RESUMO
CRISPR-Cas technology has rapidly changed life science research and human medicine. The ability to add, remove, or edit human DNA sequences has transformative potential for treating congenital and acquired human diseases. The timely maturation of the cell and gene therapy ecosystem and its seamless integration with CRISPR-Cas technologies has enabled the development of therapies that could potentially cure not only monogenic diseases such as sickle cell anemia and muscular dystrophy, but also complex heterogenous diseases such as cancer and diabetes. Here, we review the current landscape of clinical trials involving the use of various CRISPR-Cas systems as therapeutics for human diseases, discuss challenges, and explore new CRISPR-Cas-based tools such as base editing, prime editing, CRISPR-based transcriptional regulation, CRISPR-based epigenome editing, and RNA editing, each promising new functionality and broadening therapeutic potential. Finally, we discuss how the CRISPR-Cas system is being used to understand the biology of human diseases through the generation of large animal disease models used for preclinical testing of emerging therapeutics.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Humanos , Sistemas CRISPR-Cas/genética , Ecossistema , Terapia Genética , EpigenomaRESUMO
53BP1 regulates DNA end-joining in lymphocytes, diversifying immune antigen receptors. This involves nucleosome-bound 53BP1 at DNA double-stranded breaks (DSBs) recruiting RIF1 and shieldin, a poorly understood DNA-binding complex. The 53BP1-RIF1-shieldin axis is pathological in BRCA1-mutated cancers, blocking homologous recombination (HR) and driving illegitimate non-homologous end-joining (NHEJ). However, how this axis regulates DNA end-joining and HR suppression remains unresolved. We investigated shieldin and its interplay with CST, a complex recently implicated in 53BP1-dependent activities. Immunophenotypically, mice lacking shieldin or CST are equivalent, with class-switch recombination co-reliant on both complexes. ATM-dependent DNA damage signalling underpins this cooperation, inducing physical interactions between these complexes that reveal shieldin as a DSB-responsive CST adaptor. Furthermore, DNA polymerase ζ functions downstream of shieldin, establishing DNA fill-in synthesis as the physiological function of shieldin-CST. Lastly, 53BP1 suppresses HR and promotes NHEJ in BRCA1-deficient mice and cells independently of shieldin. These findings showcase the resilience of the 53BP1 pathway, achieved through the collaboration of chromatin-bound 53BP1 complexes and DNA end-processing effector proteins.
RESUMO
Protein ubiquitination at sites of DNA double-strand breaks (DSBs) by RNF168 recruits BRCA1 and 53BP11,2, which are mediators of the homologous recombination and non-homologous end joining DSB repair pathways, respectively3. Non-homologous end joining relies on 53BP1 binding directly to ubiquitinated lysine 15 on H2A-type histones (H2AK15ub)4,5 (which is an RNF168-dependent modification6), but how RNF168 promotes BRCA1 recruitment and function remains unclear. Here we identify a tandem BRCT-domain-associated ubiquitin-dependent recruitment motif (BUDR) in BRCA1-associated RING domain protein 1 (BARD1) (the obligate partner protein of BRCA1) that, by engaging H2AK15ub, recruits BRCA1 to DSBs. Disruption of the BUDR of BARD1 compromises homologous recombination and renders cells hypersensitive to PARP inhibition and cisplatin. We further show that BARD1 binds nucleosomes through multivalent interactions: coordinated binding of H2AK15ub and unmethylated H4 lysine 20 by its adjacent BUDR and ankyrin repeat domains, respectively, provides high-affinity recognition of DNA lesions in replicated chromatin and promotes the homologous recombination activities of the BRCA1-BARD1 complex. Finally, our genetic epistasis experiments confirm that the need for BARD1 chromatin-binding activities can be entirely relieved upon deletion of RNF168 or 53BP1. Thus, our results demonstrate that by sensing DNA-damage-dependent and post-replication histone post-translation modification states, BRCA1-BARD1 complexes coordinate the antagonization of the 53BP1 pathway with promotion of homologous recombination, establishing a simple paradigm for the governance of the choice of DSB repair pathway.
Assuntos
Recombinação Homóloga , Lisina/química , Lisina/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Adulto , Motivos de Aminoácidos , Proteína BRCA1/química , Proteína BRCA1/metabolismo , Cromatina/metabolismo , Cisplatino/farmacologia , Quebras de DNA de Cadeia Dupla , Dano ao DNA/efeitos dos fármacos , Feminino , Células HCT116 , Células HEK293 , Histonas/química , Histonas/metabolismo , Humanos , Masculino , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Domínios Proteicos , Reparo de DNA por Recombinação , Proteínas Supressoras de Tumor/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/deficiênciaRESUMO
Genotoxic DNA double-strand breaks (DSBs) can be repaired by error-free homologous recombination (HR) or mutagenic non-homologous end-joining1. HR supresses tumorigenesis1, but is restricted to the S and G2 phases of the cell cycle when a sister chromatid is present2. Breast cancer type 1 susceptibility protein (BRCA1) promotes HR by antagonizing the anti-resection factor TP53-binding protein 1(53BP1) (refs. 2-5), but it remains unknown how BRCA1 function is limited to the S and G2 phases. We show that BRCA1 recruitment requires recognition of histone H4 unmethylated at lysine 20 (H4K20me0), linking DSB repair pathway choice directly to sister chromatid availability. We identify the ankyrin repeat domain of BRCA1-associated RING domain protein 1 (BARD1)-the obligate BRCA1 binding partner3-as a reader of H4K20me0 present on new histones in post-replicative chromatin6. BARD1 ankyrin repeat domain mutations disabling H4K20me0 recognition abrogate accumulation of BRCA1 at DSBs, causing aberrant build-up of 53BP1, and allowing anti-resection activity to prevail in S and G2. Consequently, BARD1 recognition of H4K20me0 is required for HR and resistance to poly (ADP-ribose) polymerase inhibitors. Collectively, this reveals that BRCA1-BARD1 monitors the replicative state of the genome to oppose 53BP1 function, routing only DSBs within sister chromatids to HR.
Assuntos
Proteína BRCA1/metabolismo , Cromátides/metabolismo , Histonas/metabolismo , Recombinação Homóloga , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Proteína BRCA1/genética , Linhagem Celular Tumoral , Cromátides/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Fase G2/genética , Células HCT116 , Células HeLa , Humanos , Lisina/metabolismo , Metilação , Fase S/genética , Homologia de Sequência de Aminoácidos , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
53BP1 controls a specialized non-homologous end joining (NHEJ) pathway that is essential for adaptive immunity, yet oncogenic in BRCA1 mutant cancers. Intra-chromosomal DNA double-strand break (DSB) joining events during immunoglobulin class switch recombination (CSR) require 53BP1. However, in BRCA1 mutant cells, 53BP1 blocks homologous recombination (HR) and promotes toxic NHEJ, resulting in genomic instability. Here, we identify the protein dimerization hub-DYNLL1-as an organizer of multimeric 53BP1 complexes. DYNLL1 binding stimulates 53BP1 oligomerization, and promotes 53BP1's recruitment to, and interaction with, DSB-associated chromatin. Consequently, DYNLL1 regulates 53BP1-dependent NHEJ: CSR is compromised upon deletion of Dynll1 or its transcriptional regulator Asciz, or by mutation of DYNLL1 binding motifs in 53BP1; furthermore, Brca1 mutant cells and tumours are rendered resistant to poly-ADP ribose polymerase (PARP) inhibitor treatments upon deletion of Dynll1 or Asciz. Thus, our results reveal a mechanism that regulates 53BP1-dependent NHEJ and the therapeutic response of BRCA1-deficient cancers.
Assuntos
Proteína BRCA1/genética , Dineínas do Citoplasma/metabolismo , Reparo do DNA por Junção de Extremidades/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Feminino , Instabilidade Genômica/genética , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
53BP1 governs a specialized, context-specific branch of the classical non-homologous end joining DNA double-strand break repair pathway. Mice lacking 53bp1 (also known as Trp53bp1) are immunodeficient owing to a complete loss of immunoglobulin class-switch recombination1,2, and reduced fidelity of long-range V(D)J recombination3. The 53BP1-dependent pathway is also responsible for pathological joining events at dysfunctional telomeres4, and its unrestricted activity in Brca1-deficient cellular and tumour models causes genomic instability and oncogenesis5-7. Cells that lack core non-homologous end joining proteins are profoundly radiosensitive8, unlike 53BP1-deficient cells9,10, which suggests that 53BP1 and its co-factors act on specific DNA substrates. Here we show that 53BP1 cooperates with its downstream effector protein REV7 to promote non-homologous end joining during class-switch recombination, but REV7 is not required for 53BP1-dependent V(D)J recombination. We identify shieldin-a four-subunit putative single-stranded DNA-binding complex comprising REV7, c20orf196 (SHLD1), FAM35A (SHLD2) and FLJ26957 (SHLD3)-as the factor that explains this specificity. Shieldin is essential for REV7-dependent DNA end-protection and non-homologous end joining during class-switch recombination, and supports toxic non-homologous end joining in Brca1-deficient cells, yet is dispensable for REV7-dependent interstrand cross-link repair. The 53BP1 pathway therefore comprises distinct double-strand break repair activities within chromatin and single-stranded DNA compartments, which explains both the immunological differences between 53bp1- and Rev7- deficient mice and the context specificity of the pathway.
Assuntos
Reparo do DNA por Junção de Extremidades , DNA/química , DNA/metabolismo , Proteínas Mad2/metabolismo , Complexos Multiproteicos/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Switching de Imunoglobulina/genética , Proteínas Mad2/deficiência , Proteínas Mad2/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multiproteicos/química , Mutação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Recombinação V(D)J/genéticaRESUMO
Overexpression of the flap endonuclease FEN1 has been observed in a variety of cancer types and is a marker for poor prognosis. To better understand the cellular consequences of FEN1 overexpression we utilized a model of its Saccharomyces cerevisiae homolog, RAD27. In this system, we discovered that flap endonuclease overexpression impedes replication fork progression and leads to an accumulation of cells in mid-S phase. This was accompanied by increased phosphorylation of the checkpoint kinase Rad53 and histone H2A-S129. RAD27 overexpressing cells were hypersensitive to treatment with DNA damaging agents, and defective in ubiquitinating the replication clamp proliferating cell nuclear antigen (PCNA) at lysine 164. These effects were reversed when the interaction between overexpressed Rad27 and PCNA was ablated, suggesting that the observed phenotypes were linked to problems in DNA replication. RAD27 overexpressing cells also exhibited an unexpected dependence on the SUMO ligases SIZ1 and MMS21 for viability. Importantly, we found that overexpression of FEN1 in human cells also led to phosphorylation of CHK1, CHK2, RPA32 and histone H2AX, all markers of genome instability. Our data indicate that flap endonuclease overexpression is a driver of genome instability in yeast and human cells that impairs DNA replication in a manner dependent on its interaction with PCNA.
Assuntos
Dano ao DNA , Endonucleases Flap/metabolismo , Instabilidade Genômica , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Neoplasias Pulmonares/enzimologia , Carcinoma de Pequenas Células do Pulmão/enzimologia , Sumoilação , UbiquitinaçãoRESUMO
The transposons of Drosophila melanogaster are regulated by small RNAs that interact with the Piwi family of proteins. These piRNAs are generated from transposons inserted in special loci such as the telomere-associated sequences at the left end of the X chromosome. Drosophila's P transposons can also be regulated by a polypeptide encoded by the KP element, a 1.15-kb-long member of the P family. Using piRNA-generating telomeric P elements (TPs) and repressor-producing transgenic KP elements, we demonstrate a functional connection between these two modes of regulation. By themselves, the TPs partially repress gonadal dysgenesis, a trait caused by rampant P-element activity in the germ line. This repression is manifested as a strictly maternal effect arising from the cytoplasmic transmission of P-specific piRNAs from mother to offspring. The repression is enhanced by genetic interactions between the TPs and other, non-telomeric P elements-a phenomenon attributable to ping-pong amplification of maternal piRNAs. KP elements, like other kinds of non-telomeric P elements, enhance regulation anchored in the TPs. However, with some TPs, the enhanced regulation is manifested as a strictly zygotic effect of the KP element. This effect is seen when the TP has few sequences in common with the KP element, a condition not conducive to ping-pong amplification of piRNAs; it can be attributed to the action of the KP repressor polypeptide. Because the effect is seen only when a TP was present in the mother's genotype, maternally generated P-element piRNAs could facilitate regulation by the KP repressor polypeptide.