Mono- and Biallelic Replication-Coupled Gene Editing Discriminates Dominant-Negative and Loss-of-Function Variants of...

Area of Science:

• Molecular Biology
• Genetics
• Cancer Research

Background:

• DNA mismatch repair (MMR) gene variants of uncertain significance pose diagnostic challenges for Lynch syndrome.
• Locked nucleic acid-modified single-stranded DNA oligonucleotides (LMOs) enable precise, replication-coupled gene editing at the single nucleotide level.
• Previous studies utilized hemizygous mouse embryonic stem cells (mESCs) for oligonucleotide-directed mutation screening (ODMS).

Purpose of the Study:

• To adapt and validate ODMS for use in wild-type mESCs harboring two functional alleles of MMR genes.
• To determine if ODMS can differentiate between dominant-negative and loss-of-function MMR gene variants.
• To enhance the efficiency of LMO-mediated gene targeting events.

Main Methods:

• Application of ODMS in wild-type mESCs carrying functional MMR gene alleles.
• Selection for 6-thioguanine resistance to indicate defective MMR and pathogenicity.
• Biallelic targeting of MMR genes to distinguish variant types.
• Simultaneous selectable LMO targeting to enrich nonselectable LMO-mediated gene modification events.

Main Results:

• ODMS is feasible in wild-type mESCs, enabling the assessment of MMR gene variants.
• The method successfully discriminates between dominant-negative and loss-of-function MMR variants based on resistance patterns.
• Biallelic targeting in wild-type cells improves the recovery of LMO-mediated gene modification events.

Conclusions:

• ODMS in wild-type mESCs provides a robust platform for classifying MMR gene variants and assessing their pathogenicity in Lynch syndrome.
• The developed protocol enhances the efficiency of LMO-mediated gene editing, facilitating genetic engineering applications.
• This advancement offers improved diagnostic capabilities for hereditary cancer syndromes.