Concurrent genome and epigenome editing by CRISPR-mediated sequence replacement.

Area of Science:

• Molecular Biology
• Epigenetics
• Genomics

Background:

• Genome editing advances enable manipulation of both genome and epigenome.
• CRISPR/Cas9 typically creates a double-strand break (DSB) for repair.
• Epigenome editing, like CpG methylation, often uses catalytically inactive Cas9 (dCas9) fused to methyltransferases, but has resolution limits.

Purpose of the Study:

• To develop an alternative epigenome editing strategy for high-resolution methylation studies.
• To investigate the role of DNA methylation in gene silencing.
• To achieve concurrent genome and epigenome editing in a single event.

Main Methods:

• Utilized CRISPR/Cas9 dual cutting of the genome.
• Introduced in vitro methylated exogenous DNA during the cutting process.
• Aimed to drive DNA sequence replacement between dual cuts via non-homologous end joining (NHEJ).

Main Results:

• Demonstrated successful replacement events at the HPRT1 promoter using methylated DNA.
• Achieved functional silencing of the HPRT1 gene.
• Showcased concurrent epigenome and genome editing, although efficiency requires improvement.

Conclusions:

• This method enables functional consequence investigations of methylation patterns at single CpG resolution.
• Promoter methylation is sufficient for functional gene expression silencing.
• Opens new avenues for studying epigenetics and gene regulation.