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Deactivated CRISPR Associated Protein 9 for Minor-Allele Enrichment in Cell-Free DNA.
Aalipour, Amin; Dudley, Jonathan C; Park, Seung-Min; Murty, Surya; Chabon, Jacob J; Boyle, Evan A; Diehn, Maximilian; Gambhir, Sanjiv S.
Afiliación
  • Aalipour A; Department of Bioengineering, Stanford University School of Medicine, Stanford, CA.
  • Dudley JC; Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, CA.
  • Park SM; Department of Pathology, Stanford University School of Medicine, Stanford, CA.
  • Murty S; Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, CA.
  • Chabon JJ; Department of Radiology, Stanford University School of Medicine, Stanford, CA.
  • Boyle EA; Department of Bioengineering, Stanford University School of Medicine, Stanford, CA.
  • Diehn M; Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, CA.
  • Gambhir SS; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA.
Clin Chem ; 64(2): 307-316, 2018 02.
Article en En | MEDLINE | ID: mdl-29038154
BACKGROUND: Cell-free DNA (cfDNA) diagnostics are emerging as a new paradigm of disease monitoring and therapy management. The clinical utility of these diagnostics is relatively limited by a low signal-to-noise ratio, such as with low allele frequency (AF) mutations in cancer. While enriching for rare alleles to increase their AF before sample analysis is one strategy that can greatly improve detection capability, current methods are limited in their generalizability, ease of use, and applicability to point mutations. METHODS: Leveraging the robust single-base-pair specificity and generalizability of the CRISPR associated protein 9 (Cas9) system, we developed a deactivated Cas9 (dCas9)-based method of minor-allele enrichment capable of efficient single-target and multiplexed enrichment. The dCas9 protein was complexed with single guide RNAs targeted to mutations of interest and incubated with cfDNA samples containing mutant strands at low abundance. Mutation-bound dCas9 complexes were isolated, dissociated, and the captured DNA purified for downstream use. RESULTS: Targeting the 3 most common epidermal growth factor receptor mutations (exon 19 deletion, T790M, L858R) found in non-small cell lung cancer (NSCLC), we achieved >20-fold increases in AF and detected mutations by use of qPCR at an AF of 0.1%. In a cohort of 18 NSCLC patient-derived cfDNA samples, our method enabled detection of 8 out of 13 mutations that were otherwise undetected by qPCR. CONCLUSIONS: The dCas9 method provides an important application of the CRISPR/Cas9 system outside the realm of genome editing and can provide a step forward for the detection capability of cfDNA diagnostics.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Ácidos Nucleicos Libres de Células / Proteína 9 Asociada a CRISPR / Frecuencia de los Genes Tipo de estudio: Risk_factors_studies Límite: Humans Idioma: En Revista: Clin Chem Asunto de la revista: QUIMICA CLINICA Año: 2018 Tipo del documento: Article Pais de publicación: Reino Unido
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Ácidos Nucleicos Libres de Células / Proteína 9 Asociada a CRISPR / Frecuencia de los Genes Tipo de estudio: Risk_factors_studies Límite: Humans Idioma: En Revista: Clin Chem Asunto de la revista: QUIMICA CLINICA Año: 2018 Tipo del documento: Article Pais de publicación: Reino Unido