2.1. Strains and reagents:
The T4 DNA ligase (400000 U/mL, NEB #M0202S), and the BbsI restriction enzyme (10000 U/mL, NEB # R0539S) was purchased from the New England Biolabs (NEB) company to carry out the ligation and digestions respectively. The pSpCas9 (BB)-2A-GFP (PX458) (Plasmid # 48138) was supplied by Prof. A. Mani (Yale Cardiovascular Research Center, University of Yale, New Haven, Connecticut, USA). Human embryonic stem cell (HEK) 293T was provided from the biotechnology research center in Shiraz University of Medical Sciences, Iran. Lipofectamine 2000 (Invitrogen # 11668027) and Opti-MEM (Gibco # 31985) was purchased from Thermofisher Company to meet the requirement of an efficient transfection process.
Designing guide RNAs and constructing the gRNAs expressing vector
The online CRISPR tool of http://crispor.tefor.net/ was used to design a pair of guide RNAs targeting separate sites within B2M exon 1 (BME) (Table 1). The two gRNAs were designed to target distinct sites separated approximately 2.2 kb within the B2M gene located in chromosome 15. The gRNA_ 1 and gRNA_2 harbored complementary sequences to the sense strand of exon 1 and antisense strand of intron 1 respectively. In order to facilitate the cloning process of gRNAs, an adapter sequence was incorporated according to the requirement of the vector and restriction enzyme. Furthermore, several factors were taken into account, upon designing gRNAs sequences including on-target activity, specificity, and the presence of 5'-NGG sequence at the 3' end of gRNAs sequences to meet the PAM requirement of Cas9 enzyme. The double-stranded form of each guide RNA was cloned into the PX458 expression plasmid via BbsI restriction sites. To monitor the transfection efficacy and the transcription of the two guide RNAs, the Green fluorescent protein (GFP), which served as a reporter protein was exploited. Digestion and ligation were performed simultanously in a total of 10 μl reaction mixture per each gRNA. In order to do so, 1 μl annealed gRNAs oligos, 400 ng circular PX458 vector, 0.5 μl BbsI restriction enzyme (10000 U/mL), 1μl of 10x NEB buffer, 0.5 μl T4 DNA ligase (400,000 units/ml), and 1 μl of 10× NEB T4 DNA ligase buffer were supplemented by H2O to the total volume of 10 μl. Finally, the test tubes were placed in a thermal cycler with the following pre-set settings: the first step entailed 3 cycles of two-part temperature regulation, the first part was at 37° C for 20 min, and the second part was at 16° C for 15 min, the second step entailed one cycles of two-part temperature regulation, the first part was at 37° C for 10 min, and the second part was at 55° C for 15 min. Proper vector construction was confirmed by PCR using CrB2M_1 and CrB2M_2 primers (Table2). The results were further confirmed by sequencing.
Cell culture and transfection
The HEK293T cell line was selected for genetic manipulation of the B2M gene due to its high-rate transfection efficiency, docility, and fast growth. The transfection was mediated by the intrinsic properties of Lipofectamine. To do so, the cloned gRNAs were encapsulated in the Lipofectamine reagent, and the cells were simultaneously supplemented with both gRNAs. In order to investigate the transfection efficiency, the FACSAria™ cell sorter was exploited. HEK293T cell line was cultured in Dulbecco’s modified Eagle’s medium (Gibco), supplemented with 10% Fetal bovine serum (Gibco) and 500μl Penicillin-Streptomycin. The cell passaging was performed upon reaching 90% confluency every 2 to 3 days. Twenty-four hours prior to transfection, a six-welled plate was seeded with 5×105 cells in each well, and cultivated in 2 mL growth media devoid of the Penicillin-Streptomycin antibiotics. The transfections were mediated by Lipofectamine 2000 (Invitrogen) encapsulating the cloned gRNAs according to the procedure published by Pankaj K. Mandal (Mandal et al., 2014). To obtain the optimum result the final concentration of each gRNA was reduced to half.
Isolation of transfected cells via fluorescence-activated cell sorting (FACS) and single cell preparation
Forty-eight hours ensuing the transfection, the GFP expressing cells (GFP+) were separated from others via the FACS, and subsequently were cultivated in complete growth media for 3 to 4 days to expand into 80% of confluency, and then the cell suspension was subjected to serial dilution to determine the zygosity of single cells. To do so, the cell suspension with a concentration of 106 cells per 1 mL was provided and subsequently was serially diluted up until reaching the final concentration of 1 cell per 100 μL. Every single cell was supplemented with an additional 100 μL growth media to propagate.
Zygosity determination via polymerase chain reaction and sequencing confirmation
The distinct clones of cells each derived from single-cell expansion were subjected to genomic DNA extraction. The extracted DNAs were amplified by PCR using primers depicted in (Table 2). The PCR product was analyzed by gel electrophoresis and further purification. The gel-purified PCR products were cloned into a TA vector using the TOPO TA cloning kit (Thermo Fisher Scientific) according to the manufacturer's instruction for sequencing analysis. The results demonstrated the zygosity state of each clone, including homozygous, heterozygous, and wild-type states, each signifying single allele mutation, double allele mutation, and no mutation respectively.