New Study Focusing on Discovering A New Approach to RNA Splicing Gene Editing

A recent study focusing on genetic editing being published online at the international academic journal Molecular Cell. It has been demonstrated that TAM (Targeted-AID induced mutagenesis) gene editing can be used to target RNA splicing cis-elements on DNA, thereby efficiently regulating RNA splicing for the study of RNA alternative splicing and for the treatment of human genetic diseases.

In eukaryotic cells, RNA splicing is an important part of gene expression. It is estimated that more than 75% of human genes involve more than one mRNA splicing pattern (variable splicing), most of which can be translated into functional proteins. However, comparing to the function of genes, the understanding of the physiological functions of alternative splicing is still very limited, which is mainly caused by the limited experimental means of regulating endogenous RNA splicing. Abnormal RNA splicing is also a direct cause of many diseases, it is estimated that 35 to 50% of human diseases are caused by abnormalities in gene splicing. Therefore, there is an urgent need to develop new methods for gene editing that regulate RNA splicing whether from academic research or clinical application.

The research team used to develop TAM (Targeted AID-induced mutagenesis), which is a Cas9 protein and cytosine deaminase AID with defective nuclease activity, and there are two characteristics of it being discovered. First, the C/G base can be randomly mutated to other bases on the sgRNA-targeted DNA, which can be used to analyze tumor resistance mutations and induce protein in vitro evolution. Second, after coupling the UNG inhibitor UGI, C can be efficiently mutated to T within a small window (5-6 bases) in the sgRNA targeting region.

In this new study, the researchers first noticed that more than 98% of the introns have conserved GU (intron start) and AG (intron tail) sequences. In this new study, the researchers first noticed that more than 98% of the introns have conserved GU (intron start) and AG (intron tail) sequences. With this strategy, taking TAM to induce G>A mutations in the splice site DNA, it is possible to induce alternative exon and exemplified exon skipping, altering the alternative splice site, and adjusting the choice of mutually exclusive exons (mutually exclusive exons), as well as inducing intron retention of small introns. Furthermore, if the C-containing T contained in the polypyrimidine tract upstream of the 3' splice site is mutated by TAM, the inclusion of the downstream exon can be promoted. Therefore, the "loss of function" and "gain of function" regulation for RNA splicing can be successfully achieved using TAM.

Finally, the researchers explored the feasibility of using TAM to repair Duchenne muscular dystrophy (DMD), which is a deadly genetic disease (1/4000 in men). The cause of this disease is that genetic mutations cause complete loss of Dystrophin protein, trigger muscle atrophy and spasm, and ultimately lead to failure of heart or lung function. If exon skipping is performed, an internal truncated Dystrophin protein can be produced, which can achieve a therapeutic effect on DMD. Therefore, the researchers constructed inducible pluripotent stem cells derived from patients with DMD who had a complete loss of Dystrophin protein due to exon deletion. By inducing a mutation in G>A at the splice site, the researchers achieved complete skipping of the target exon, restored Dystrophin protein expression in all TAM-expressing cells, and repaired myocardial cell defects.

https://www.creative-biogene.com/Services/Gene-expression-service.html

https://www.creative-biogene.com/Product/Gene-Editing-Kits.html

Share interesting but useful information!