Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles

Area of Science:

• Genetics
• Molecular Biology
• Bioengineering

Background:

• The International Mouse Phenotyping Consortium aims to create null allele mice for all protein-coding genes.
• CRISPR/Cas9 is efficient for null alleles but less so for conditional alleles.
• Conditional allele generation is crucial for large-scale genetic studies.

Purpose of the Study:

• To evaluate CRISPR/Cas9-mediated homology-driven repair (HDR) for generating conditional knockout mice.
• To compare the efficacy of short (ssODNs) and long (lssDNAs) single-stranded oligodeoxynucleotides for conditional allele production.

Main Methods:

• Utilized Cas9-initiated HDR with ssODNs and lssDNAs to introduce loxP sites.
• Employed pairs of single guide RNAs for targeted gene editing.
• Assessed integration of loxP sites and screened for mutations and random integrations.

Main Results:

• Conditional allele founders were generated for 23 out of 30 genes using ssODNs.
• LoxP sites integrated in cis for 18 of 23 genes, with some mutations observed.
• All four genes targeted with lssDNAs yielded conditional allele founders, despite some sequence errors.
• Random integration events were detected with both ssODNs and lssDNAs.

Conclusions:

• Cas9-mediated HDR with ssODNs can generate conditional null alleles but shows inefficiencies at scale.
• LssDNAs are more suitable for high-throughput conditional allele production.
• Screening for sequence errors at HDR sites and random donor integrations is essential for both methods.