1. Minigene Assay to Evaluate CRISPR/Cas9-based Excision of Intronic Mutations that Cause Aberrant Splicing in Human Cells
- Author
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Patrick T. Harrison and David J. Sanz
- Subjects
Cas9 ,Strategy and Management ,Mechanical Engineering ,Metals and Alloys ,Intron ,Computational biology ,Biology ,Industrial and Manufacturing Engineering ,genomic DNA ,Exon ,RNA splicing ,Methods Article ,CRISPR ,splice ,Minigene - Abstract
The construction of Hybrid minigenes provides a robust and simple strategy to study the effects of disease-causing mutations on mRNA splicing when biological material from patient cells is not available. Hybrid minigenes can be used as splicing reporter plasmids allow RNA expression and heterologous splicing reactions between synthetic splicing signals in the vector and endogenous splicing signals in a cloned genomic DNA fragment that contains one or more introns and exons. Minigene-based assay has been used extensively to test the effect of mutations in the splicing of a target sequence. They can also be used to test the ability of CRISPR/Cas9 and one or more associated gRNAs to target specific sequences in the minigene, and determine the effect of these editing events on splicing. As an example, it is shown that CRISPR/Cas9-based, targeted excision of short intronic sequences containing mutations which create cryptic splice signals, can restore normal splicing in a CFTR Hybrid minigene.
- Published
- 2019