CRISPR/Cas9 gene correction of HbH-CS thalassemia-induced pluripotent stem cells

Abstract

Haemoglobin (Hb) H-constant spring (CS) alpha thalassaemia (- -/-α^CS) is the most common type of nondeletional Hb H disease in southern China. The CRISPR/Cas9-based gene correction of patient-specific induced pluripotent stem cells (iPSCs) and cell transplantation now represent a therapeutic solution for this genetic disease. We designed primers for the target sites using CRISPR/Cas9 to specifically edit the HBA2 gene with an Hb-CS mutation. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm the mutation correction, we verified that the purified clones retained full pluripotency and exhibited a normal karyotype. This strategy may be promising in the future, although it is far from representing a solution for the treatment of HbH-CS thalassemia now.

Keywords: CRISPR/Cas9; HbH-CS thalassemia; Induced pluripotent stem cells (iPSCs).

Fig. 1

Validation of iPSCs (SU-iPS-1). a Immunocytochemistry of SU-iPS-1 cells for the detection of TRA-1-81, SSEA4, SOX2, and OCT4. b The iPSCs spontaneously differentiated into cells that highly expressed AFP, SMA, and NESTIN. c Teratoma formation. d The results of pluripotent validation included quantitative PCR analysis of the expression of OCT4, SOX2, and NANOG

Fig. 2

Specific RNA-guided Cas9 nuclease-mediated HBA2 gene correction. a Schematic overview of the gene-targeting strategy for the human HBA2 c.427T>C mutation. b Sequencing results of the HBA2 c.427T>C mutation site before and after gene correction

Fig. 3

Pluripotency of gene-corrected α-Thal iPSCs (C-SU-iPS-1). a Immunostaining for the pluripotent markers SSEA-4, TRA-1-81, SOX2, and OCT4. b The SCID mouse results of the initiation of teratoma formation for all three germ layers. c STR analysis showed the same origin of C-SU-iPS-1 and SU-iPS cells with PBMCs

Fig. 4

No obvious genome change was detected in SU-iPS-1 and C-SU-iPS-1 cells. a The gene-corrected clones maintained pluripotent genes, such as NANOG, OCT4, and SOX2. b Copy number variations (CNVs), as well as single-nucleotide variations (SNVs) (c) and indels (d), were detected in the SU-iPS-1 and C-SU-iPS-1 cells (e) with exome sequencing, which showed no obvious mutations in these sites

Fig. 5

Differentiation of C-iPSCs into HSCs and erythrocytes. a Flow cytometry analysis of CRISPR/Cas9-corrected C-SU-iPS-1 cells, the parental cell line (SU-iPS-1) and wild-type iPSCs using the surface marker CD34 after differentiation for 14 days. b, c Compared with the Hb-CS iPS cell lines, the gene-repaired Hb-CS cell lines did not show any significant difference in haematopoietic differentiation efficiency based on the colony-forming assay but showed differences using real-time quantitative PCR (d)