Amin Aalipour1,2, Jonathan C Dudley3, Seung-Min Park2,4, Surya Murty1,2, Jacob J Chabon5,6, Evan A Boyle7, Maximilian Diehn5,6,8, Sanjiv S Gambhir9,2,4,10. 1. Department of Bioengineering, Stanford University School of Medicine, Stanford, CA. 2. Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, CA. 3. Department of Pathology, Stanford University School of Medicine, Stanford, CA. 4. Department of Radiology, Stanford University School of Medicine, Stanford, CA. 5. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA. 6. Stanford Cancer Institute, Stanford University, Stanford, CA. 7. Department of Genetics, Stanford University, Stanford, CA. 8. Department of Radiation Oncology, Stanford University, Stanford, CA. 9. Department of Bioengineering, Stanford University School of Medicine, Stanford, CA; sgambhir@stanford.edu. 10. Canary Center at Stanford for Cancer Early Detection, Stanford University School of Medicine, Palo Alto, CA.
Abstract
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.
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 NSCLCpatient-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.
Authors: Seung-Min Park; Daeyoun D Won; Brian J Lee; Diego Escobedo; Andre Esteva; Amin Aalipour; T Jessie Ge; Jung Ha Kim; Susie Suh; Elliot H Choi; Alexander X Lozano; Chengyang Yao; Sunil Bodapati; Friso B Achterberg; Jeesu Kim; Hwan Park; Youngjae Choi; Woo Jin Kim; Jung Ho Yu; Alexander M Bhatt; Jong Kyun Lee; Ryan Spitler; Shan X Wang; Sanjiv S Gambhir Journal: Nat Biomed Eng Date: 2020-04-06 Impact factor: 25.671
Authors: Jinzhao Song; Jorrit W Hegge; Michael G Mauk; Junman Chen; Jacob E Till; Neha Bhagwat; Lotte T Azink; Jing Peng; Moen Sen; Jazmine Mays; Erica L Carpenter; John van der Oost; Haim H Bau Journal: Nucleic Acids Res Date: 2020-02-28 Impact factor: 16.971
Authors: Ieva Keraite; Virginia Alvarez-Garcia; Isaac Garcia-Murillas; Matthew Beaney; Nicholas C Turner; Clare Bartos; Olga Oikonomidou; Maïwenn Kersaudy-Kerhoas; Nicholas R Leslie Journal: Sci Rep Date: 2020-10-13 Impact factor: 4.379