Precisely defining disease variant effects in CRISPR-edited single cells.

Area of Science:

• Genomics
• Molecular Biology
• Immunology

Background:

• Thousands of disease-associated non-coding alleles are known, but identifying causal ones and their functions is challenging.
• CRISPR-Cas editing allows DNA modification but faces limitations like inefficient editing and nonspecific transcriptional changes.

Purpose of the Study:

• To develop and apply a multi-omic single-cell sequencing approach to identify causal disease alleles and their functional consequences.
• To overcome the limitations of current CRISPR editing techniques for functional genomic studies.

Main Methods:

• Developed a multi-omic single-cell sequencing strategy integrating DNA sequencing, transcriptome analysis, and cell-surface protein expression.
• Applied the method to study gene disruption, regulatory region deletions, single-nucleotide polymorphism (SNP) alleles, and multiplexed editing.
• Investigated the effects of an IL2RA autoimmune variant in primary human T cells.

Main Results:

• Successfully identified the functional effects of individual single-nucleotide polymorphisms (SNPs).
• Demonstrated state-specific effects of an IL2RA autoimmune variant in primary human T cells.
• Validated the utility of multimodal functional genomic single-cell assays for identifying causal variation.

Conclusions:

• This multimodal single-cell approach enables precise identification of causal genetic variations in primary human cells.
• The method bridges a critical gap in understanding the functional impact of genetic variations in complex human diseases.
• Advances the functional characterization of non-coding disease alleles.