|ZFIN ID: ZDB-PUB-180814-20|
Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection
Kc, R., Srivastava, A., Wilkowski, J.M., Richter, C.E., Shavit, J.A., Burke, D.T., Bielas, S.L.
|Source:||Scientific Reports 6: 32048 (Journal)|
|Registered Authors:||Richter, Catherine, Shavit, Jordan|
|PubMed:||27557703 Full text @ Sci. Rep.|
Kc, R., Srivastava, A., Wilkowski, J.M., Richter, C.E., Shavit, J.A., Burke, D.T., Bielas, S.L. (2016) Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection. Scientific Reports. 6:32048.
ABSTRACTCRISPR/Cas9 genome-editing has emerged as a powerful tool to create mutant alleles in model organisms. However, the precision with which these mutations are created has introduced a new set of complications for genotyping and colony management. Traditional gene-targeting approaches in many experimental organisms incorporated exogenous DNA and/or allele specific sequence that allow for genotyping strategies based on binary readout of PCR product amplification and size selection. In contrast, alleles created by non-homologous end-joining (NHEJ) repair of double-stranded DNA breaks generated by Cas9 are much less amenable to such strategies. Here we describe a novel genotyping strategy that is cost effective, sequence specific and allows for accurate and efficient multiplexing of small insertion-deletions and single-nucleotide variants characteristic of CRISPR/Cas9 edited alleles. We show that ligation detection reaction (LDR) can be used to generate products that are sequence specific and uniquely detected by product size and/or fluorescent tags. The method works independently of the model organism and will be useful for colony management as mutant alleles differing by a few nucleotides become more prevalent in experimental animal colonies.