Methods

CRISPR enriches for cells with mutations in a p53-related interactome, and this can be inhibited

Cells

The Hox bone marrow cell line was generated by transducing bone marrow cells of C57BL/6 Cas9+ GFP+ mice (The Jackson Laboratory, #026179) with an estrogen inducible retroviral construct expressing HoxB8 (ER-Hoxb8, a kind gift from Mark P. Kamps, University of California, San Diego) as described in1,2. Cells were cultured in 1 μM β-estradiol (BE, Sigma-Aldrich #E2758) and 25 nM mouse Stem Cell Factor (SCF, Peprotech #250-03) for several weeks with HoxB8 expression turned on to establish a cell line like population. Hox cells were cultured in RPMI-1640 (Sigma-Aldrich #R0883) with 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin-glutamine (Gibco #10378016), 1 μM BE, and 25 nM SCF.

B16-F10 cell is a mouse melanoma cell line, purchased from ATCC (#CRL6475) and used at a low passage number. Cas9 expressing cells were generated by transducing B16-F10 cells with lentiCas9-Blast (Addgene #52962) lentiviral particles. B16 cells were cultured in RPMI-1640 (Sigma-Aldrich #R0883) with 10% heat-inactivated fetal bovine serum, and 1% penicillin-streptomycin-glutamine (Gibco #10378016)

Viral preparation and transduction

Lentiviral particles were generated by seeding 2×106 HEK293T cells in 10 cm plate in 10 ml of DMEM (Sigma-Aldrich #D6546) with 10% heat-inactivated fetal bovine serum and 1% L-glutamine (Gibco #A2916801). After ~24 h of culture, the cells reached ~60-70% confluency, and the medium was replaced by 5 ml of prewarmed fresh media. Transfer plasmids (lentiCas9-Blast, Addgene #52962; or LentiGuide-Puro-P2A-EGFP_mRFPstuf, Addgene #137730), pMD2.G (Addgene #12259), and psPAX2 (Addgene #12260) were mixed at 4:5:1 ratio (10 μg : 12.5 μg : 2.5 μg for 10 cm plate), and transfected into HEK293T cells using Lyovec (Invivogen #lyec) according to the manufacturer’s protocol. After 12 h, the medium was replaced by 8 ml of DMEM with 30% heat-inactivated fetal bovine serum and 1% L-glutamine. After another 36 h, the supernatant containing the virus was collected and centrifuged to remove the cell debris and used to spin infect cells.

For ER-Hoxb8 retrovirus preparation, plasmids including ER-Hoxb8 and the EcoPac gag-pol-env plasmid (both are kind gifts from Mark P. Kamps, University of California, San Diego) were mixed at 1:1 ratio (12 μg:12 μg for 10 cm plate) for transfection into HEK293T following the same approach as for generating lentiviral particles.

To transduce Hox or B16 cells with LentiGuide-Puro-P2A-EGFP_mRFPstuf, the multiplicity of infection (MOI) of the viral particles was tested by infection with serial dilutions of virus particles, to find a dilution resulting in a suitable MOI described below. Virus supernatant was added to each well of 6-well plate containing cells (4×105 for Hox cells with SCF and BE, and 1×105 for B16 cells) with 8 μg/ml polybrene (Sigma-Aldrich #H9268). The plate was centrifuged at 37°C, 1200g (120 min for Hox cells, and 45 min for B16 cells). After 24h, the virus-containing medium was replaced with fresh medium, and the infection rate was measured by the percentage of GFP+ cells if the vector contains GFP. Puromycin (Invivogen #ant-pr) selection (10 μg/ml for HoxB8 cells, and 5ug/ml for B16 cells) or Blasticidin (Invivogen #ant-bl) selection (10 μg/ml for B16 cells) was performed for 24 h to remove the non-infected cells.

Electroporation and transfection

sgRNAs were designed using the Green Listed software3,4 using sgRNA design from the Doench mouse library5. 2’-O-methyl and phosphorothioate stabilized sgRNAs (Supplementary Table 8) were ordered from Sigma-Aldrich. Off-target activity was calculated using Cas-OFFinder (http://www.rgenome.net/cas-offinder/)6, and On-target activity was extracted from CHOPCHOP (https://chopchop.cbu.uib.no/)7 by searching for Ccr1, or entering the EGFP FASTA sequence from the LentiGuide-Puro-P2A-EGFP_mRFPstuf (Addgene #137730).

For Hox cells, Neon Transfection System (Invitrogen #MPK5000) was used to perform the electroporation following the manufacturer’s instructions (Pulse voltage: 1700, Pulse width: 20 ms, Pulse number: 1, for Hox cells). 100 pmol of sgRNA were electroporated into 2×105 Hox cells for each electroporation experiment using Neon Transfection System 10 μL Kit (Invitrogen #MPK1096). For B16 cells, Lipofectamine 2000 Transfection Reagent (Invitrogen, #11668019) was used following the recommended protocol. 100pmol of sgRNA were transfected into 1×105 B16 cells for each transfection experiment.

Trp53 ON-TARGETplus siRNA SMARTPool was ordered from Horizon. For Hox cells, 100 pmol of siRNA was electroporated into 2×105 Hox cells for each electroporation experiment. For B16 cells, 100 pmol of siRNA was transfected into 1×105 B16 cells for each transfection experiment. The Trp53 siRNA was typically delivered in the same reaction as the sgRNAs.

CRISPR KO genotyping

1×105 cells were collected for genomic DNA extraction using DNeasy Blood & Tissue Kit (Qiagen #69504) following the recommended protocol. Primers (Sigma-Aldrich) were designed using Primer-BLAST (Supplementary Table 8), aiming for a 400-1000 bp amplicon with the sgRNA target in the middle. Amplicons were gel purified and recovered using Zymoclean Gel DNA Recovery Kit (Zymo Research #D4007/D4008). The PCR products were quantified using Nanodrop and sequenced by Eurofins Genomics. The Sanger sequencing data was subsequently analyzed by ICE (Synthego, https://ice.synthego.com).

Growth curve characterization

Hox cells were cultured with the following interventions: electroporation with a GFP targeting sgRNA; 0.5 μg/ml Etoposide (Sigma-Aldrich #E1383); 3 μM AMG232 (Axon Medchem #2639). The Etoposide and AMG232 were removed after 8 h by removing the medium and adding fresh medium without Etoposide or AMG232. The cells were cultured in 6 well plates with 4ml medium and passaged every day at the ratio of 1:3. 80 ul of cells were taken every day for flow cytometry (BD Accuri) with the existence of CountBright Absolute Counting Beads (Invitrogen #C36950). The absolute viable cell number was calculated by comparing the events number of viable cells and counting beads in the flow cytometry data.

Real-Time PCR

TRIzol Reagent (Invitrogen #15596026) and Direct-zol RNA MiniPrep Kit (Zymo Research #R2051) was used to extract RNA. RNA was then converted into cDNA using High Capacity RNA-to cDNA kit (Applied Biosystem #4388950). The expression of Cdkn1a was quantified with a CFX 384 Real-Time PCR machine (Bio-Rad) using TaqMan gene expression FAM assays for Cdkn1a (Mm00432448_m1) with the TaqMan Gene Expression Master Mix (Applied Biosystem #4369542) as suggested by the manufacturer. Expression was normalized by TaqMan gene expression VIC assays for β-actin (Mm00607939) and Gene expression was quantified using the ddCT method.

Apoptosis TUNEL assay

Cells were collected and fixed by PFA at different time point, and FlowTAC Apoptosis Detection Kit (R&D Systems #4817-60-K) was used to stain apoptotic cells following the manufacturer’s instructions. Stained cells were analyzed by flow cytometry (BD Accuri).

Cloning of sgRNAs into lentiviral transfer plasmid, and CRISPR screens

sgRNAs with overhangs for the plasmids (Supplementary Table 1) were designed using the Green Listed software3,4 using sgRNA design from the Doench mouse library5 and, for intergenic controls, the Wang mouse library8. Individual sgRNAs were ordered from Sigma-Aldrich, and the sgRNA library was ordered from CustomArray as a DNA oligo pool. Cloning was performed using 150 ng of BsmBI (New England Biolabs #R0739) cleaved lentiGuide-Puro-P2A-EGFP_mRFPstuf plasmid (Addgene #137730), and 10 ng of the library oligo pool, using NEBuilder HiFi DNA assembly master mix (New England Biolabs #E2621). Endura ElectroCompetent Cells (Lucigen #60242), were subsequently transformed with the cloned plasmid pool using electroporation (1.0 mm cuvette, 10 μF, 600 Ohms, 1800 Volts) following the suggested protocol. The electroporated cells were combined and seeded on ten 20 cm LB agar plates with 100 μg/ml carbenicillin and grown at 37°C overnight. The lentiGuide-Puro-P2A-EGFP_mRFPstuf plasmid (Addgene #137730) makes bacteria red if the stuffer has not been exchanged by a sgRNA, and a few red clones were removed before collecting all other white clones. Then the plasmids were purified using EndoFree Plasmid Maxi Kit (Qiagen #12362).

The sgRNA cloned lentiGuide-Puro-P2A-EGFP_mRFPstuf was used as transfer plasmid for lentiviral preparation and transduction. The total amount of transduced cells was calculated based on MOI (0.25 for B16 cells, and 0.05-0.1 for Hox cells, which were difficult to transduce), based on the % GFP+ cells before puromycin selection, aiming for 1000 transduced cells for each sgRNA.

The CRISPR library transduced cells were exposed to GFP targeting sgRNA electroporation with or without Trp53 siRNA, 0.5 μg/ml Etoposide (Sigma-Aldrich #E1383) 8 h pulse stimulation, or 3 μM AMG232 (Axon Medchem #2639) 8 h pulse stimulation.

Cells were collected for genomic DNA extraction using DNeasy Blood & Tissue Kit (Qiagen #69504). Genomic DNA was then amplified using Q5 High-Fidelity DNA Polymerase (New England Biolabs #M0491) while introduced sample-specific barcodes and adapters for Illumina Sequencing similar as described in Joung J et al.9 using primers specified in Supplementary Table 9. The final PCR products were gel purified and recovered using Zymoclean Gel DNA Recovery Kit (Zymo Research #D4007/D4008), and quantified with Qubit 4 Fluorometer (Invitrogen #Q33238) using Qubit dsDNA HS Assay Kit (Invitrogen #Q32851) and pooled for next-generation sequencing (Illumina MiSeq v3 run, 2×75bp reads). The raw FASTQ data were analyzed by MAGeCK10. Read counts from CRISPR screens found in Supplementary Table 10-11.

JAK1/STAT1 signaling assay

Hox cells were cultured with or without mouse Interferon Beta (IFNβ, Nordic BioSite #12405-1) and the Jak1 inhibitor Solcitinib (Selleckchem #S5917) for 7 days. Cells were passaged at the ratio of 1:8 every other day. 100 ul of cells were taken on day 7 for flow cytometry (BD Accuri) with the existence of CountBright Absolute Counting Beads (Invitrogen #C36950). The absolute viable cell number was calculated by comparing the events number of viable cells and counting beads in the flow cytometry data.

Competitive co-culture assay

Trp53 KO and WT cells were mixed at 1:4 ratio, and were electroporated with sgRNA or transduced with lentivirus (lentiGuide-Puro-P2A-EGFP_mRFPstuf) and cultured for 7 days; or cultured with Etoposide (Sigma-Aldrich, #E1383, 0.05 μg/ml) or AMG232 (Axon Medchem #2639, 0.5 μM for Hox cells, 4 μM for B16 cells) for 7 days; or cultured with Cobalt(II) chloride (CoCl2, Sigma-Aldrich #232696, 10-20 μg/ml) for 7 days. Different p53 related inhibitors were added during culture: Trp53 ON-TARGETplus siRNA SMARTPool (Horizon), KU55933 (Sigma-Aldrich #SML1109, 100 ng/ml for B16, 10 ng/ml for Hox), VE821 (Sigma-Aldrich #SML1415, 50 ng/ml), Pifithrin-μ (Sigma-Aldrich #P0122, 2 μg/ml for B16, 200 ng/ml for Hox), Cyclic Pifithrin-α (Sigma-Aldrich #P4236, 700 ng/ml for B16, 70 ng/ml for Hox), C646 (Sigma-Aldrich, #SML0002, 1 μg/ml), AZD2461 (Selleckchem #S7029, 250 ng/ml), LJI308 (Sigma-Aldrich #SML1788, 2 ng/ml), Z-VAD-FMK (Selleckchem #S7023, 50 μM). siRNA was delivered to cells 1 day before CRISPR/Etoposide/AMG232 exposure (100 pmol of siRNA electroporated into 2×105 Hox cells, or transfected into 1×105 B16 cells), or together with sgRNA for the transfection/electroporation groups. Other inhibitors were added to cell culture media 1 day before CRISPR/Etoposide/AMG232 exposure and cultured for 7 days. Cells were then collected for Trp53 KO genotyping. Supplementary Table 7 further describes how stock solutions of inhibitors were generated and stored.

Flow Cytometry Analysis

Fresh bone marrow cells from C57BL/6 Cas9+ GFP+ mice and Hox cells were stained with the following antibodies: FITC Rat anti-Mouse CD34 (BD Biosciences #553733, 1:500), PE anti-mouse CD150 (BioLegend #115904, 1:200), PerCP/Cyanine5.5 anti-mouse Ly-6A/E (BioLegend #122524, 1:200), APC anti-mouse CD117 (BioLegend #105812, 1:500), APC/Cyanine7 anti-mouse CD16/32 (BioLegend #156612, 1:500), Biotin anti-mouse Lineage Panel (BioLegend #133307, 1:100), BV421 Streptavidin (BD Biosciences #563259, 1:1000), LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen #L34957, 1:2000). After 30 min of staining, the cells were washed and analyzed by flow cytometry (BD FACSVerse). FACS FCS files were analyzed by FlowJo version 10 (FlowJo, LLC).

Analysis of data from the Depmap portal

sgRNA enrichment (CRISPR (Avana) Public 20Q4 release), mutation profile (Mutation Public 20Q4 release), drug sensitivity (PRISM Repurposing Primary Screen 19Q4 release), and mRNA expression levels (Expression Public 20Q4 release) was extracted December 13th 2020 from the Depmap portal (https://depmap.org/portal/)1113. Connectivity maps were generated using the geneMANIA plugin for Cytoscape14,15. tSNE plots were made with the Rtsne package (https://github.com/jkrijthe/Rtsne) to analyze the cluster and ggplot2 (https://github.com/tidyverse/ggplot2) to visualize the data, or tSNE-online (https://github.com/jefworks/tsne-online). The “ENCODE and ChEA Consensus TFs from ChIP-X” functionality of Enrichr (https://maayanlab.cloud/Enrichr/index.html)16,17 was used to identify transcription factor binding to gene sets.

Article TitleCRISPR enriches for cells with mutations in a p53-related interactome, and this can be inhibited

Abstract

CRISPR/Cas9 can be used to inactivate or modify genes by inducing double-stranded DNA breaks1–3. As a protective cellular response, DNA breaks result in p53-mediated cell cycle arrest and activation of cell death programs4,5. Inactivating p53 mutations are the most commonly found genetic alterations in cancer, highlighting the important role of the gene6–8. Here, we show that cells deficient in p53, as well as in genes of a core CRISPR-p53 tumor suppressor interactome, are enriched in a cell population when CRISPR is applied. Such enrichment could pose a challenge for clinical CRISPR use. Importantly, we identify that transient p53 inhibition suppresses the enrichment of cells with these mutations. Furthermore, in a data set of >800 human cancer cell lines, we identify parameters influencing the enrichment of p53 mutated cells, including strong baseline CDKN1A expression as a predictor for an active CRISPR-p53 axis. Taken together, our data identify strategies enabling safe CRISPR use.


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