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Exome sequencing in the knockin mice generated using the CRISPR/Cas system.
Kazuo Nakajima1, An-A Kazuno1, John Kelsoe2
1Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan.
Scientific Reports
|October 5, 2016
Summary
CRISPR/Cas technology enables creation of knockin (KI) mice for disease research. However, unintended insertions/deletions (InDels) can occur, requiring careful assessment in KI mouse models.
Area of Science:
- Genetics and Genomics
- Molecular Biology
- Animal Models
Background:
- Knockin (KI) mice with specific point mutations are crucial for disease modeling.
- CRISPR/Cas systems offer an alternative method for generating KI mice.
- Unintended insertions and deletions (InDels) in KI mice can complicate research findings.
Purpose of the Study:
- To evaluate the occurrence of unintended InDels in CRISPR/Cas-generated Ntrk1 KI mice.
- To compare the exomes of KI mice with wild-type controls.
- To investigate the origins of detected InDels.
Main Methods:
- Whole exome sequencing of F1 heterozygous Ntrk1 KI mice and wild-type controls.
- In vitro digestion assays to analyze InDel origins.
Main Results:
- Three InDels were identified in four KI mouse lines, absent in the control.
- InDels likely arose from de novo mutations, parental carry-over, or Cas9 off-target effects.
- The frequency of InDels was not high but significant.
Conclusions:
- CRISPR/Cas technology can introduce unintended InDels in KI mice.
- Thorough assessment for InDels is essential when using CRISPR/Cas for KI mouse generation.
- These findings highlight the need for rigorous quality control in genetically engineered mouse models.
