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Correction of RNA splicing defect in β654-thalassemia mice using CRISPR/Cas9 gene-editing technology

β654-thalassemia is a prominent Chinese subtype of β-thalassemia, representing 17% of all cases of β-thalassemia in China. The molecular mechanism underlying this subtype involves the IVS-2-654 C→T mutation leading to aberrant β-globin RNA splicing. This results in an additional 73-nucleotide exon between exons 2 and 3 and leads to a severe thalassemia syndrome. Herein, we explored a CRISPR/Cas9 genome editing approach to eliminate the additional 73-nucleotide by targeting both the IVS-2-654 C→T and a cryptic acceptor splice site at IVS-2-579 in order to correct aberrant β-globin RNA splicing and ameliorate the clinical β-thalassemia syndrome in β654 mice. Gene-edited mice were generated by microinjection of sgRNA and Cas9 mRNA into one-cell embryos of β654 or control mice: 83.3% of live-born mice were gene-edited, 70% of which produced correctly spliced RNA. No off-target events were observed. The clinical symptoms, including hematologic parameters and tissue pathology of all of the edited β654 founders and their offspring were significantly improved compared to those of the non-edited β654 mice, consistent with the restoration of wild-type β-globin RNA expression. Notably, the survival rate of gene-edited heterozygous β654 mice increased significantly, and live-born homozygous β654 mice were observed. Our study demonstrated a new and effective gene-editing approach that may provide groundwork for the exploration of β654-thalassemia therapy in the future.


In vitro transcription of sgRNAs
The DNA templates were prepared by PCR of pX459-sgRNA(G1/G2) plasmids as templet with specific primers (Table S1). The sgRNAs were in vitro transcribed with HiScribe™T7 Quick High Yield RNA Synthesis Kit (New England Biolabs) and purified with the MEGAclear kit (Life Technologies), according to manufacturer's instructions.

Cell culture and transfection
293T cells were cultured using Gibco® DMEM, high glucose, supplemented with 10% fetal bovine serum. 293T cells were seeded into 12-well plates to which a total of 1 μg of DNA plasmid pairs (pX459-sgRNA(G1/G2) plasmids, 0.5 μg for each plasmid) mixed with Lipofectamine 3000 (Invitrogen) were added according to the manufacturer's instructions. After 72 hours, genomic DNA was extracted from these cell lines.

Sub-cloning and genotyping
The PCR product was purified and ligated to pGEM-T vector and transformed to competent E. coli strain DH5α. After overnight culture at 37°C, randomly selected clones were sequenced by the Sanger method. The genotypes were determined by PCR of genomic DNA extracted from cells. ExTaq was activated at 95°C for 5 min, and PCR was performed for 34 cycles at 95°C for 30 sec, 58°C for 30 sec, and 72°C for 40 sec, with a final extension at 72°C for 7 min.

Targeted deep sequencing
DNA fragments containing the off-target sites were amplified from genomic DNA using KOD DNA polymerase (TOYOBP). Primers of targeted deep sequencing are listed in Table S1. Following amplification, the paired-end sequencing of PCR amplicons were gel-purified using QIAquick Gel Extraction Kit (Qiagen) and used for sequencing on Illumina Nextseq 500 (2×150) platform at Mingma, China. Data were analyzed using CRISPResso2. 2

HPLC
The samples for HPLC were prepared by collecting 50 μL whole blood and dissolving in 1mL pure water after filtration. 10 μL samples were loaded into ChromCore 300 C4 reversed-phase columns for polypeptides (300 Å, 5 μm, 4.6 mm × 250 mm). Individual globin chain levels were quantified on an Agilent 1260 instrument. A 40%-80% gradient mixture of 0.1% trifluoroacetic acid in water/acetonitrile was applied at a rate of 1 mL/min.

Whole-genome sequencing and data analysis
Genomic DNA was extracted from cells by using the DNeasy Blood and tissue kit (catalog number 69504, Qiagen) according to the manufacturer's instructions. WGS was performed at mean coverages of 50x by Illumina HiSeq X Ten. BWA (v0.7.12) was used to map qualified sequencing reads to the reference genome (mm10). The workflow of "Best Practice of GATK" 3 was used for sequence alignment to the reference genome (mm10) and variant (SNVs and indels) calling. The software involved includes BWA, 4 SAMtools, 5 and Genome Analysis Toolkit (GATK 4). 6 Structural variants (SV) were detected with Manta. 7 For analysis of sequence variations in β 654-ER mice, the Cas-OFFinder 8 Web tool was used to identify candidate off-target sites with up to 3 mismatches.

Statistical analysis
All experimental data were analyzed using GraphPad Prism 5. A Student's t-test was used for intergroup comparisons. Probability (P) values < 0.05 was considered statistically significant.   The red letters in the sequences represent mismatched bases.   Figure S1. Deep sequencing analysis of potential off-target sites in 293T cells edited by sgRNA G1+G2. Predicted off-target sites OT1 to OT20 are listed in Table S2.