Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression.

BCL11A-XL directly binds and represses the fetal globin (HBG1/2) gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for ß-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (HSPC) engraftment and erythroid maturation. Intriguingly, neurodevelopmental patients with ZnF domain mutations have elevated HbF with normal hematological parameters. Inspired by this natural phenomenon, we used both CRISPR-Cas9 and base editing at specific ZnF domains and assessed the impacts on HbF production and hematopoietic differentiation. Generating indels in the various ZnF domains by CRISPR-Cas9 prevented the binding of BCL11A-XL to its site in the HBG1/2 promoters and elevated the HbF levels but affected normal hematopoiesis. Far fewer side effects were observed with base editing- for instance, erythroid maturation in vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B cell development in vivo, presumably because current base editing is not capable of precisely recapitulating the mutations found in patients with BCL11A-XL-associated neurodevelopment disorders. Overall, our results reveal that disrupting different ZnF domains has different effects. Disrupting ZnF4 elevated HbF levels significantly while leaving many other erythroid target genes unaffected, and interestingly, disrupting ZnF6 also elevated HbF levels, which was unexpected because this region does not directly interact with the HBG1/2 promoters. This first structure/function analysis of ZnF4-6 provides important insights into the domains of BCL11A-XL that are required to repress fetal globin expression and provide framework for exploring the introduction of natural mutations that may enable the derepression of single gene while leaving other functions unaffected.

Efficient gene editing in induced pluripotent stem cells enabled by an inducible adenine base editor with tunable expression.

The preferred method for disease modeling using induced pluripotent stem cells (iPSCs) is to generate isogenic cell lines by correcting or introducing pathogenic mutations. Base editing enables the precise installation of point mutations at specific genomic locations without the need for deleterious double-strand breaks used in the CRISPR-Cas9 gene editing methods. We created a bulk population of iPSCs that homogeneously express ABE8e adenine base editor enzyme under a doxycycline-inducible expression system at the AAVS1 safe harbor locus. These cells enabled fast, efficient and inducible gene editing at targeted genomic regions, eliminating the need for single-cell cloning and screening to identify those with homozygous mutations. We could achieve multiplex genomic editing by creating homozygous mutations in very high efficiencies at four independent genomic loci simultaneously in AAVS1-iABE8e iPSCs, which is highly challenging with previously described methods. The inducible ABE8e expression system allows editing of the genes of interest within a specific time window, enabling temporal control of gene editing to study the cell or lineage-specific functions of genes and their molecular pathways. In summary, the inducible ABE8e system provides a fast, efficient and versatile gene-editing tool for disease modeling and functional genomic studies.

Protocol for arrayed gRNA screening by base editors in mammalian cell lines using lentiviral system.

Base editing, a CRISPR-based genome engineering technique, enables precise single-nucleotide modifications while minimizing double-strand breaks. Here, we present a protocol for arrayed mutagenesis using base editors to identify regulatory elements within the gamma-globin locus. We describe steps for guide RNA (gRNA) cloning into lentiviral vectors, establishing stable cell lines with base editor expression, transducing gRNAs, and assessing editing efficiency. This protocol can be applied to diverse genomic regions and cell lines for arrayed screening, facilitating genetic research, and target discovery. For complete details on the use and execution of this protocol, please refer to Ravi et al. (2022)1.

Using Fetal Fibronectin Test to Reduce Hospital Admissions with Diagnosis of Preterm Labor: An Economic Evaluation Study.

Background: Preterm labor and delivery remain a major problem in obstetrics accounting for perinatal morbidity and mortality. The challenge is to identify those with true preterm labor to avoid unnecessary hospital admissions. The fetal fibronectin (FFN) test is a strong predictor of preterm birth and can help identify women with true preterm labor. However, its cost-effectiveness as a strategy for triaging women with threatened preterm labor is still debatable. Objective: To evaluate the effect of FFN test implementation on hospital resources by reducing the admission rate of threatened preterm labor in a tertiary hospital, Latifa Hospital, UAE. Methods: A retrospective cohort study of singleton pregnancies between 24 and 34 weeks of gestation who attended Latifa Hospital in the period of September 2015-December 2016, complaining of threatened preterm labor after the availability of an FFN test, and a historical cohort study for those who attended with threatened preterm labor before the availability of an FFN test. Data analysis was performed using a Kruskal-Wallis test, Kaplan-Meier, Fischer exact chi-square and cost analysis. The significance was set at p-value < 0.05. Results: In total, 840 women met the inclusion criteria and were enrolled. The relative risk of FFN for delivery at term was 4.35 times higher among the negative-tested compared to preterm delivery (p-value < 0.001). A total of 134 (15.9%) women were unnecessarily admitted (FFN tested negative, delivered at term) which yielded $107,000 in extra costs. After the introduction of an FFN test, a 7% reduction of threatened preterm labor admissions was recorded.

Heavy metal contamination in soil and groundwater around industrial areas of Kollam District, Kerala, India.

The present study was intended for assessing the contamination level of various heavy metals in surface soil and groundwater around the automobile workshops situated at different locations in the Kollam District of Kerala state, India. The procured soil and groundwater samples were analyzed for cadmium, zinc, iron, lead, nickel, chromium, copper, manganese, and arsenic using atomic absorption spectrophotometer by following standard procedures. The contamination level of these metals was assessed using the pollution indices like enrichment factor (EF), geo-accumulation index (Igeo), contamination factor (CF), and pollution load index (PLI). The results revealed that the concentrations of all analyzed metals in the surface soils of the automobile workshops were higher than the control. On the contrary, the concentration of no heavy metal in the groundwater was either equal to or higher than the limit prescribed by WHO. However, the concentration levels of Fe, Pb, Cu, and Zn were either equal to or higher than the control values. Based on the Igeo, CF, and EF, it was found that the contamination intensity of the heavy metals in soil decreased in the following order: Fe > Pb > Cd > As > Cr > Zn > Cu > Ni > Mn. From the results of PLI, it was interpreted that the sampling sites S2, S4, and S5 were highly polluted. Non-contamination of underground water from the age-old workshops is the uniqueness of the present study against the other studies, which were completed in alluvial formations with inverse results. In the studied region, the groundwater is stored in the hard rock formations and its hydraulics remains different from alluvial aquifers.

Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin.

Naturally occurring point mutations in the HBG promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous HBG proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of HBG promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T > C and -124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.

Efficient and error-free correction of sickle mutation in human erythroid cells using prime editor-2.

Sickle cell anaemia (SCA) is one of the common autosomal recessive monogenic disorders, caused by a transverse point mutation (GAG > GTG) at the sixth codon of the beta-globin gene, which results in haemolytic anaemia due to the fragile RBCs. Recent progress in genome editing has gained attention for the therapeutic cure for SCA. Direct correction of SCA mutation by homology-directed repair relies on a double-strand break (DSB) at the target site and carries the risk of generating beta-thalassaemic mutations if the editing is not error-free. On the other hand, base editors cannot correct the pathogenic SCA mutation resulting from A > T base transversion. Prime editor (PE), the recently described CRISPR/Cas 9 based gene editing tool that enables precise gene manipulations without DSB and unintended nucleotide changes, is a viable approach for the treatment of SCA. However, the major limitation with the use of prime editing is the lower efficiency especially in human erythroid cell lines and primary cells. To overcome these limitations, we developed a modular lenti-viral based prime editor system and demonstrated its use for the precise modelling of SCA mutation and its subsequent correction in human erythroid cell lines. We achieved highly efficient installation of SCA mutation (up to 72%) and its subsequent correction in human erythroid cells. For the first time, we demonstrated the functional restoration of adult haemoglobin without any unintended nucleotide changes or indel formations using the PE2 system. We also validated that the off-target effects mediated by the PE2 system is very minimal even with very efficient on-target conversion, making it a safe therapeutic option. Taken together, the modular lenti-viral prime editor system developed in this study not only expands the range of cell lines targetable by prime editor but also improves the efficiency considerably, enabling the use of prime editor for myriad molecular, genetic, and translational studies.

Utility of ferritin and inflammatory biomarkers in the diagnosis of different stages of breast cancer.

OBJECTIVES: To assess the utility of ferritin and inflammatory biomarkers in the diagnosis of stages of breast cancer. METHODS: The study consisted of 4 groups of 20 women, including the healthy control. The patients of group 1 comprised of stages I and II, group 2: stage III and group 3: stage IV of the disease. High sensitive C-reactive protein (hsCRP) and hepcidin were estimated by enzyme linked immunosorbent assay and ferritin by chemiluminescence immunoassay. The study was carried out in 2018 at Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry-6, India. RESULTS: The breast cancer disease progression correlated negatively with hemoglobin (Hb) (r= -0.6937, p<0.0001) and with ferritin levels, had a positive correlation (r=0.8444, p<0.0001). Ferritin negatively correlated with Hb among the subjects of the study (r= -0.6130, p<0.0001). Hepcidin correlated positively with hsCRP (r=0.998, p<0.0001). The ferritin/Hb ratio >3.9516 was used to identify the possibility of breast cancer utilizing the receiver operator curve values (area under curve [AUC]=0.997, sensitivity: 96.7, specificity:100). Ferritin values >103.4 were used to differentiate stage 4 from stage 3 of the disease (AUC: 0.893, sensitivity: 100, specificity: 85). CONCLUSION: Ferritin and ferritin/Hb are helpful in the differential diagnosis of stages of breast cancer.

CRISPR/Cas based gene editing: marking a new era in medical science.

CRISPR/Cas9 system, a bacterial adaptive immune system developed into a genome editing technology, has emerged as a powerful tool revolutionising genome engineering in all branches of biological science including agriculture, research and medicine. Rapid evolution of CRISPR/Cas9 system from the generation of double strand breaks to more advanced applications on gene regulation has made the wide-spread use of this technology possible. Medical science has benefited greatly from CRISPR/Cas9; being both a versatile and economical tool, it has brought gene therapy closer to reality. In this review, the development of CRISPR/Cas9 system, variants thereof and its application in different walks of medical science- research, diagnostics and therapy, will be discussed.