RESUMEN
The major challenges in pancreatic ductal adenocarcinoma (PDAC) management are local or distant metastasis and limited targeted therapeutics to prevent it. To identify a druggable target in tumor secretome and to explore its therapeutic intervention, we performed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis of tumors obtained from a patient-derived xenograft model of PDAC. Galectin-3 binding protein (Gal-3BP) is identified as a highly secreted protein, and its overexpression is further validated in multiple PDAC tumors and primary cells. Knockdown and exogenous treatment of Gal-3BP showed that it is required for PDAC cell proliferation, migration, and invasion. Mechanistically, we revealed that Gal-3BP enhances galectin-3-mediated epidermal growth factor receptor signaling, leading to increased cMyc and epithelial-mesenchymal transition. To explore the clinical impact of these findings, two antibody clones were developed, and they profoundly abrogated the metastasis of PDAC cells in vivo. Altogether, our data demonstrate that Gal-3BP is an important therapeutic target in PDAC, and we propose its blockade by antibody as a therapeutic option for suppressing PDAC metastasis.
Asunto(s)
Antígenos de Neoplasias , Antineoplásicos Inmunológicos , Biomarcadores de Tumor , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/inmunología , Antineoplásicos Inmunológicos/uso terapéutico , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/secundario , Carcinoma Ductal Pancreático/terapia , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Cromatografía Liquida , Transición Epitelial-Mesenquimal , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/terapia , Proteómica , Secretoma , Espectrometría de Masas en Tándem , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CRISPR-based base editors (BEs) are widely used to induce nucleotide substitutions in living cells and organisms without causing the damaging DNA double-strand breaks and DNA donor templates. Cytosine BEs that induce C:G to T:A conversion and adenine BEs that induce A:T to G:C conversion have been developed. Various attempts have been made to increase the efficiency of both BEs; however, their activities need to be improved for further applications. Here, we describe a fluorescent reporter-based drug screening platform to identify novel chemicals with the goal of improving adenine base editing efficiency. The reporter system revealed that histone deacetylase inhibitors, particularly romidepsin, enhanced base editing efficiencies by up to 4.9-fold by increasing the expression levels of proteins and target accessibility. The results support the use of romidepsin as a viable option to improve base editing efficiency in biomedical research and therapeutic genome engineering.
Asunto(s)
Adenina , Sistemas CRISPR-Cas , Edición Génica , Inhibidores de Histona Desacetilasas/farmacología , Depsipéptidos/farmacología , Doxiciclina/farmacología , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Células HeLa , Humanos , Sustancias Luminiscentes/análisis , Biosíntesis de Proteínas , ARN/biosíntesisRESUMEN
Although prime editors are a powerful tool for genome editing, which can generate various types of mutations such as nucleotide substitutions, insertions, and deletions in the genome without double-strand breaks or donor DNA, the conventional prime editors are still limited to their target scopes because of the PAM preference of the Streptococcus pyogenes Cas9 (spCas9) protein. Here, we describe the engineered prime editors to expand the range of their target sites using various PAM-flexible Cas9 variants. Using the engineered prime editors, we could successfully generate more than 50 types of mutations with up to 51.7% prime-editing activity in HEK293T cells. In addition, we successfully introduced the BRAF V600E mutation, which could not be induced by conventional prime editors. These variants of prime editors will broaden the applicability of CRISPR-based prime editing technologies in biological research.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Ingeniería Genética , Motivos de Nucleótidos , Alelos , Sustitución de Aminoácidos , Sitios de Unión , Proteína 9 Asociada a CRISPR , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Ingeniería Genética/métodos , Células HEK293 , Humanos , Mutación , Proteínas Proto-Oncogénicas B-raf/genéticaRESUMEN
KRAS is the most commonly mutated RAS family gene and is a primary cause of the occurrence of several types of cancer. However, KRAS mutations have several unique and diverse molecular identities, making it difficult to find specific treatments. Here, we developed universal pegRNAs which can correct all types of G12 and G13 oncogenic KRAS mutations with CRISPR-mediated prime editors (PEs). The universal pegRNA successfully corrected 12 types of KRAS mutations, accounting for 94% of all known KRAS mutations, by up to 54.8% correction frequency in HEK293T/17 cells. We also applied the universal pegRNA to correct endogenous KRAS mutations in human cancer cells and found that G13D KRAS mutation was successfully corrected to wild-type KRAS sequences with up to 40.6% correction frequency without indel mutations. We propose prime editing with the universal pegRNA as a 'one-to-many' potential therapeutic strategy for KRAS oncogene variants.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Células HEK293 , Proteínas Proto-Oncogénicas p21(ras)/genética , Mutación INDEL , MutaciónRESUMEN
Various CRISPRâCas9 orthologs are used in genome engineering. One of the smallest Cas9 orthologs is cjCas9 derived from Campylobacter jejuni, which is a highly specific genome editing tool. Here, we developed cjCas9-based base editors including a cytosine base editor (cjCBEmax) and an adenine base editor (cjABE8e) that can successfully induce endogenous base substitutions by up to 91.2% at the HPD gene in HEK293T cells. Analysis of the base editing efficiency of 13 endogenous target sites showed that the active windows of cjCBEmax and cjABE8e are wider than those of spCas9-based base editors and that their specificities are slightly lower than that of cjCas9. Importantly, engineered cjCas9 and gRNA scaffolds can improve the base editing efficiency of cjABE8e by up to 6.4-fold at the HIF1A gene in HEK293T cells. Due to its small size, cjABE8e can be packaged in a single adeno-associated virus vector with two tandem arrays of gRNAs, and the delivery of the resulting AAV could introduce base substitutions at endogenous ANGPT2 and HPD target sites. Overall, our findings have expanded the potential of the use of base editors for in vivo or ex vivo therapeutic approaches.
Asunto(s)
Campylobacter jejuni , Edición Génica , Humanos , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Células HEK293 , ARN Guía de Sistemas CRISPR-CasRESUMEN
Lesch-Nyhan syndrome (LNS) is inherited as an X-linked recessive genetic disorder caused by mutations in hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1). Patients with LNS show various clinical phenotypes, including hyperuricemia, gout, devastating behavioral abnormality, intellectual disability, and self-harm. Although uric acid overproduction can be modulated with the xanthine oxidase inhibitor allopurinol, there exists no treatment for behavioral and neurological manifestations of LNS. In the current study, CRISPR-mediated base editors (BEs) and prime editors (PEs) were utilized to generate LNS-associated disease models and correct the disease models for therapeutic approach. Cytosine BEs (CBEs) were used to induce c.430C>T and c.508C>T mutations in HAP1 cells, and then adenine BEs (ABEs) were used to correct these mutations without DNA cleavage. PEs induced a c.333_334ins(A) mutation, identified in a Korean patient with LNS, in HAP1 cells, which was corrected in turn by PEs. Furthermore, improved PEs corrected the same mutation in LNS patient-derived fibroblasts by up to 14% without any unwanted mutations. These results suggest that CRISPR-mediated BEs and PEs would be suggested as a potential therapeutic strategy of this extremely rare, devastating genetic disease.
RESUMEN
Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C>T, c.154C>T, c.3847C>T, c.5056C>T, and c.4986+5G>A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.