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Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection
R Kc1, A Srivastava1, J M Wilkowski1
1Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Scientific Reports
|August 26, 2016
Summary
CRISPR/Cas9 genome editing creates challenges for genotyping. A new ligation detection reaction (LDR) method offers a cost-effective, sequence-specific solution for managing CRISPR-edited alleles in model organisms.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR/Cas9 genome editing enables precise mutant allele creation in model organisms.
- Genotyping and colony management are complicated by the precision of CRISPR/Cas9 mutations.
- Traditional genotyping methods are less effective for alleles generated by non-homologous end-joining (NHEJ) repair.
Purpose of the Study:
- To develop a novel, cost-effective, and sequence-specific genotyping strategy.
- To enable accurate and efficient multiplexing of small insertion-deletions and single-nucleotide variants from CRISPR/Cas9 editing.
- To provide a robust method for managing colonies with CRISPR-edited alleles.
Main Methods:
- Utilized ligation detection reaction (LDR) for genotyping.
- Generated sequence-specific products detectable by size and/or fluorescent tags.
- Applied the method to alleles created by CRISPR/Cas9 genome editing.
Main Results:
- Demonstrated that LDR can generate sequence-specific products.
- Showcased the ability to uniquely detect products by size and/or fluorescent tags.
- Validated the method's effectiveness for genotyping small insertion-deletions and single-nucleotide variants.
Conclusions:
- Ligation detection reaction (LDR) is a viable and efficient genotyping strategy for CRISPR/Cas9 edited alleles.
- The developed method is organism-independent and suitable for colony management.
- This approach addresses the challenges of genotyping precise mutations generated by CRISPR/Cas9 technology.
