Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Multiplexed genome editing with Cas9 is often hindered by the requirement for multiple or large expression constructs.
• Efficiently targeting several genomic locations simultaneously presents a significant challenge in genetic research and therapeutic development.

Purpose of the Study:

• To develop a simplified and efficient method for multiplexed genome editing.
• To leverage the self-processing capability of Cpf1 for streamlined CRISPR array design.

Main Methods:

• Utilized the Cpf1 nuclease system for its intrinsic CRISPR RNA (crRNA) processing ability.
• Designed a single, customized CRISPR array for multiplexed gene targeting.
• Applied the system to edit multiple genes in mammalian cell lines and in vivo in the mouse brain.

Main Results:

• Demonstrated successful simultaneous editing of up to four genes in mammalian cells using a single Cpf1-based CRISPR array.
• Achieved simultaneous editing of three genes in the mouse brain, showcasing in vivo applicability.
• The Cpf1-mediated processing of crRNA significantly simplified the construct design for multiplexed editing.

Conclusions:

• The Cpf1 system offers a streamlined approach to multiplexed genome editing, overcoming limitations associated with Cas9.
• This method provides a powerful tool for complex genetic modifications in various biological systems, including in vivo applications.