investigating the correction of ivs ii-1 (g> a) mutation in hbb gene in tls-12 cell line using crispr/cas9 system

Objective: beta-thalassemia is a group of inherited hematologic. the most hbb gene variant among iranian beta-thalassemia patients is related to two mutations of ivsii-1 (g>a) and ivsi-5 (g>c). therefore, our aim of this study is to use the knock in capability of crispr cas9 system to investigate the correction of ivsii-1 (g>a) variant in iran. materials and methods: in this experimental study, following bioinformatics studies, the vector containing puromycin resistant gene (px459) was cloned individually by designed rna-guided nucleases (grnas), and cloning was confirmed by sequencing. proliferation of tls-12 was done. then, the transfect was set up by the vector with gfp marker (px458). the px459 vectors carrying the designed grnas together with single-stranded oligodeoxynucleotides (ssodns) as healthy dna pattern were transfected into tls-12 cells. after taking the single cell clones, molecular evaluations were performed on single clones. sanger sequencing was then performed to investigate homology directed repair (hdr). results: the sequencing results confirmed that all three grnas were successfully cloned into px459 vector. in the transfection phase, the tls-12 containing px459-grna/ssodn was selected. molecular evaluations showed that the hbb gene was cleaved by the crispr/cas9 system, that indicates that the performance of non-homologous end joining (nhej) repair system. sequencing in some clones cleaved by the t7e1 enzyme showed that hdr was not confirmed in these clones. conclusion: ivs-ii-1 (g> a) mutation, which is the most common thalassemia mutation especially in iran, the crispr/ cas9 system was able to specifically target the hbb gene sequence. this could even lead to a correction in the mutation and efficiency of the hdr repair system in future research.