Lymphoblastoid and Jurkat cell lines are useful surrogate in developing a CRISPR-Cas9 method to correct Leukocyte adhesion deficiency genomic defect.

Ahmad Ramadan ¹ Noureddine Ben Khalaf ² Khaled Trabelsi ³ HeLa Bakhiet ⁴ Imen Ben Mustapha ¹ Mohamed Ridha Barbouche ⁵ M-Dahmani Fathallah ^6*

• ¹ Laboratory of Transmission, Control and Immunobiology of Infections (LR11IPT02), Institute Pasteur Tunis, Faculty of Medicine, Tunis El Manar University, Tunis, Tunisia
• ² Department of Molecular Medicine, College of Medicine and Health Sciences, Department of Life Sciences, King Fahd Chair of Medical Biotechnology, College of graduate studies Arabian Gulf University., Manama, Bahrain
• ³ Laboratory of Molecular Microbiology, Vaccinology and Biotechnology Development. Institute Pasteur Tunis. Tunis El Manar University, Tunis, Tunisia
• ⁴ Department of Molecular Medicine, College of Medicine and Health Sciences, Arabian Gulf University, Manama, Bahrain
• ⁵ Department of Microbiology, Immunology and Infectious Diseases, College of Medicine and Health Sciences, Arabian Gulf University, Manama, Bahrain
• ⁶ Department of Life Sciences, King Fahd Chair of Medical Biotechnology, College of graduate studies Arabian Gulf University, Manama, Manama, Bahrain

We herein describe a method that uses Jurkat and patient-derived lymphoblastoid cell lines as surrogates to progenitor cells to correct a gene defect in the ITGB2 gene causing severe leukocyte adhesion deficiency type one (LAD1). LAD1 is an inborn error of immunity caused by small mutations in the ITBG2 gene, which codes for the beta 2-integrin subunit (CD18). These defects translate into a lack or deficient expression of the leukocyte adhesion molecules beta 2 integrin heterodimers CD18/CD11a, b and c. Stable restoration of the expression levels of these molecules is the ultimate cure for LAD1. Therefore, LAD1 is a good candidate for gene editing to correct the genomic defects underlying the disease. In this CRISPR-Cas9 method, we first used an in silico tool (CRISPOR) to predict three good gRNAs and tested them experimentally in Jurkat cell line that expresses wild-type ITGB2 gene. Thus, we selected the gRNA that showed the highest level of genomic DNA cleavage. We used this gRNA with espCas9 to test five HDRs (single-stranded donor oligonucleotides or ssODNs) for the repair of genomic defects in two LAD1 patients’ lymphoblastoid cell lines. We used cytofluorometry to show that the level of CD18 expression by the patients-edited cells reached 23% which in clinic can alleviate the clinical symptoms of LAD1 from severe to moderate. Whole-genome sequencing revealed accurate, “scareless” genomes with no off-target DNA insertions. This method is useful for determining the best gRNA/HDR combination to correct genomic defects in the patients’ hematopoietic stem cells or CD34+ peripheral blood cells.