Integrate CRISPR/Cas9 for protein expression of HLA-B*38:68Q via precise gene editing

The determination of null- or low-expressed HLA alleles is clinically relevant in both hematopoietic stem cell transplantation and solid organ transplantation. We studied the expression level of a questionable (Q) HLA-B*38:68Q allele, which carries a 9-nucleotide (nt) deletion at codon 230–232 in exon 4 of HLA-B*38:01:01:01 using CRISPR/Cas9 gene editing technology. CRISPR/Cas9 gene editing of HLA-B*38:01:01:01 homozygous EBV B cell line resulted in one HLA-B*38:68Q/B*38:01:01:01 heterozygous and one HLA-B*38:68Q homozygous clone. Flow cytometric analysis of monoclonal anti-Bw4 antibody showed the protein expression of HLA-B*38:01:01:01 in homozygous cells was 2.2 fold higher than HLA-B*38:68Q/B*38:01:01:01 heterozygous cells, and the expression of HLA-B*38:68Q/B*38:01:01:01 heterozygous cells was over 2.0 fold higher than HLA-B*38:68Q homozygous cells. The HLA-B*38:68Q expression was further confirmed using anti-B38 polyclonal antibody. Similarly, the expression of the HLA-B*38:01:01:01 homozygous cells was 1.5 fold higher than that of HLA-B*38:68Q/B*38:01:01:01 heterozygous cells, and the HLA-B*38:68Q/B*38:01:01:01 heterozygous cells was over 1.6 fold higher than that of HLA-B*38:68Q homozygous cells. The treatment of HLA-B*38:68Q homozygous cells with IFN-γ significantly increased its expression. In conclusion, we demonstrate that HLA-B*38:68Q is a low-expressing HLA allele. The CRISPR/Cas9 technology is a useful tool to induce precise gene editing in HLA genes to enable the characterization of HLA gene variants on expression and function.

remains challenging. Therefore, it is important to determine the expression patterns of abnormally expressed HLA variants 7 .
The CRISPR (clustered regularly interspaced short palindromic repeats) is an adoptive immune system in bacteria that protects the bacterium from invading foreign genetic elements such as plasmid and bacteriophages 10 . The CRISPR/Cas9 system contains two components: a guide RNA (gRNA) and a CRISPR-associated endonuclease (Cas protein) 11,12 . The gRNA is a short RNA composed of a scaffold sequence needed for Cas-binding and a user-defined ∼20 nucleotide spacer that defines the genomic target to be modified 13 . The gRNA spacer sequence could be designed to target DNA sites with Protospacer Adjacent Motif (PAM) 14,15 . The most common PAM sequence recognized by Cas9 is NGG that is found directly downstream of the target DNA. The CRISPR/Cas9 cuts double strand DNA to generate double strand breaks (DSBs) between 3-4 bp upstream of the NGG PAM under the guidance of gRNA 16 . The DSBs can be repaired by non-homologous end joining (NHEJ), which is an error-prone process that introduces unpredictable insertions and deletions (indels); DSBs can also be repaired by homology directed repair (HDR) with the presence of DNA template, which induces desired DNA editing 11,12,17 . Two types of the DNA template can be used for HDR: a small single stranded DNA (ssDNA) oligonucleotide with 30-67 nt homology arms flanking the gene editing site 18 or a double stranded DNA (dsDNA) plasmid with long homology arms of 1-3 kb 19 .
The recent discovery of CRIPSR/Cas9 system provides a faster and more economical approach to gene editing 20 compared to the traditional zinc-finger nucleases (ZFNs) 21 and transcription activator-like effector nuclease (TALEN) methods 22 . The goal of this study was to generate homozygous and heterozygous cells carrying the HLA-B*38:68Q with deletion at codon 230-232 at exon 4 using CRISPR/Cas9 gene editing to study the effect of this mutation on HLA-B*38:01:01:01 expression.

Results
crRNA design and selection. We identified a new HLA B allele that is similar to HLA-B*38:01:01:01 except for a nine-nucleotide deletion (5′-CTTGTGGAG-3′) at codon 230 to 232 that results in a coding shift at α3 domain of HLA-B38 (Fig. 1A). The sequence was submitted to the GenBank database (accession number MF069211) and IMGT/HLA databases (submission number HWS10028807). Since the expression level of this novel B*38 allele is unknown, it was named HLA-B*38:68Q. To determine if the deletion at codon 230-232 affected the level of protein expression, the homozygous HLA-B*38:01:01:01 EBV B cell line TEM665 was used to generate homozygous HLA-B*38:68Q alleles to study its expression (Fig. 1B). GeneArt TM CRISPR Search and Design Tool was used to design crRNAs targeting the DNA sequences close to the 9-nt deletion site at exon 4 of   1C). crRNA1 (5′-GGATGGCGAGGACCAAACTC-3′) was designed to recognize −12 to −31 bp upstream of the 9-nt deletion, and crRNA2 (5′-TGGTCTGGTCTCCACAAGCT-3′) was designed to recognize −2 to +9 bp sequence of the 9-nt deletion. Both crRNAs share a ssDNA target with a 67-nt of left arm and a 30-nt of right arm of the deletion site (Fig. 1C). The crRNA1 and crRNA2 were then mixed with universal tracrRNA to form gRNA1 and gRNA2. Next, Cas9 protein (1.5 µg) and gRNA1/gRNA2 (360 ng) were mixed to form Cas9 ribonucleoprotein (RNP), respectively 18 . Twenty four electroporation conditions were tested to optimize transportation efficiency using Neon transfection system 23 (Supplemental Table 1). The program of the highest transportation efficiency (58.4%) was selected for transfecting HLA-B*38:01:01:01 homozygous EBV B cell line. Our results showed that gRNA1 induced 22.6% DSB cleavage and gRNA2 induced 13.8% DSB cleavage using GeneArt ® Genomic Cleavage Detection assay. The gRNA1 was chosen for transfection with ssDNA target due to its high efficiency.
Allograft rejection often correlates with increased cytokine production including IFN-γ 26 . We therefore tested if the expression of HLA-B*38:68Q will be stimulated by IFN-γ under inflammatory environment. Our results showed that in the HLA-B*38:68Q homozygous cells, the treatment of IFN-γ significantly increased the HLA-B expression and resulted in a binding of 36,036 ± 887 MFI, 1.9 fold higher than the HLA-B*38:68Q homozygous cells without IFN-γ treatment (19,379 ± 900 MFI, P < 0.0001, Fig. 6). Similarly, in the HLA-B*38:01:01:01 homozygous cells, the expression of HLA-B treated with IFN-γ was at 57,646 ± 357 MFI, which was 1.2 fold higher than the untreated group (46,642 ± 231 MFI, P < 0.0001). The results showed that although the expression of HLA-B*38:68Q can be upregulated by IFN-γ treatment, the upregulation of HLA-B expression was much lower compared to IFN-γ treated HLA-B*38:01:01:01 homozygous cells (Fig. 6).

Discussion
In this study, we reported efficient HLA-B*38:01:01:01 gene modification and expression in an EBV B cell line using the CRISPR/Cas9 system. We successfully introduced gene editing in 84% of clones and achieved precise deletion at codon 230-232 at exon 4 in 5 alleles. Similar to other publications, the CRISPR/Cas9 gene editing of HLA-B*38:01:01:01 involved DNA repair via either NHEJ or HDR pathway 27 . However, even in the presence of guided DNA templates, 72% of gene editing was through the NHEJ repair pathway compared to 10% in HDR pathway. HDR pathway provides desired repair of the target DNA in the presence of template DNA. The low incidence of HDR makes the selection of precise gene editing challenging. In order to achieve higher HDR gene editing efficiency, the DSBs induced by CRISPR/ Cas9 nuclease should be in close proximity to the edit site 18 . The homologous recombination rate could be increased with larger flanking sequences, therefore standard gene deletion/disruption protocols typically use flanking regions over 1 kb on either side of the target gene to increase HDR 28 . The evidence of using cell lines deficient in NHEJ pathways increased the levels of HDR suggesting these two pathways are competitive 29 . Recent studies have demonstrated the use of KU70, KU80 or DNA ligase IV to suppress key NHEJ molecule can increase HDR pathway 30 .
Our study successfully demonstrated that the deletion at  Currently, there are 44 questionable HLA alleles (Q) in IMGT/HLA data base 2 . The frequency of these questionable alleles has not been well established, particularly the HLA allele frequency is largely based on the Caucasian population. Therefore, the frequency of these questionable alleles may be underestimated in other populations. With the advancement of the full gene HLA sequencing by NGS technology, the laboratory is able to obtain high resolution HLA typing with minimum ambiguities. However, due to additional sequencing information on exons outside the antigen recognition sites (ARS) and introns, it is likely that more questionable alleles will be discovered. The knowledge of the expression level of these questionable alleles may be important for donor selection in HSCT. Petersdorf et al. demonstrated that increasing expression level of the patient's mismatched HLA-C allele was associated with increased risk of grades III-IV acute GVHD with odds ratio of 1.34 in HSCT 34 . In addition, the understanding of the expression level can also help to identify donors with the least immunogenic mismatches, or select donors to cross permissive immunological barriers for highly sensitized patients in solid organ transplantation. These questionable alleles could also be potential null alleles. Failure to identify HLA null alleles in donors may cause severe GVHD in HSCT 7 . Increasing knowledge of the expression level of HLA variant alleles will help to improve the understanding of HLA allogenicity in both HSCT and solid organ transplantation. The CRISPR/Cas9 system provides an effective tool to study the expression level of these variant HLA alleles. In addition, CRISPR/Cas9 can also introduce insertions and deletions at the UTRs, exons and introns to study the regulations and functions of HLA genes.
CRISPR/Cas9 has been used to facilitate correction of mutated genes in various diseases. Recently, CRISPR/ Cas9 has been used to treat single nucleotide polymorphism in the β-globulin gene to treat sickle cell diseases in a mouse model 35 . The chimeric antigen receptor (CAR) modified T cells have been applied to various cancers, especially in B cell hematologic malignancies 36 . With the application CRISPR/Cas9 system, Liu et al. 37 have successfully down regulated the expression of HLA class I and TCRα to generate a universal chimeric antigen receptor (CAR) T cells. The CRISPR/Cas9 technology has also come under the spotlight in transplantation. Entry of human immunodeficiency virus (HIV) into target cells requires both CD4 and CCR5 receptors 38 . A 32-base pair deletion in CCR5 (CCR5-Δ32) is associated with reduced HIV transmission risk and delayed disease progression 39 . In HIV+ patients with hematological malignancies, gene editing using CRISPR/Cas9 40 has been used to generate homozygous CCR5-Δ32 deletion in CD34+ cells to introduce HIV resistance. Currently, there are several ongoing clinical trials evaluating the safety of transplantation of CRISPR modified CCR5-Δ32 CD34+ cells in HIV+ patients with hematological malignances 41,42 . In conclusion, the CRISPR/Cas9 system is a powerful gene editing tool that can be used to study HLA gene expression and function and improve HLA matching in hematopoietic stem cell and solid organ transplantation. Modification of HLA gene expression by CRISPR/Cas9 also promises to provide new approaches for cellular therapies in transplantation. www.nature.com/scientificreports www.nature.com/scientificreports/

Material and Methods
HLA-B*38:68Q was submitted to the GenBank database (accession number MF069211) and IMGT/HLA database (submission number HWS10028807) with full genomic allele sequence as a questionable allele due to its unknown surface expression by our center in 2017 43 . Research approval for performing CRISPR/CAS9 on the sample was granted by the UCLA Institutional Review Board (IRB#14-000516).  Fig. 1B) was selected for gene editing, and in addition, a Bw6 homozygous EBV B cell line AOH749 (from AOH Workshop, AOH9004) and/or K562 (from UCLA Immunogenetics Center) which lacks HLA expression were used as negative controls for monoclonal or polyclonal antibody test. AOH749 expresses HLA A31, B65, Bw6, C8, DR1, DQ5, DP3 and DP0401, which does not cross reactive to the anti-Bw4 antibody. All cell lines were cultured in RPMI-1640 (GE Healthcare Life Sciences, USA) containing 20% FBS (Omega, USA), 1%
ssDNA target design/Homologous recombination assays. To create homologous recombination (HR) assays, two gRNAs target the sequence near the deletion site within the HLA-B gene were designed and synthesized 18 . The Cas9 RNPs were then used to transfect cells via Neon ® electroporation (Invitrogen, USA) for 24-well format electroporation transfection testing for B cell line. The genomic cleavage efficiency was then evaluated using the GeneArt ® Genomic Cleavage Detection kit (ThermoFisher, USA) at 48 h post transfection. The cleavage efficiencies were calculated based on the relative agarose gel band intensity, which was quantified using a high sensitivity DNA chip on TapeStation 2200 (Agilent, USA). Cleaved efficiency was calculated following the manufacturer's instruction. A program with voltage set at 1700 V, pulse width set at 20 ms, and one pulse was used for the subsequent study. The gRNA with highest editing was selected for the subsequent HR assays. For ssDNA target design, typically the mutation site was positioned at the center flanked by 67-nt to 30-nt on each side (Fig. 1C). To measure homologous recombination efficiency, the ssDNA target was co-transfected with Cas9 RNPs into cells via electroporation. The genomic loci were PCR-amplified using the corresponding primers and then subjected to GeneArt ® Genomic Cleavage Detection assay (ThermoFisher, USA) for restriction enzyme digestion.
Culture of single cell derived clones. Transfected cells were washed with 500 μL of PBS buffer (Corning, USA) and resuspended at the density of 8 cells/mL with a total volume of 50 mL. 100 μL of the cell suspension was transferred into the wells of the 96 well tissue culture plates to ensure each well contained a single cell. The plates were incubated at 37 °C, 5% CO 2 incubator (Thermo Scientific, USA). The plates were then scanned for single cell colonies as soon as small aggregates of cells are visible under a 4× microscope (usually after first week, depending on the growth rate of the cell line) to ensure the cell colonies were derived from a single cell. The cells were incubated for an additional 2-3 weeks to expand the clonal populations for further analysis and characterization.
Harvest single cell derived clones. Single cell derived clones were washed with 100 µL of 1× PBS buffer (Corning, USA). 1 × 10 5 of the cells from each clones were transferred into the PCR plate containing 25 µL "Direct lysis buffer". The "Direct lysis buffer" was made by adding 10 μL of Proteinase K (Thermo Scientific, USA) to 1 mL DirectPCR Lysis Reagent (Thermo Scientific, USA). The PCR plate was incubated at 55 °C for 30 mins to lyse the cells and followed by 95 °C for 45 mins to denature the Proteinase K.
HLA gene amplification and next generation sequencing. Multiplex long-range PCR were employed using AllType NGS assay (One Lambda, USA) to co-amplify 11 HLA loci including HLA-A, -B, -C, -DRB1,3,4,5 -DQB1 -DPB1, -DQA1 and -DPA1. HLA-A, -B, -C, -DQA1, and -DPA1 were amplified from 5′UTR to 3′UTR, and remaining loci are beginning at intron 1 to 3′UTR. Library construction was automated on the Biomek FX (Beckman coulter, USA). Sequence-ready libraries were validated and quantitated on the High Sensitivity D1000 ScreenTape (Agilent Technologies, USA) to allow for library normalization and equimolar pooling of all study samples on the Biomek FX (Beckman coulter, USA). Pooled libraries were diluted and loaded at Ion Chef (Thermo Scientific, USA) for template amplification. Sequencing on Ion S5 XL is followed the manufacture instruction (Thermo Scientific, USA). When the sequencing was done, the TSVEngine v1.2.0 (One Lambda, USA) was employed to analyze the data.
Single antigen antibody testing. Neat and serum at 1:2 dilution were treated with DTT and tested for HLA antibodies using the IgG-SAB Assay from One Lambda (Canoga Park, CA) as previously described 44 . Antibodies were considered positive if the MFI >1000 for HLA-A, -B, -DR, -DQ and >2000 was used for HLA-C and -DP to correct for the enhanced amount of HLA-C and -DP antigens conjugated to the Luminex beads compared to the lower cell surface expression on lymphocytes.
www.nature.com/scientificreports www.nature.com/scientificreports/ Flow cytometric analysis of HLA protein expression. Expression of HLA-B locus antigens in the edited cell lines containing the HLA-B*38:68Q allele and control cells were determined by flow cytometry using a FITC-conjugated monoclonal anti-IgG Bw4 antibody (One Lambda, USA). Approximately 10 5 cells were incubated with 0.5, 1, and 2 μL (10 mg/mL) anti-Bw4 monoclonal antibody on ice for 30 mins. Isotype control cells were incubated with 0.5 μL of FITC-conjugated mouse IgG secondary antibody (Jackson ImmunoResearch, USA). After staining, cells were washed with 1× PBS buffer (Corning, USA) containing 2% FBS (Omega, USA) and suspended in 300 μL of PBS/2% FBS. TEM665 homozygous HLA-B*38:01:01:01 EBV transformed B cell line was used as the positive control, AOH749 and K562 were used as the negative control cells. Samples were tested in triplicate. Analysis of HLA B locus expression was performed using FlowJo software version 10 (BD, USA).
Expression of HLA-B38 in the edited cell lines containing the HLA-B*38:68Q allele was determined using UCLA serum exchange sample that contains anti-B38 antibody but lacks Bw4 activity. Approximately 1.5 × 10 5 cells/tube were incubated with 25 μL UCLA serum exchange serum at neat and 1:2 dilution for 30 mins at room temperature. After incubation, cells were washed 4 times with PBS/2% FBS followed by labeling for 20 mins at 4 °C with an anti-human IgG FITC-conjugated antibody (Jackson ImmunoResearch, USA). Negative sera routinely used in the clinical lab were used as controls. Samples were tested in triplicate. Analysis of HLA-B38 expression was performed using FlowJo software version 10 (BD, USA). Statistical analysis. Each experiment of protein expression was tested in triplicate and the expression level is shown as mean fluorescence intensity (MFI) ± SD. Statistical analysis was performed the Student's t test or ANOVA on GraphPad Prism 7 (GraphPad, USA). P < 0.05 was denoted as significant.

Data Availability
All data generated or analyzed during this study are included in this article.