Electroporation-based CRISPR gene editing in adult buffalo fibroblast cells.

Electroporation is a widely used method for delivering CRISPR components into cells; however, it presents challenges when applied to difficult-to-transfect cells like adult buffalo fibroblasts. In this study, the ITGB2 gene (encoding the CD18 protein), plays vital for cellular adhesion and immune responses, was selected for editing experiments. To optimize electroporation conditions, we investigated parameters such as electric field strength, pulse duration, plasmid DNA amount, cuvette type, and cell type. The best transfection rates were obtained in a 4 mm gap cuvette with a single 20-millisecond pulse of 300 V using a 10 µg of all-in-one CRISPR plasmid for 106 cells in 100 µL of electroporation buffer. Increasing DNA quantity enhanced transfection rates but compromised cell viability. The 4 mm cuvette gap had high transfection rates than the 2 mm gap, and newborn cells exhibited higher transfection rates than adult cells. We achieved transfection rates of 10-12% with a cell viability of 25-30% for adult fibroblast cells. Subsequently, successfully edited the ITGB2 gene with a 30% editing efficiency, confirmed through various analysis methods, including T7E1 assay, TIDE and ICE analysis, and TA cloning. In conclusion, electroporation conditions reported here can edit buffalo gene(s) for various biotechnological research applications.

Report of a Chinese Cohort with Leukocyte Adhesion Deficiency-I and Four Novel Mutations.

PURPOSE: We aimed to report the characteristics of leukocyte adhesion deficiency-I (LAD-I) and four novel mutations in the ITGB2 gene in a Chinese cohort. METHODS: Seven patients with LAD-I were reported in our study. Clinical manifestations and immunological phenotypes were reviewed. The expression of CD18 was detected by flow cytometry. Next-generation sequencing (NGS) and Sanger sequencing were performed to identify gene mutations. RESULTS: The mean onset age of all the patients was 1.3 months. Recurrent bacterial infections of the skin and lungs were the most common symptoms. Most patients (6/7) had delayed cord separation. The number of white blood cells (WBC) was increased significantly, except that two patients had a mild increase in the number of WBC during infection-free periods. The expression of CD18 was very low in all patients. Homozygous or compound heterozygous mutations in the ITGB2 gene were identified in each patient. Four mutations were novel, including c.1794dupC (p.N599Qfs*93), c.1788C>A (p.C596X), c.841-849del9, and c.741+1delG. Two patients had large deletions of the ITGB2 gene. Five patients were cured by hematopoietic stem cell transplantation (HSCT). CONCLUSIONS: This study reported the clinical and molecular characteristics of a Chinese patient cohort. It is helpful in understanding the current status of the disease in China.

Molecular characterization of leukocyte adhesion deficiency-I in Indian patients: identification of 9 novel mutations.

PURPOSE: Leukocyte adhesion deficiency type-I (LAD-I) is caused by mutations in the ITGB2 gene, encoding the ß2-subunit of ß2-integrin (CD18) which leads to markedly reduced expression of CD18 on leukocytes resulting into recurrent life threatening infections. Here we aim to identify the molecular defects underlying LAD-I in Indian patients and correlate with the clinical presentation. METHODS: Blood was collected from 30 patients and their parents for absolute neutrophil count, expression of CD18 and CD11 by flow cytometry and DNA extraction. PCR and DNA sequencing of the ITGB2 gene was done for mutation characterization. RESULTS: Phenotypically, 22 patients were LAD-I(0), 1 was LAD-I(-) and 7 were LAD-I(+) showing no expression and reduced expression of CD18 respectively. Nine novel mutations in 15 patients and 11 known mutations in 16 patients were detected. Prenatal diagnosis was performed for 5 families. CONCLUSION: In this study 30 patients were phenotypically and genotypically evaluated for a less known disease LAD-I. Unavailability of curative options to majority of the patients and high cost of supportive care emphasize the need to increase awareness about a suspicious case so that timely management can be given to the patient and prenatal diagnosis can be offered to their families.