CRISPR/Cas9-mediated gene disruption determines the roles of MITF and CITED2 in human mast cell differentiation

The differentiation of hematopoietic stem and progenitor cells into the various cell lineages is regulated by cell-intrinsic factors and the progenitors' microenvironment. Experiments in mice have allowed the identification of transcriptional modulators that control mast cell differentiation. However, a comprehensive approach that allows efficient disruption of individual transcriptional modulators in primary human hematopoietic progenitors coupled with a mast cell formation readout has not been described. Here, we report a simple electroporation- and ribonucleoprotein-based knockout system that allows the identification of genes that are required and dispensable for human mast cell differentiation. We show that the transcription factor MITF is upregulated in human mast cell progenitors and reveal that the loss of MITF results in the suppressed formation of mast cells. By contrast, CITED2, another transcriptional modulator that is upregulated along the mast cell differentiation trajectory, was dispensable for human mast cell differentiation. Taken together, we report a CRISPR/Cas9-based framework that serves to identify genes involved in regulating the formation of human mast cells, and the results uncover the role of two transcriptional modulators in controlling human mast cell differentiation.


Ethics statement
The Swedish Ethical Review Authority approved the study (2019-01729).

sgRNA and primer design
The sgRNAs were designed using the websites http://crispor.tefor.net/, 1 http://greenlisted.cmm.ki.se/, 2 and https://chopchop.cbu.uib.no/. 3The sgRNAs were designed targeting a sequence near the 5' end of the gene.We combined information from the three websites and specifically selected sgRNAs with the highest on-target activity while having a low off-target score.The negative control sgRNA (NC sgRNA) was pre-designed by Sigma-Aldrich.The CD45 sgRNA was designed by Gundry et al. 4 All sgRNAs with stabilizing 2'-Omethyl and phosphorothioate linkages were ordered from Sigma-Aldrich.The sgRNA sequences are shown in supplemental Table 1 and supplemental Figures 2-3.
PCR primers to amplify the sgRNA target region were designed using SnapGene software, and the primers were subsequently ordered from Sigma-Aldrich.The primer sequences are shown in supplemental Table 1.

Cell isolation, genetic editing, and cell culture
Blood mononuclear cells were extracted from buffy coats of anonymous healthy blood donors using Ficoll centrifugation (Cytiva).The CD34 MACS Microbead Kit (Miltenyi Biotec) was used to isolate CD34 + hematopoietic progenitors.Lin -CD34 + c-Kit + FcεRI -progenitors (hereafter referred to as FcεRI -progenitors) and Lin -CD34 + c-Kit + FcεRI + mast cell progenitors (referred to as MCPs) were isolated as described in Wu et al. 5 Briefly, the CD117 MACS Microbead Kit (Miltenyi Biotec) was used to enrich c-Kit + cells.FcεRI -progenitors and MCPs were then sorted on the FACS Aria Fusion system (BD Biosciences).The hematopoietic progenitor cells were cultured in complete StemPro-34 serum-free medium (Thermo Fisher Scientific) containing 2 mM L-glutamine (Cytiva HyClone), 100 U/ml penicillin (Cytiva HyClone), and 0.1 mg/ml streptomycin (Cytiva HyClone).No antibiotics were added to the culture medium immediately after electroporation.Instead, antibiotics were added 2-5 days after the electroporation.The culture medium for the primary cells was supplemented with 5 ng/mL SCF (Swedish Orphan Biovitrum), 50 ng/mL IL-6 (PeproTech), and 10 ng/mL IL-3 (PeproTech) during the first week.The cells were cultured without IL-3 during the second week unless otherwise specified in the figure legend.
To form the ribonucleoprotein (RNP), TrueCut™ Cas9 Protein v2 (Thermo Fisher Scientific) and synthetic sgRNA were added to a PCR tube at a molar ratio of 1:3, and the mixture was incubated in a PCR machine at 25°C for 30 minutes.The Amaxa™ Nucleofector™ II System (Lonza) was used to electroporate cells to deliver the Cas9 RNPs.The Amaxa human CD34+ cell nucleofector kit (Lonza) and the program X-001 were used to electroporate the hematopoietic progenitors.The Amaxa cell line nucleofector Kit V (Lonza) and the program T-020 were used to electroporate the cell lines.

Quality control
FACS isolation of a least 10 000 primary hematopoietic progenitors from a given sample was used as a result analysis criterion.One control sample failed to produce a population of mast cells following culture, which made it impossible to study the effect of gene disruption on the formation of mast cells in this sample, and was therefore excluded.

Sanger sequencing, Inference of CRISPR Edits (ICE) analysis
The cells of interest were sorted into 250 µL or 500 µL of lysis buffer for the extraction of genomic DNA using the QuickExtract™ DNA kit (Lucigen).To amplify the sgRNA target region, 5 µL genomic DNA template was used in the PCR process.The PCR amplicons were purified using the Wizard SV Gel Clean-up system (Promega) and subsequently submitted to Eurofins Genomics for Sanger sequencing.The obtained Sanger sequencing data was analyzed using ICE (Inference of CRISPR Edits) (Synthego, https://ice.synthego.com) 6to assess the editing efficiency.

Single-cell RNA-sequencing analysis
Single-cell RNA-sequencing data generated in Wu et al, 5 as deposited in the Gene Expression Omnibus database (GSE184351), was processed and the expression of individual genes was visualized using the UMAP embedding.

Statistical analysis
Statistical analysis was performed using Prism 9 software (Graphpad software, La Jolla, CA).

Supplemental Figure 3 .
(A) Schematic illustration of the CITED2 sgRNA location.(B) Flow cytometry plots showing the gating strategy of cultured CD34 + cells electroporated with negative control (NC) sgRNA and three CITED2 sgRNAs.Cells gated on live singlets.(Ci) Editing efficiency of sorted live singlets 5 days after electroporation.Two independent experiments were performed for CITED2 sgRNA1 and CITED2 gRNA2.One experiment was performed for CITED2 sgRNA3.The sgRNA1 data points are also shown in Figure 2B.(Cii) Editing efficiency of sorted mast cells 12 days after electroporation.(D) Fraction of mast cells normalized to NC sgRNA 11 days after electroporation.The sgRNA1 samples presented here are also shown in Figure 2D.The NC sgRNA condition refers to non-targeting sgRNA.Twotailed one sample t test, hypothetical value is 1. ns = non-significant.
P < 0.05 was considered significant.The statistical tests used are specified in the figure legends.The error bars in the figures indicate Standard Deviation (SD).