Application of CRISPR-Cas12a technology in zebrafish to simulate ß-globin gene (HBB) mutations
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Background
The 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.
Objectives
This study aimed to evaluate the feasibility of zebrafish hbbe1.1 gene editing via CRISPR-Cas12a gene-technology.
Methods
In current study, thalassemic mutations were replicated in zebrafish to improve gene editing. the embryonic hemoglobin gene hbbe1.1 was preferred over adult hemoglobin hbß-a1 due 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.
Results
These 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.
Conclusion
Our 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.
