRISPR/Cas9 technology can efficiently engineer megabase deletions in human cell lines.
• The study reports the first fully haploid human cell line, eHAP.
• This new cell line significantly advances capabilities for genetic screens and genome engineering.