Combining single-strand oligodeoxynucleotides and CRISPR/Cas9 to correct gene mutations in ß-thalassemia-induced Pluripotent Stem Cells

Xiaohua Niu, Wenyin He, Bing Song, Zhanhui Ou, Di Fan, Yuchang Chen, Yong Fan, Xiaofang Sun
Source: J Biol Chem
Publication Date: (2016)
Issue: 291(32): 16576-85
Research Area:
Immunotherapy / Hematology
Stem Cells
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
4D-Nucleofector® X-Unit
Electroporation and Drug Selection——The sequence of the ssODNs used for correcting the HBB gene 41/42 mutation was as follows: before electroporation, ß-iPS-41/42 cells were grown in feeder-free adherent culture in chemically defined mTeSR (STEM CELL Technologies, Vancouver, Canada) on plates coated with Matrigel (BD Bioscience). Transfections were done using P3 Primary Cell 4D Nucleofector X Kit (Lonza). Specifically, the cells were pretreated with 10µM ROCK Inhibitor for 2 hours and dissociated into a single cell suspension with 1 mg/mL Accutase (Invitrogen). Subsequently, 8µg of Cas9 and gRNA plasmid and 2µg of ssODNs were mixed with 5x106 ß-iPS-41/42 cells and transferred into 100 µl of Nucleocuvette and conducted nucleofection using CB150 program. Cells were plated on Matrigel-coated plates in mTeSR1 medium supplemented with ROCK inhibitor for the first 24h. 0.5µg/mL puromycin was used to select cells for 2 days and changed to mTeSR1 medium without puryomycin until clones picking.
ß-Thalassemia (ß-Thal) is one of the most common genetic diseases in the world. The generation of patient-specific ß-Thal-induced pluripotent stem cells (iPSCs), correction of the disease-causing mutations in those cells, and then differentiation into hematopoietic stem cells offers a new therapeutic strategy for this disease. Here, we designed a CRISPR/Cas9 to specifically target the Homo sapiens hemoglobin ß (HBB) gene CD41/42(-CTTT) mutation. We demonstrated that the combination of single strand oligodeoxynucleotides with CRISPR/Cas9 was capable of correcting the HBB gene CD41/42 mutation in ß-Thal iPSCs. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm mutation correction, we verified that the purified clones retained full pluripotency and exhibited normal karyotyping. Additionally, whole-exome sequencing showed that the mutation load to the exomes was minimal after CRISPR/Cas9 targeting. Furthermore, the corrected iPSCs were selected for erythroblast differentiation and restored the expression of HBB protein compared with the parental iPSCs. This method provides an efficient and safe strategy to correct the HBB gene mutation in ß-Thal iPSCs