ABSTRACT
Genomic instability can trigger cancer-intrinsic innate immune responses that promote tumor rejection. However, cancer cells often evade these responses by overexpressing immune checkpoint regulators, such as PD-L1. Here, we identify the SNF2-family DNA translocase SMARCAL1 as a factor that favors tumor immune evasion by a dual mechanism involving both the suppression of innate immune signaling and the induction of PD-L1-mediated immune checkpoint responses. Mechanistically, SMARCAL1 limits endogenous DNA damage, thereby suppressing cGAS-STING-dependent signaling during cancer cell growth. Simultaneously, it cooperates with the AP-1 family member JUN to maintain chromatin accessibility at a PD-L1 transcriptional regulatory element, thereby promoting PD-L1 expression in cancer cells. SMARCAL1 loss hinders the ability of tumor cells to induce PD-L1 in response to genomic instability, enhances anti-tumor immune responses and sensitizes tumors to immune checkpoint blockade in a mouse melanoma model. Collectively, these studies uncover SMARCAL1 as a promising target for cancer immunotherapy.
Subject(s)
B7-H1 Antigen , DNA Helicases , Immunity, Innate , Melanoma , Tumor Escape , Animals , Mice , B7-H1 Antigen/metabolism , Genomic Instability , Melanoma/immunology , Melanoma/metabolism , DNA Helicases/metabolismABSTRACT
Mutations in DNA damage response (DDR) genes endanger genome integrity and predispose to cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editing screens, we identify > 2,000 sgRNAs that generate nucleotide variants in 86 DDR genes, resulting in altered cellular fitness upon DNA damage. Among those variants, we discover loss- and gain-of-function mutants in the Tudor domain of the DDR regulator 53BP1 that define a non-canonical surface required for binding the deubiquitinase USP28. Moreover, we characterize variants of the TRAIP ubiquitin ligase that define a domain, whose loss renders cells resistant to topoisomerase I inhibition. Finally, we identify mutations in the ATM kinase with opposing genome stability phenotypes and loss-of-function mutations in the CHK2 kinase previously categorized as variants of uncertain significance for breast cancer. We anticipate that this resource will enable the discovery of additional DDR gene functions and expedite studies of DDR variants in human disease.
Subject(s)
DNA Damage , Gene Editing , Genetic Testing , Amino Acid Sequence , Ataxia Telangiectasia Mutated Proteins/metabolism , Base Sequence , CRISPR-Cas Systems/genetics , Camptothecin/pharmacology , Cell Line , DNA Damage/genetics , DNA Repair/genetics , Female , Humans , Mutation/genetics , Phenotype , Protein Binding , Protein Domains , RNA, Guide, Kinetoplastida/genetics , Topoisomerase Inhibitors/pharmacology , Tumor Suppressor p53-Binding Protein 1/chemistry , Tumor Suppressor p53-Binding Protein 1/genetics , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%-99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP.
Subject(s)
CRISPR-Associated Proteins/genetics , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Codon, Terminator , Gene Editing/methods , Gene Silencing , Animals , Arabidopsis/genetics , Arabidopsis/metabolism , CRISPR-Associated Proteins/metabolism , Codon, Nonsense , Computational Biology , DNA Restriction Enzymes/genetics , DNA Restriction Enzymes/metabolism , Databases, Genetic , Gene Expression Regulation, Fungal , Gene Expression Regulation, Neoplastic , Gene Expression Regulation, Plant , HEK293 Cells , Humans , Mice , Neoplasms/genetics , Neoplasms/metabolism , Polymorphism, Restriction Fragment Length , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism , Rats , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , TransfectionABSTRACT
The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise in recent clinical trials. Nuclease-driven genome editing provides a method in which to precisely target genetic changes to further enhance T cell function in vivo. We describe the development of a highly efficient method to genome edit both primary human CD8 and CD4 T cells by homology-directed repair at a pre-defined site of the genome. Two different homology donor templates were evaluated, representing both minor gene editing events (restriction site insertion) to mimic gene correction, or the more significant insertion of a larger gene cassette. By combining zinc finger nuclease mRNA delivery with AAV6 delivery of a homologous donor we could gene correct 41% of CCR5 or 55% of PPP1R12C (AAVS1) alleles in CD8(+) T cells and gene targeting of a GFP transgene cassette in >40% of CD8(+) and CD4(+) T cells at both the CCR5 and AAVS1 safe harbor locus, potentially providing a robust genome editing tool for T cell-based immunotherapy.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Dependovirus/genetics , Endonucleases/genetics , Genetic Vectors , Genome, Human , RNA, Messenger/genetics , Transfection , Zinc Fingers , CD4-Positive T-Lymphocytes/enzymology , CD8-Positive T-Lymphocytes/enzymology , HumansABSTRACT
CRISPR-dependent genome editing enables the study of genes and mutations on a large scale. Here we review CRISPR-based functional genomics technologies that generate gene knockouts and single nucleotide variants (SNVs) and discuss how their use has provided new important insights into the function of homologous recombination (HR) genes. In particular, we highlight discoveries from CRISPR screens that have contributed to define the response to PARP inhibition in cells deficient for the HR genes BRCA1 and BRCA2, uncover genes whose loss causes synthetic lethality in combination with BRCA1/2 deficiency, and characterize the function of BRCA1/2 SNVs of uncertain clinical significance. Further use of these approaches, combined with next-generation CRISPR-based technologies, will aid to dissect the genetic network of the HR pathway, define the impact of HR mutations on cancer etiology and treatment, and develop novel targeted therapies for HR-deficient tumors.
Subject(s)
Gene Regulatory Networks , Poly(ADP-ribose) Polymerase Inhibitors , Genomics , Homologous Recombination/genetics , Mutation , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic useABSTRACT
Genome editing technologies have transformed our ability to engineer desired genomic changes within living systems. However, detecting precise genomic modifications often requires sophisticated, expensive, and time-consuming experimental approaches. Here, we describe DTECT (Dinucleotide signaTurE CapTure), a rapid and versatile detection method that relies on the capture of targeted dinucleotide signatures resulting from the digestion of genomic DNA amplicons by the type IIS restriction enzyme AcuI. DTECT enables the accurate quantification of marker-free precision genome editing events introduced by CRISPR-dependent homology-directed repair, base editing, or prime editing in various biological systems, such as mammalian cell lines, organoids, and tissues. Furthermore, DTECT allows the identification of oncogenic mutations in cancer mouse models, patient-derived xenografts, and human cancer patient samples. The ease, speed, and cost efficiency by which DTECT identifies genomic signatures should facilitate the generation of marker-free cellular and animal models of human disease and expedite the detection of human pathogenic variants.
Subject(s)
Gene Editing , Genetic Variation , Genomics , Animals , BRCA1 Protein/genetics , BRCA2 Protein/genetics , Base Sequence , DNA/genetics , Disease Models, Animal , Genetic Loci , Genetic Markers , Genotype , HEK293 Cells , Humans , Mice , Mutation/genetics , NIH 3T3 Cells , Neoplasms/genetics , Nucleotides/genetics , Oncogenes , Recombinational DNA Repair/genetics , Restriction MappingABSTRACT
Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. Here we characterize C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we report that MCM8IP directly associates with MCM8-9, a helicase complex mutated in primary ovarian insufficiency, and RPA1. We additionally show that the interactions of MCM8IP with MCM8-9 and RPA facilitate HR and promote replication fork progression and cellular viability in response to treatment with crosslinking agents. Mechanistically, MCM8IP stimulates the helicase activity of MCM8-9. Collectively, our work identifies MCM8IP as a key regulator of MCM8-9-dependent DNA synthesis during DNA recombination and replication.
Subject(s)
DNA Damage , DNA Replication , DNA-Binding Proteins/metabolism , Minichromosome Maintenance Proteins/metabolism , Recombinational DNA Repair , Cell Line, Tumor , Cell Survival/genetics , Chromatin/genetics , Chromatin/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/genetics , HCT116 Cells , HEK293 Cells , Humans , Minichromosome Maintenance Proteins/genetics , Mutation , Protein Binding , Rad51 Recombinase/metabolism , Replication Protein A/genetics , Replication Protein A/metabolismABSTRACT
Precise editing of genomic DNA can be achieved upon repair of CRISPR-induced DNA double-stranded breaks (DSBs) by homology-directed repair (HDR). However, the efficiency of this process is limited by DSB repair pathways competing with HDR, such as non-homologous end joining (NHEJ). Here we individually express in human cells 204 open reading frames involved in the DNA damage response (DDR) and determine their impact on CRISPR-mediated HDR. From these studies, we identify RAD18 as a stimulator of CRISPR-mediated HDR. By defining the RAD18 domains required to promote HDR, we derive an enhanced RAD18 variant (e18) that stimulates CRISPR-mediated HDR in multiple human cell types, including embryonic stem cells. Mechanistically, e18 induces HDR by suppressing the localization of the NHEJ-promoting factor 53BP1 to DSBs. Altogether, this study identifies e18 as an enhancer of CRISPR-mediated HDR and highlights the promise of engineering DDR factors to augment the efficiency of precision genome editing.
Subject(s)
DNA-Binding Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats , DNA Damage , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Gene Editing , Humans , Protein Domains , Protein Engineering , Recombinational DNA Repair , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/geneticsABSTRACT
Gene therapy for HIV-1 infection is a promising alternative to lifelong combination antiviral drug treatment. Chemokine receptor 5 (CCR5) is the coreceptor required for R5-tropic HIV-1 infection of human cells. Deletion of CCR5 renders cells resistant to R5-tropic HIV-1 infection, and the potential for cure has been shown through allogeneic stem cell transplantation with naturally occurring homozygous deletion of CCR5 in donor hematopoietic stem/progenitor cells (HSPC). The requirement for HLA-matched HSPC bearing homozygous CCR5 deletions prohibits widespread application of this approach. Thus, a strategy to disrupt CCR5 genomic sequences in HSPC using zinc finger nucleases was developed. Following discussions with regulatory agencies, we conducted IND-enabling preclinical in vitro and in vivo testing to demonstrate the feasibility and (preclinical) safety of zinc finger nucleases-based CCR5 disruption in HSPC. We report here the clinical-scale manufacturing process necessary to deliver CCR5-specific zinc finger nucleases mRNA to HSPC using electroporation and the preclinical safety data. Our results demonstrate effective biallelic CCR5 disruption in up to 72.9% of modified colony forming units from adult mobilized HSPC with maintenance of hematopoietic potential in vitro and in vivo. Tumorigenicity studies demonstrated initial product safety; further safety and feasibility studies are ongoing in subjects infected with HIV-1 (NCT02500849@clinicaltrials.gov).
ABSTRACT
Genome editing with targeted nucleases and DNA donor templates homologous to the break site has proven challenging in human hematopoietic stem and progenitor cells (HSPCs), and particularly in the most primitive, long-term repopulating cell population. Here we report that combining electroporation of zinc finger nuclease (ZFN) mRNA with donor template delivery by adeno-associated virus (AAV) serotype 6 vectors directs efficient genome editing in HSPCs, achieving site-specific insertion of a GFP cassette at the CCR5 and AAVS1 loci in mobilized peripheral blood CD34+ HSPCs at mean frequencies of 17% and 26%, respectively, and in fetal liver HSPCs at 19% and 43%, respectively. Notably, this approach modified the CD34+CD133+CD90+ cell population, a minor component of CD34+ cells that contains long-term repopulating hematopoietic stem cells (HSCs). Genome-edited HSPCs also engrafted in immune-deficient mice long-term, confirming that HSCs are targeted by this approach. Our results provide a strategy for more robust application of genome-editing technologies in HSPCs.