Methods

Pseudogene-mediated gene conversion after CRISPR-Cas9 editing demonstrated by partial CD33 conversion with SIGLEC22P

Cell Lines and Antibodies

U937 and HEK293 cell lines were obtained from American Type Culture Collection (ATCC). U937 cells were cultured in RPMI 1640 with HEPES (Gibco 22400-089) supplemented with 10% fetal bovine serum, defined (HyClone, GE Healthcare SH30070.03); 50 U/mL penicillin, 50 μg/mL streptomycin (Gibco 15070-063); and 2 μM L-glutamine (Gibco A2916801). HEK293 cells were cultured in EMEM, ATCC formulation (ATCC 30-2003) supplemented with 10% fetal bovine serum, defined (HyClone, GE Healthcare SH30070.03); 50 U/mL penicillin, 50 μg/mL streptomycin (Gibco 22400-089). Cells were maintained at 37°C in a 5% CO2 in air atmosphere. The U937 cell line has been reported as either diploid or triploid at chromosome 19 which contains CD33.43, 44Antibodies, concentrations, and CD33 domains targeted are shown in Table 1.

CRISPR-Cas9 Gene Editing

CRISPR reagents were purchased from Integrated DNA Technologies (IDT). Single-guide RNAs (sgRNAs) and S. pyogenes Cas9 protein (IDT 1081059) were incubated at a 1:1 molar ratio (0.5 nmol each) at room temperature for 10 minutes to form ribonucleotide-protein complexes (RNPs). The sgRNA sequences targeting CD33 exon 2 were 5’-TCCATAGCCAGGGCCCCTGT and 5’-GCATGTGACAGGTGAGGCAC.25 U937 cells were washed three times in PBS (Gibco 10010-023) and resuspended in complete Nucleofector Kit C (Lonza Biosciences VCA-1004) media (106 cells per transfection) with 5 μL electroporation enhancer (IDT 1075916) and RNPs. Cells were electroporated using a Nucleofector IIb device (Lonza Biosciences) under protocol V-001 and immediately added to a 12 well plate with 1.5 mL complete media and cultured for two weeks.

Cell Sorting and Flow Cytometry

Edited U937 cells were washed in PBS with 5% heat-inactivated fetal bovine serum (Gibco 10082-147), resuspended at 106 cells/mL and then treated with Human TruStain FcX blocker (BioLegend 422302). Cell sorting was carried out in azide-free buffers; for flow cytometry, 0.02% sodium azide was included in all buffers. Cells were stained with HIM3-4-FITC and P67.6-BV711 for one hour on ice, washed twice with HBSS, then stained with Fixable Viability Dye eFluor780 (Invitrogen 65-0865-18). Cells were resuspended in HBSS (Gibco 24020-117) with 5% heat-inactivated fetal bovine serum (Gibco 10082-147) for sorting. Viable cells were gated using scatter and viability exclusion stain, sorted as either HIM3-4+ P67.6+, HIM3-4+ P67.6-, or HIM3-4- P67.6- and collected in complete media. At 48 hours post-sort, cells were split using limiting dilution into a 96 well plate at an average density of 0.5 cells/well and expanded until sufficient cell numbers for analysis were achieved, approximately 8 weeks. Clones were screened by flow cytometry again prior to PCR and sequence analysis.

PCR Screening and Cloning

Genomic DNA from CRISPR-edited U937 clones was isolated with a DNeasy kit (Qiagen 69506) per manufacturer instructions. A portion of CD33 was amplified with Q5 High-Fidelity DNA Polymerase (New England BioLabs M0439L) using forward primer 5’-CACAGGAAGCCCTGGAAGCT and reverse primer 5’-GAGCAGGTCAGGTTTTTGGA (Invitrogen). SIGLEC22P was amplified similarly with forward primer 5’-GCACCTCAGAGTGGAAGGAC and reverse primer 5’-GAAGGGGTGACTGAGGTACA. Thermocycling parameters were as follows: 98°C 1 min; 98°C 15 s, 66°C 15 s, 72°C 45 s, 32 cycles; 72°C 2 min, 25°C hold. PCR products were separated on a 0.8% agarose-TBE gel, purified using a Monarch gel extraction kit (New England BioLabs T1020L), and sequenced by a commercial company (ACGT, Wheeling, IL). The three missense mutations identified were introduced into a previously described pcDNA3.1-CD33-V5/HIS vector using a QuikChange XL kit (Agilent 200517) with forward primer 5’-GCACTTGCAGCCGGATTTTTGGATCCATAGCCAGGGCC and reverse primer 5’-GGCCCTGGCTATGGATCCAAAAATCCGGCTGCAAGTGC (Invitrogen) to generate the pcDNA3.1-KIR-CD33-V5/HIS vector, transformed into TOP10 E. coli (Invitrogen C404003), isolated using a Plasmid Plus Midiprep Kit (Qiagen 12945) and verified by sequencing (ACGT).8

Gene expression by qPCR

Quantitative PCR (qPCR) was used to quantify expression of total CD33 and D2-CD33 as previously described.14 Briefly, primers corresponding to sequences within exons 4 and 5 were used to quantify total CD33 expression (forward, 5’-TGTTCCACAGAACCCAACAA-3’; reverse, 5’-GGCTGTAACACCAGCTCCTC-3’), as well as primers corresponding to sequences at the exon 1–3 junction and exon 3 to quantify the D2-CD33 isoform (forward, 5’-CCCTGCTGTGGGCAGACTTG-3’; reverse, 5’-GCACCGAGGAGTGAGTAGTCC-3’). PCR was conducted using an initial 2 min incubation at 95°, followed by cycles of 10 s at 95°C, 20 s at 60°C, and 20 s at 72°C. The 20 μl reactions contained 1 μM each primer, 1X PerfeCTa SYBR Green Super Mix (Quanta Biosciences), and 20 ng of cDNA. Experimental samples were amplified in parallel with serially diluted standards that were generated by PCR of cDNA using the indicated primers, followed by purification and quantitation by UV absorbance. Results from samples were compared relative to the standard curve to calculate copy number in each sample. Assays were performed in triplicate and normalized to expression of ribosomal protein L32 (RPL32) as the housekeeping gene.14, 45

HEK293 Transfection

HEK293 cells were seeded at approximately 70% confluency 24 hours before transfection. Cells were then transfected with Lipofectamine 3000 with Plus Reagent (Invitrogen L3000001) per manufacturer instructions, 250 ng plasmid per well in 8 well glass chamber slides (MatTek CCS-8) for immunofluorescence or 1000 ng per well in 12 well plates (Corning 3513) for flow cytometry. Cells were transfected with either the previously described wild-type CD33 vector (pcDNA3.1-CD33-V5/HIS), pcDNA3.1-KIRCD33-V5/HIS, or no vector control. Cells were incubated for 24 hours before analysis by flow cytometry or immunofluorescence and confocal microscopy.

Confocal Immunofluorescence Microscopy

Transfected HEK293 cells were fixed with 10% neutral buffered formalin (Fisher Scientific SF100-4) for 30 minutes then blocked and permeabilized for 30 minutes with 10% goat serum (Sigma S26-LITER), 0.1% Triton X-100 (Fisher Scientific BP151-500) in PBS (Fisher BioReagents BP665-1). Primary and secondary antibodies were diluted in the same blocking and permeabilization buffer and incubated at room temperature for 90 minutes. Cells were washed three times in blocking and permeabilization buffer between primary and secondary antibodies, and three times in PBS prior to coverslip mounting with Prolong Glass with NucBlue mounting media (Invitrogen P36981) and high-tolerance No. 1.5 coverglass (ThorLabs CG15KH1). Images were acquired using a Nikon A1R HD inverted confocal microscope with a 60X oil objective and NIS Elements AR software.

Statistical analyses

Analyses were performed using GraphPad Prism 8.4.2. Gene expression data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons to unedited U937 cells.

Article TitlePseudogene-mediated gene conversion after CRISPR-Cas9 editing demonstrated by partial CD33 conversion with SIGLEC22P

Abstract

Although gene editing workflows typically consider the possibility of off-target editing, pseudogene-directed homology repair has not, to our knowledge, been reported previously. Here, we employed a CRISPR-Cas9 strategy for targeted excision of exon 2 in CD33 in U937 human monocyte cell line. Candidate clonal cell lines were screened by using a clinically relevant antibody known to label the IgV domain encoded by exon 2 (P67.6, gemtuzumab). In addition to the anticipated deletion of exon 2, we also found unexpected P67.6-negative cell lines which had apparently retained CD33 exon 2. Sequencing revealed that these lines underwent gene conversion from the nearby SIGLEC22P pseudogene during homology repair that resulted in three missense mutations relative to CD33. Ectopic expression studies confirmed that the P67.6 epitope is dependent upon these amino acids. In summation, we report that pseudogene-directed homology repair can lead to aberrant CRISPR gene editing.


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