A pre-screening FISH-based method to detect CRISPR/Cas9 off-targets in mouse embryonic stem cells

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system offers efficient DNA targeting for gene engineering.
• Investigating the specificity of CRISPR/Cas9 is crucial for its safe application.

Purpose of the Study:

• To assess the frequency and location of non-specific DNA integrations mediated by the CRISPR/Cas9 system.
• To evaluate the accuracy of off-target prediction software for CRISPR/Cas9 applications.

Main Methods:

• CRISPR/Cas9 was used to integrate exogenous DNA into the Hprt gene of mouse embryonic stem cells.
• Fluorescence in situ hybridization (FISH) was employed to detect and quantify non-specific plasmid integrations.

Main Results:

• FISH analysis identified non-specific plasmid integrations.
• These observed integrations did not correspond to the loci predicted by off-target analysis software.
• The frequency of CRISPR-mediated off-target DNA cuts was found to be negligible compared to spontaneous double-strand breaks.

Conclusions:

• The CRISPR/Cas9 system demonstrates high specificity in DNA targeting.
• FISH is an effective method for evaluating non-specific integrations after CRISPR/Cas9 use.
• Current off-target prediction tools may not fully capture all potential integration sites.