Application of CRISPR-Cas12a technology in zebrafish to simulate ß-globin gene (HBB) mutations

Abstract

AbstractBackgroundThe human ß-globin (HBB) gene translated into the hemoglobin subunit beta (ß) of the hemoglobin protein. When mutated, it can lead to the blood disorder ß-thalassemia. Recently, the CRISPR-Cas12 technology has shown promising potential in treating different genetic abnormalities.ObjectivesThis study aimed to evaluate the feasibility of zebrafish hbbe1.1 gene editing via CRISPR-Cas12a gene-technology.MethodsIn current study, thalassemic mutations were replicated in zebrafish to improve gene editing. the embryonic hemoglobin genehbbe1.1was preferred over adult hemoglobinhbß-a1due to its early detection at larval stages. The CRISPR-Cas12 technology was utilised in combination with phosphorothioated “rescue template” (ssDNA L33P) to introduce base edits to the DNA sequence of zebrafish.ResultsThese experiments indeed resulted in the alteration of a single amino acid at the protein level. With the help of this genetic editing method, we were able to generate a novel zebrafish strain that carried specific amino acid alterations resembling the pathogenic mutations found in HBB for ß-thalassemia disease. However, in several cases additional indels or base alterations were observed.ConclusionOur findings suggests that MMEJ double stranded break repair mechanism causes more knock-in events and germline inheritance than HR-mediated events. Modifying the technique could improve results by reducing MMEJ frequency.