Efficient and specific generation of knockout mice using Campylobacter jejuni CRISPR/Cas9 system

Area of Science:

• Molecular Biology
• Genetics
• Genomics

Background:

• The Streptococcus pyogenes CRISPR/Cas9 (SpCas9) system is widely used for mouse genome engineering.
• Concerns exist regarding SpCas9's potential for off-target mutations.

Purpose of the Study:

• To evaluate the Campylobacter jejuni Cas9 (CjCas9) system for generating knockout mice.
• To assess the precision and efficiency of CjCas9 in mouse genome editing.

Main Methods:

• Designed single-guide RNAs (sgRNAs) targeting mouse Tyr or Foxn1 genes.
• Microinjected CjCas9 mRNA and sgRNAs into mouse zygotes.
• Analyzed newborn mice for biallelic mutations using targeted deep sequencing and whole genome sequencing.

Main Results:

• Achieved efficient genome targeting with CjCas9, resulting in 50% biallelic mutations in Tyr and 38.5% in Foxn1.
• No off-target mutations were detected at predicted sites.
• Whole genome sequencing confirmed the absence of unexpected random mutations in founder mice.

Conclusions:

• The CjCas9 system demonstrates high efficiency and precision for generating genome-edited mice.
• CjCas9 offers a reliable and safer alternative to SpCas9 for mouse genetic engineering, minimizing off-target effects.