LION: a simple and rapid method to achieve CRISPR gene editing

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• CRISPR-Cas9 technology enables efficient gene editing but requires robust methods for guide RNA (gRNA) generation and validation.
• Previous work demonstrated Golden Gate Assembly (GGA) for multiplexed CRISPR gRNA expression arrays.
• A need exists for faster, more efficient methods for creating and validating CRISPR gene targeting vectors.

Purpose of the Study:

• To present an optimized and rapid method, termed LION (Ligation Independent Open-reading frame cloning), for generating and validating CRISPR gene targeting vectors.
• To demonstrate that the LION method can achieve vector generation and validation in less than one day.
• To confirm the functional activity and equivalence of LION-generated vectors compared to traditionally produced ones.

Main Methods:

• The LION method utilizes Golden Gate Assembly (GGA) for ligating double-stranded gRNA oligos into CRISPR vectors, followed by nucleic acid purification.
• Functional validation employed a dual-fluorescent reporter vector (C-Check), T7E1 assay, TIDE assay, and a traffic light reporter assay.
• Activity of LION-generated CRISPR vectors was tested in various human cell types.

Main Results:

• The LION method successfully generated functional CRISPR vectors within a day.
• LION-generated CRISPR vectors demonstrated equivalent activity to those produced by traditional methods, confirmed by multiple validation assays.
• LION CRISPR vectors showed activity across different human cell types, indicating broad applicability.

Conclusions:

• The LION method significantly advances the rapid generation and functional validation of CRISPR gene editing vectors.
• This technique simplifies CRISPR gene editing procedures, making it more accessible and efficient.
• LION offers a streamlined approach for researchers utilizing CRISPR technology for gene editing applications.