Zinc finger nucleases for targeted mutagenesis and repair of the sickle-cell disease mutation: An in-silico study

First, using the complete 1606 genomic DNA base pair (bp) sequences of the normal hemoglobin-beta (βA) chain gene, and the ZiFiT-CoDA-ZFA software preset at default, 57 three-finger arrays (ZFAs) that specifically bind 9 base-pair sequences within the normal hemoglobin-beta chain, were computationally assembled. Second, by serial linkage of these ZFAs to the Flavobacterium okeanokoites endonuclease Fok I― four ZFNs with unique specificity to >24？bp target-sequences at the genomic contextual positions 82, 1333, 1334, and 1413 of the βA chain-gene were constructed in-silico. Third, localizing the point-mutation of SCD at genomic contextual position ？69-70-71- bp (a position corresponding to the 6th codon) of the βA chain-gene, inspired the final design of five more ZFNs specific to >24？bp target-sequences within the 8,954？bp that are genomically adjacent to the 5′ end of the βA chain-gene.This set of 57 ZFAs and 9 ZFNs offers us gene-therapeutic precursors for the targeted mutagenesis and repair of the SCD mutation or genotype.