Study On Genetic Correction Of β-thalassemia Patient-specific IPS Cell Lines By Single-strand Oligodeoxynucleotides Combined With Small Molecule Compounds

β-Thalassemia is caused by the human HBB gene mutation located on chromosome 11.β-Thalassemia,which is one of the most commonmonogenic disorders(MGDs),leads to deficiency or absence of hemoglobin synthesis.The technologies of induced pluripotent stem cells(iPSCs)andprovide insight into the treatments for MDGs including β-thalassemia.However,traditional genetic correctioncombined with the target vectors can be used to repair the gene mutation,which are of both low efficiency and leaveing a residualfootprint,including small insertions or deletions.This may caused some safety concerns in clinical applications.The development of artificial nucleoside technology,especially CRISPR / Cas9(Clustered Regulatory Interspaced Short Palindromic Repeat associated endonuclease 9)technology,provides great convenience for gene editing,which is simple,inexpensive,and can be done in general laboratories,greatly improving the gene operational efficiency and simplicity.In our study,we derived patient-specific iPSCs from a β-thalassemiahomologues patient.Then,we utilized single-strand oligodeoxynucleotides(ssODNs),high-fidelity CRISPR–Cas9nuclease and small molecules to achieve a seamless correction of the β-41/42(TCTT)deletion mutation in β-thalassemia patient-specific i PSCs with remarkable efficiency.Additionally,off-target effects analysis and whole-exome sequencing revealed that correctedcells exhibited minimal mutational load and no off-target mutagenesis.Afterwards,hematopoietic differentiation was performed on patient-specific iPSCs and genetically corrected iPSCs,the latter was able to expressnormal β-globin transcripts.Our study shows that ssODNs together with small molecule compounds is more effective in improving HDR-mediated gene repair efficiency,which provides a better theoretical basis for future clinical gene therapies.