Characterization of CRISPR-Cas9 target sites in the MHC locus

Murine RAW264.7 macrophages, a well-characterized antigen-presenting cell line derived from the Balb/c mouse strain, offer a suitable model system to evaluate reprogramming in the highly polymorphic MHC locus. Our overall strategy was based on using CRISPR-Cas9 to generate two DSBs in the native MHC-I H2-Kd gene while also providing a replacement donor template containing an orthogonal MHC-I allele of H2-Kb (derived from the C57BL/6 mouse strain) and homology regions flanking either side of the DSBs (Fig. 1a). HDR would then facilitate the exchange of the >3 kb MHC alleles. No selection marker (i.e., fluorescence reporter gene or antibiotic resistance gene) is required to be incorporated into the HDR donor because cells surface express MHC, thus fluorescence labeling with H2-Kd- and H2-Kb-specific antibodies can be used for selection. In order to find gRNA sites in the MHC locus of RAW264.7 macrophages, we first amplified and Sanger sequenced the H2-Kd gene and corresponding flanking regions of up to 1 kb. The exons of the H2-Kd allele share 91% nucleotide (nt.) identity with those of the H2-Kb allele from the C57BL/6 genome, with the most diversity located in the peptide binding regions encoded in exons 2 and 3 (Supplementary Fig. S1a). The CRISPR design tool (http://crispr.mit.edu23) was used to identify gRNA sites (compatible with S. Pyogenes Cas9 and its protospacer adjacent motif (PAM, 5′-NGG)) in the sequenced H2-Kd gene and the C57BL/6 genome was used as a reference for off-target events (a reference BALB/c genome was not available). Due to the inherent high degree of similarity between MHC genes, finding unique gRNA sites in this locus is especially challenging. We identified a selection of potentially H2-Kd-exclusive gRNAs sites within the exons at the 5′ and 3′ portion of the gene (Fig. 1b). To minimize the size of the required donor template, we limited the search for gRNA sites to the region between exons 2 and 7. Thus, we were able to exclude exon 1 as a target as it encodes the MHC signal peptide that is cleaved upon expression, and exon 8, as it shares 100% nt. identity between the two alleles. Each gRNA was cloned into the CRISPR-Cas9 plasmid pX458 (pSpCas9(BB)-2A-GFP), where Cas9 and green fluorescence protein (GFP) are expressed under the control of the same promoter, but as two separate proteins due to the self-cleaving T2A peptide22. The resulting pX458 plasmids were individually electroporated (nucleofection protocol) into wildtype RAW264.7 cells; at ~24 h, cells expressing Cas9 (via 2A-GFP) were isolated by FACS and expanded. The activity of Cas9 at each gRNA site was determined by measuring NHEJ via Surveyor nuclease assays27. We observed strong Cas9-induced cleavage (with the expected fragment sizes) at each of the gRNA sites tested (Fig. 1b,c). Based on their strong NHEJ activity, gRNA10 and gRNA13 were evaluated for multiplexed targeting. After simultaneous electroporation with pX458-G10 and –G13, deletion of the genomic region in between these two gRNA was verified by PCR (Fig. 1d).

Figure 1: CRISPR-Cas9 targeting of the MHC locus. (a) Overall schematic of the experimental approach for the exchange of MHC alleles by CACE. Immortalized RAW264.7 cells carrying the H2-Kd MHC I allele are cleaved by Cas9 in the presence of a donor template containing the 3.4 kb H2-Kb allele. HDR mediates seamless exchange of the template allowing for the presentation of new peptides (e.g., SIINFEKL) in the MHC complex. (b) The location of gRNAs in H2-K1 locus, exons are highlighted in green and PCR primers used for amplification of the cleaved loci are shown as black arrows. The size in bp for expected cleavage products are indicated for each guide site. Deletion at the locus is detected with the flanking primer pair p1/p4. (c) Surveyor assay cleavage products following electroporation of cells with CRISPR-Cas9 plasmid (px458) with corresponding gRNA (10–13). Fragments were run with (+S) and without (-S) the addition of Surveyor enzyme. Red arrows indicate the detected cleavage products. (d) Agarose gel of genomic PCR products from RAW264.7 cells transfected simultaneously with px458-G10 and px458–G13 show deletion at the H2-K1 locus. Full size image

Reprogramming MHC specificity in RAW264.7 macrophages

To reprogram the MHC H2-Kd allele of RAW264.7 cells, a ~4 kb exchange template was constructed based on the H2-Kb allele (exons 2–7), which was derived from genomic DNA of the JAWSII cell line (originating from the C57BL/6 mouse strain). The H2-Kb region was flanked by left and right homology arms (~350 bp each) corresponding to RAW264.7 BALB/c genome (Fig. 1a). In order to prevent Cas9 cleavage of the exchange template, the PAM site was modified (NGG > NGA) or silent mutations were created within the gRNA recognition sequence (Supplementary Fig. S1b,c). The donor template, generated by PCR and thus in a linear format, was electroporated with pX458-G10 and pX458-G13 plasmids into RAW264.7 macrophages. After ~24 h, cells were isolated by FACS for Cas9 expression (via 2A-GFP) and expanded in culture. In order to detect cells that had undergone MHC replacement we used a flow cytometry-based assay that relied on differential monoclonal antibody labeling of H2-Kb and H2-Kd (Supplementary Fig. S1d). To further improve the specificity of detection for H2-Kb expression, we used a monoclonal antibody that had specificity for the H2-Kb allele only when bound to a cognate peptide derived from ovalbumin (OVA 257 – 264 or SIINFEKL)27. This peptide bound selectively and with high affinity to the H2-Kb MHC allele but not the H2-Kd allele (Supplementary Fig. S1d). In RAW264.7 cells that received pX458-G10 and –G13 plasmids alone (without donor template), we observed substantial knockout of the endogenous MHC gene, as 34% of cells no longer surface expressed H2-Kd (Fig. 2a). When the exchange template was included, an additional small population of cells (0.48%) appeared which were positive for H2-Kb-SIINFEKL and negative for H2-Kd. Single-cell clones were sorted from this population and expanded for further analysis.

Figure 2: Generation and characterization of MHC-reprogrammed RAW264.7 cells. (a) Representative flow cytometry dot plot shows cells 24 h after electroporation with px458-G10, px458-G13 and linear donor H2-Kd template, cells were sorted for Cas9-2A-GFP (488 nm) expression (Far left). Representative flow cytometry dot plots show expression of H2-Kd and H2-Kb/SIINFEKL. Cells electroporated with px458-G10, px458-G13 and linear donor H2-Kb template show a population of H2-Kb positive cells in the Q1 gate, these cells were single-cell sorted (Far right). (b) Representative dot plot shows the single-cell sorted F4 clone is negative for wildtype H2-Kd expression and positive for H2-Kb/SIINFEKL expression, while showing a similar H2-Kb/SIINFEKL expression level of control JAWSII cells. (c) The location of primer sets used to interrogate the presence of mRNA derived from both H2-Kd (p5/p6) and H2-Kb (p7/p8) alleles. (d) Agarose gels show PCR products from mRNA, indicating expression from the H2-K locus for both H2-K alleles. RAW264.7 (H2-Kd+) and JAWSII (H2-Kb+) cells were used as controls. (e) Sequencing of the generated PCR product from clone F4 shows correct splicing of exons 3 and 4 that are separated by a ~1700 bp intron. Full size image

Flow cytometry analysis on several of the single-cell colonies revealed high expression levels of the new H2-Kb protein with no detectable expression of native H2-Kd. The reprogrammed RAW264.7 cells expressed H2-Kb at similar levels to that of JAWSII dendritic cells (Fig. 2b and Supplementary Fig. S1e). To further confirm expression of the newly introduced H2-Kb allele, total mRNA was isolated and used to generate cDNA from each of the single-cell derived clones. The resulting cDNA was used in a PCR reaction with primers flanking exons 3 and 4 of the H2-Kb and H2-Kd genes, respectively (Fig. 2c). PCR products of 153 bp should only be generated if successful splicing of exons 3 and 4 occurs. All reprogrammed cell lines showed robust expression of H2-Kb transcripts, while residual H2-Kd transcripts were not detected (Fig. 2d). Sanger sequencing of these PCR products verified correct allelic expression and showed the correct splicing junction sequence for each clone, matching that of wildtype H2-Kd expression in JAWSII cells (Fig. 2e).

Reprogrammed MHC cell lines activate T cells

We next confirmed that the newly expressing MHC H2-Kb alleles in reprogrammed RAW264.7 cells were capable of providing functional immune activity. To upregulate MHC expression and ensure a mature cell phenotype, LPS and IL-4 cytokines were added to cultures for 24 hours. Flow cytometry analysis revealed a moderate upregulation (~2-fold) of the maturation markers CD80 and CD86, while there was a nearly 2-fold upregulation in H2-Kb expression with LPS (10 μg/ml) (Supplementary Fig. S2a). The addition of IL-4 (10 ng/ml) had a less pronounced but detectable increase in the expression of these markers. The matured cells were incubated with the SIINFEKL peptide (H2-Kb-specific) and X-ray irradiated to prevent proliferation. The irradiated RAW264.7 macrophages were then co-cultured with a CD8+ T cell hybridoma reporter cell line (B3Z) (Fig. 3a). B3Z T cells express a TCR that is specific for the MHC-peptide complex of H2-Kb-SIINFEKL, they have also been engineered to secrete β-galactosidase upon TCR engagement21. All of the reprogrammed RAW264.7 clones were able to induce strong β-galactosidase expression in B3Z T cells, which was comparable to the positive control of JAWSII dendritic cells (Fig. 3b and Supplementary Fig. S2b). Activation of B3Z T cells was completely dependent on the presence of SIINFEKL peptide in the co-culture. The parental RAW264.7 macrophage cells promoted very little β-galactosidase expression from B3Z cells (comparable to B3Z cells alone) indicating that the H2-Kd allele cannot serve to activate these T cells even with SIINFEKL present.

Figure 3: MHC-reprogrammed RAW264.7 cells activate T cells. (a) Experimental design of CD8+ T cell activation assay. Cell lines are activated with IL-4 and GM-CSF cytokines overnight, irradiated and combined with the B3Z CD8 T cell hybridoma cell line, which was engineered to express β-galactosidase upon TCR activation by H2-Kb/SIINFEKL peptide-MHC complexes. The cells are lysed and the amount of expressed β-galactosidase is detected by colorimetric turnover of CPRG substrate. (b) Representative plot of three independent experiments of B3Z activation by control and MHC-reprogrammed cell lines. The individual clones C4, F4, F5, and G5 were grown from single cells sorted from a bulk transfection. Each cell line was tested with and without SIINFEKL peptide and the absorbance at 570 nm measured after 3 hours of incubation (N = 3). Error bars indicate 95% confidence intervals for technical replicates. Full size image

Genotypic characterization of MHC-reprogrammed cells

While phenotypic analysis is able to show positive expression of H2-Kb and the lack of expression of the wildtype H2-Kd protein in reprogrammed macrophages, it is insufficient to determine whether: (i) MHC exchange occurred on both chromosomes (biallelic) or (ii) MHC exchange occurred on a single chromosome (monoallelic) in combination with NHEJ-induced knockout on the other chromosome. To address this, PCR assays on genomic DNA were designed to evaluate the various possibilities of modification in the MHC locus (Fig. 4a). In all single-cell lines tested, the newly introduced H2-Kb allele was detected by PCR (with primers p9/p10), but not in the parental RAW264.7 cell line, indicating that at least one of the alleles had been exchanged (Fig. 4b). We next looked for the presence of wildtype H2-Kd alleles, only a single colony (F5) showed a positive PCR product (p5/p6) that was comparable to the RAW264.7 positive control (Fig. 4c). This suggests cell lines C4, F4, and G5 all possessed only the new H2-Kb allele. This result correlates with the lower phenotypic H2-Kb expression observed by flow cytometry for clone F5 as compared to the other isolated clones (Supplementary Fig. S2b). However, the lack of H2-Kd allele present at the endogenous locus could either be due to exchange of the second allele or deletion at this locus. To control for this a PCR assay (p1/p4) was used to detect for H2-Kd deletion (Fig. 4d). Cells receiving only px458-G10 and px458-G13 were used as a positive control for genomic deletion. We found no bands indicating deletion in any of the cell lines tested. From each of the cell lines, we used a split-pool PCR approach to clone the inserted H2-Kb gene into a sequencing plasmid (Fig. 4e and Supplementary Fig. S3a). Each single-cell colony (C4, F4, F5, G5) had three of their resulting bacterial colonies Sanger sequenced. We observed multiple mutations that differed from the input donor template in all cell lines (Ø), and in some cases the mutations were present in all three colonies sequenced although these appeared not to have altered the H2-Kb phenotype. Because split-pool PCR was used for amplification, it is very unlikely that mutations arose during cloning into the sequencing vector. Sequencing errors are unlikely to be generated at the same location in all three colonies. Thus, mutations were most likely generated during the PCR amplification of the linear donor template despite the use of a high fidelity polymerase (KapaHiFi). Recent studies using high-throughput sequencing on PCR amplicons have shown that reproducible polymerase hotspot errors are more common than previously believed, even with high-fidelity polymerases28,29. Therefore, the generation of long donor templates by PCR may result in erroneous variants, which would present a major problem for future therapeutic applications.

Figure 4: Mapping MHC allelic exchange in reprogrammed RAW264.7 cells. (a) Schematic of primers used for the detection of wildtype H2-Kd at the genomic locus (p5/p6) and the confirmation of H2-Kb integration at the correct locus (p9/p10). (b) Agarose gels show genomic PCR products that verify correct integration of H2-Kb donor cassette in all cell lines tested. (c) Genomic PCR analysis shows the presence of residual wildtype H2-Kd alleles only in cell line F5. (d) To determine whether loss of H2-Kd is likely by deletion of the allele, or by biallelic replacement; primer pair p1/p4 was used. Only the control sample shows evidence of deletion suggesting all cell lines except F5 have biallelic H2-Kd replacement. (e) Sequence analysis of multiple colonies from the single-cell sorted clone F4 reveals a shared template mutation. Full size image

Comparison of HDR efficiency with various MHC exchange donor templates

To avoid PCR-based generation of donor templates, we investigated the use of alternative formats. Minicircles are a plasmid system that allows for selective removal of vector components, they rely on recombination of integrase AttB and AttP sites to remove bacterial gene elements prior to transfection in mammalian cells24. When compared to donor templates generated with standard plasmids, minicircles provide a smaller and less immunogenic format. We designed several minicircle donor templates and compared their exchange efficiency to our previously used PCR-generated exchange template (Fig. 5a and Supplementary Fig. S3b,c). Previous studies have suggested that linearized templates are more efficient for HDR integration when compared to circular templates30,31. Therefore, we created a “self-linearizing” minicircle exchange template, as it possessed the same gRNA sites (G10 and G13) present in the MHC locus, and thus this template would be linearized in situ (after transfection) by Cas9. We also included minicircles that were pre-linearized via an EcoRI restriction site. Equimolar quantities of each donor template were nucleofected into RAW264.7 cells along with the pX458-G10 and G-13 plasmids, and as before Cas9-expressing cells (via 2A-GFP) were sorted and expanded (Supplementary Fig. S3d,e). Flow cytometry analysis was used to determine the fraction of MHC-reprogrammed cells based on H2-Kb-positive/H2-Kd-negative expression. After six-independent experiments, the self-linearizing minicircle template showed the highest fold improvement in MHC exchange compared to linear template (7.9 fold on average) (Fig. 5b). No significant differences in HDR efficiency were observed for the other donor templates. PCR analysis was performed on the bulk population and revealed correct integration in the MHC locus for all samples (Supplementary Fig. S3f). To confirm the reprogramming efficiency of the self-linearizing minicircle template, we performed exchange on a second H2-Kd-expressing cell line, CT26 fibroblasts. We had previously found this cell line to be recalcitrant to MHC exchange with linear PCR templates. However, when using the self-linearizing minicircle, we observed an easily detectable level of MHC exchange by flow cytometry (Fig. 5c), which was confirmed by genomic PCR (Fig. 5d). This level of exchange was comparable to that seen with RAW264.7 macrophages, suggesting that this approach is applicable to other cell types.