Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency

Area of Science:

• Molecular Biology
• Genetics
• Bioengineering

Background:

• The CRISPR/Cas9 system is a powerful tool for genome engineering, but knockin efficiency via homology-directed repair (HDR) remains a challenge.
• Traditional methods often require prescreening of single-guide RNAs (sgRNAs), delaying transgenic animal generation.
• Nonhomologous end-joining (NHEJ) is generally more efficient for knockouts than HDR is for knockins.

Purpose of the Study:

• To enhance the efficiency of knockin animal generation using the CRISPR/Cas9 system.
• To investigate the impact of overlapping versus non-overlapping sgRNAs on HDR-mediated knockin efficiency.
• To develop a simplified and more efficient method for generating transgenic animals.

Main Methods:

• Utilized CRISPR/Cas9 technology for genome editing in mouse models.
• Employed single-stranded oligo-donor mediated HDR.
• Compared knockin efficiency using overlapping sgRNAs (sharing ≥5 bp target sites) versus non-overlapping sgRNAs.

Main Results:

• Overlapping sgRNAs demonstrated profound advantages in single-stranded oligo-donor-mediated HDR processes compared to non-overlapping sgRNAs.
• Cell line studies showed shorter sequence deletions near target mutations with overlapping sgRNAs, potentially favoring HDR.
• Achieved high efficiency (18-38%) in generating transgenic mouse lines with loxP insertions or single-nucleotide substitutions.

Conclusions:

• The use of overlapping sgRNAs is a simple and highly efficient method for generating transgenic animals with knockins.
• This approach overcomes limitations associated with sgRNA prescreening and improves knockin success rates.
• The findings provide a valuable strategy for the efficient generation of genetically modified mouse models.