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z Dear Editor, Mutations in the β-globin gene, the essential component of adult hemoglobin (HbA; α2β2), results in either a production of aberrant sickle hemoglobin (HbS) leading to sickle cell disease (SCD) or an insufficient β-globin synthesis leading to β-thalassemia. These two major forms of β-hemoglobinopathies cause impaired erythropoiesis and life-threatening anemia. Clinical evidence has suggested that reactivation of fetal γ-globin (HBG) gene expression which is normally silenced after birth by certain genetic mutations can ameliorate the clinical course of β-hemoglobinopathies. 1,2 In β-thalassemia, elevated levels of fetal γ-globin interact with α-globin to form fetal hemoglobin (HbF; α2γ2) restoring the α/β-like globin ratio and in SCD the γ-globin reduces HbS polymerization. There are two major strategies for re-activation of HBG expression: reducing the expression of critical trans-acting repression factors (such as BCL11A) 3,4 or deletion of inhibitory cis-regulatory elements in the HBG1/2 promoter region. To develop a practical strategy for clinical implementation, we leveraged electroporation of a Cas9:sgRNA RNP to edit the HBG1/2 promoter in hematopoietic stem/progenitor cells (HSPCs). We successfully achieved an average editing efficiency of 85% in β-thalassemia patient-derived HSPCs, leading to increased γ-globin mRNA expression (up to 126% relative to α-globin) and an improved terminal erythroid differentiation rate. Importantly, we discovered that the BCL11A binding site (TGACCA: −114 to −119), which is critical for the repression of γ-globin, 5 is an ideal target for base editor hA3A-BE3 induced mutation and elevation of HBG expression.To mimic the effect of the naturally occurring Δ13 bp allele in the HBG1 promoter (−102 to −114) which was identified in patients with hereditary persistence of fetal hemoglobin (HPFH), we synthesized two sgRNAs (with or without 2′-O-methyl 3′ phosphorothioate modifications at three terminal nucleotides at both the 5′ and 3′ ends) (sgRNA1, sgRNA2) targeting the HBG promoter region (Fig. 1a). After electroporation of either RNP complex into immortalized erythroid precursor HUDEP-2 cells, 4 we found that chemical modification 6 of sgRNA enhanced editing efficiency and that sgRNA1 was more efficient than sgRNA2 (Supplementary information, Fig. S1a). The enhancement effect of chemical modification on sgRNAs was also confirmed in T cell receptor (TCR) and beta-2-microglobulin (B2M) loci in human primary T cells (Supplementary information, Fig. S1b). Hence, unless specified, we used the chemically modified sgRNA1 in the following experiments. After titration of the optimal RNP concentration in the HUDEP-2 cell line and in human CD34 + HSPCs, we successfully achieved editing efficiency over 80% in both cells (Supplementary information, Fig. S1c, d). After erythroid differentiation, the γ-globin mRNA level in the edited HUDEP-2 cells reached a mean of 22.7% relative to the total β-like globin mRNA, which showed a 4-fold increase compared with controls (mean 5.6%) (Su...

Hereditary tyrosinemia type I (HTI) is a metabolic genetic disorder caused by mutation of fumarylacetoacetate hydrolase (FAH). Because of the accumulation of toxic metabolites, HTI causes severe liver cirrhosis, liver failure, and even hepatocellular carcinoma. HTI is an ideal model for gene therapy, and several strategies have been shown to ameliorate HTI symptoms in animal models. Although CRISPR/Cas9-mediated genome editing is able to correct the mutation in mouse models, WT Cas9 induces numerous undesired mutations that have raised safety concerns for clinical applications. To develop a new method for gene correction with high fidelity, we generated a mutant rat model to investigate whether Cas9 nickase (Cas9n)-mediated genome editing can efficiently correct the First, we confirmed that Cas9n rarely induces indels in both on-target and off-target sites in cell lines. Using WT Cas9 as a positive control, we delivered Cas9n and the repair donor template/single guide (sg)RNA through adenoviral vectors into HTI rats. Analyses of the initial genome editing efficiency indicated that only WT Cas9 but not Cas9n causes indels at the on-target site in the liver tissue. After receiving either Cas9n or WT Cas9-mediated gene correction therapy, HTI rats gained weight steadily and survived. Fah-expressing hepatocytes occupied over 95% of the liver tissue 9 months after the treatment. Moreover, CRISPR/Cas9-mediated gene therapy prevented the progression of liver cirrhosis, a phenotype that could not be recapitulated in the HTI mouse model. These results strongly suggest that Cas9n-mediated genome editing is a valuable and safe gene therapy strategy for this genetic disease.