Multiple homologous genes knockout (KO) by CRISPR/Cas9 system in rabbit

Area of Science:

• Genetics
• Molecular Biology
• Biotechnology

Background:

• CRISPR/Cas9 is a powerful genome editing tool for single or multiple gene mutations.
• Studying homologous genes with similar sequences and functions requires disrupting multiple genes simultaneously.
• Previous methods for multiple homologous gene disruption were limited.

Purpose of the Study:

• To evaluate the efficiency of the CRISPR/Cas9 system for simultaneous knockout of multiple homologous genes.
• To test the efficacy of a single guide RNA (sgRNA) targeting three fucosyltransferase genes (FUT1, FUT2, and SEC1).
• To establish a method for efficient multiple homologous gene knockout in mammals.

Main Methods:

• Designed a single guide RNA (sgRNA) targeting FUT1, FUT2, and SEC1 genes.
• Utilized the CRISPR/Cas9 system for genome editing in rabbits.
• Analyzed serum fucosyltransferase enzyme activity in gene-edited rabbits.

Main Results:

• Achieved simultaneous triple gene mutation of FUT1, FUT2, and SEC1 using the sgRNA-mediated CRISPR/Cas9 system.
• Observed significantly reduced serum fucosyltransferase enzyme activity in the triple gene knockout rabbits.
• Demonstrated the feasibility of targeting and disrupting multiple homologous genes concurrently.

Conclusions:

• The sgRNA-mediated CRISPR/Cas9 system enables efficient simultaneous knockout of multiple homologous genes in mammals.
• This approach provides a valuable tool for genotype-to-phenotype studies involving homologous genes.
• This study offers the first evidence for efficient multiple homologous gene knockout in mammals using this system.