Multiplexed genome regulation in vivo with hyper-efficient Cas12a

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• Multiplexed gene modulation is essential for advanced gene therapy and cell engineering.
• CRISPR-Cas12a systems offer multi-locus targeting but suffer from low in vivo efficiency.

Purpose of the Study:

• To develop a hyper-efficient CRISPR-Cas12a variant for improved in vivo gene modulation.
• To assess the efficacy and safety of the engineered hyperCas12a system.

Main Methods:

• Structure-guided protein engineering of Lachnospiraceae bacterium Cas12a.
• Development of a catalytically dead hyperdCas12a variant for gene activation.
• In vivo testing in post-natal mouse retinas for gene activation and cell differentiation studies.

Main Results:

• The hyperCas12a variant demonstrated significantly enhanced gene activation efficiency, especially at low crRNA concentrations.
• The hyperdCas12a system showed comparable off-target effects to wild-type Cas12a.
• Simultaneous activation of Oct4, Sox2, and Klf4 genes in mouse retinas altered progenitor cell differentiation.

Conclusions:

• The hyperCas12a system provides a powerful and versatile tool for in vivo gene modulation.
• This enhanced CRISPR-Cas12a variant holds promise for broad gene therapy and cell engineering applications.