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G. Dong et al.
ed by deletion, methylation and mutation. The minimal common region of the 6q21 deletion contains a number of genes (ATG1, AIM1, etc.) but apart from PRDM1, they have not been found to be mutated or methylated either by us or others. We therefore prioritized PRDM1 for fur- ther study, and functional analysis of the gene supports its role as a tumor suppressor gene.6-8 PRDM1 has also proven to be a tumor suppressor gene in diffuse large B-cell lym- phomas4 and anaplastic large T-cell lymphoma.9 Accumulating evidence supports the concept that it is not only critical for terminal effector cell differentiation in B cells10 but that it is also important in the homeostasis of T cells and in T-cell4 effector differentiation. The level of PRDM1 increased progressively with NK-cell activation with a corresponding drop in MYC level, termination of proliferation and increased cellular apoptosis.6 The precise role of PRDM1 in this process is not clear, and the inability to maintain human NK cells in long-term culture in vitro with interleukin (IL)-2 or IL-15 is a major impediment to further analysis. We are now able to perform long-term in vitro cultures of primary, normal NK cells which allow suf- ficient time for us to perform specific genetic manipula- tions and functional studies with genome-edited cells and single-cell clones using the recently developed clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system for targeted gene editing.11 Here, we report the functional consequences of PRDM1 gene knock-out (KO) in primary NK cells from healthy donors and the implication of these findings on NK-cell homeostasis and ENKTCL pathogen- esis.
While PRDM1 is a frequently mutated tumor suppressor gene in NK-cell lymphomagenesis,6-8 it is not sufficient by itself to generate a lymphoma in a murine model, and additional alterations are necessary. As many of the fre- quent genetic and epigenetic changes in ENKTCL are loss- of-function alterations, the CRISPR/Cas 9 system enables highly efficient targeted gene editing11 to investigate these abnormalities.12-16 Here, we demonstrated other potential tumor suppressor genes that are readily modified and the feasibility of inducing pairs of deletions to study coopera- tive mutations in NK-cell lymphomagenesis.
Methods
Primary NK-cell enrichment
Primary NK cells were isolated from peripheral blood mononu- clear cells of healthy donors (donor #1 and donor #2) using the EasySepTM Human NK Cell Enrichment Kit (Stemcell, USA; #19055) according to the manufacturer’s protocol. The purity of isolated NK cells was determined by flow cytometry analysis with FITC-labeled anti-human CD56 (Biolegend, USA; n. 362545) and PE-labeled anti-human CD3 (Biolegend, USA, n. 300407) double staining.
PRDM1 knockout mediated by CRISPR/Cas9 with plasmid PX458-sgRNA4
The PX458-sgRNA4 plasmid was delivered into stimulated pri- mary NK cells by electroporation using the Amaxa® Nucleofector® II Device (Lonza, France) according to the manufacturer’s suggest- ed U001 protocol (5 μg plasmid per 2x106 cells) (Figure 1A). Cloning of the modified cells is described in the Online Supplementary Methods. Sequential gene KO was processed similar- ly but on identified PRDM1-/- NK clone #3. The various guide RNA
used in these experiments are listed in Online Supplementary Tables S1 and S2.
CRISPR/Cas9-mediated disruption of PRDM1 by introduction of a fluorescent protein through homologous recombination
Cas9/sgRNA ribonucleoprotein (RNP) complexes targeting PRDM1 exon 5 (Figure 1B), together with a double-stranded DNA repairing template consisting of a fluorescent protein gene (GFP/DsRed) flanked by long homologous arms of the PRDM1 gene, were electroporated into cells, allowing the edited cells, of which both PRDM1 loci were disrupted, to be sorted by fluores- cence activated cell sorting (FACS). Details of the experiment are shown in Online Supplementary Figure S1 and Figure 1B. sgRNA2 used in this experiment is shown in Online Supplementary Table S1.
Other experimental methods
Cell lines used and cell culture methods, the CRISPR/Cas9 experiments, western blotting, cell proliferation and apoptosis assays, cell cycle analysis, quantitative real-time polymerase (qRT- PCR), RNA-sequencing, next-generation sequencing, and statisti- cal analysis, are described in the Online Supplementary Information.
Results
Generation of PRDM1-/- primary NK cells by two different CRISPR/Cas9 methods
Generation of PRDM1-/- clones #3 and #5 using PX458-sgRNA4 plasmid electroporation
With our feeder cell culture system, we successfully cloned primary NK cells after single-cell seeding by FACS. The expanded NK-cell single clones (G-2 and G-3) were observed for approximately 3 weeks (Online Supplementary Figure S2) and were enumerated for evaluation of cloning efficiency. According to our grading system, as specified in the Methods section, the GFP+ cells (plasmid-transfected cells) had significantly higher cloning efficiency (47.7% vs. 11.7%; P<0.05) than parental primary NK cells (Online Supplementary Table S3). Sequencing analysis revealed that 66% (61 of 92) of the clones showed PRDM1 frame-shift deletions around the target site within exon 4 (Online Supplementary Figure S3), with the likelihood of their having been four founders based on the pattern of deletions. Two of these four founder clones with distinct homozygous deletions (Figure 2A, B) were the most prevalent clones iso- lated by single-cell cloning, indicating that homozygous deletion of PRDM1 confers a growth advantage among these clones. Clones #3 and #5 belonged to one of the homozygous deletions and were chosen as the biological duplicates in our subsequent studies. The loss of PRDM1 protein expression was confirmed by western blot analysis in these clones (Figure 2C, upper panel). We also measured the expression of the PRDM1 target gene MYC in the edit- ed cells and demonstrated that the expression of MYC was upregulated (9-fold) upon PRDM1 KO (Figure 2C, lower panel).
Generation of bulk PRDM1-/- NK cells through fluorescent protein knock-in by homologous DNA repair using Cas9/sgRNA2 ribonucleoprotein electroporation
To avoid the selection process inherent in cloning and potential spurious results due to off-target modification by sgRNA4, we modified a recently described technology
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