CRISPR-Cas9 Targeted Enrichment and Next-Generation Sequencing for Mutation Detection

Malekshoar, Mehrdad; Azimi, Sajad Ataei; Kaki, Arastoo; Mousazadeh, Leila; Motaei, Jamshid; Vatankhah, Majid

doi:10.1016/j.jmoldx.2023.01.010

Cited by 8 publications

(4 citation statements)

References 71 publications

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“…Here we provide proof-of-concept that our single-reaction, CRISPR-Cas9 based, PCR-free enrichment approach may overcome many of the limitations of current methods such as short read NGS, SNP-based genotyping, by directly capturing both SNP level variation and complex SV/CNV in a single assay, which can be performed using multiple clinically relevant sample types such as blood and saliva. In addition, as our approach captures the entire region in continuous long reads, data generated can be used to develop more accurate reference sequences and has the potential to improve alignment and more accurate genotype and phenotype assignment ( Bu et al, 2020 ; Malekshoar et al, 2023 ).…”

Section: Discussionmentioning

confidence: 99%

Characterization of complex structural variation in the CYP2D6-CYP2D7-CYP2D8 gene loci using single-molecule long-read sequencing

Turner¹,

Derezinski²,

Gaedigk

et al. 2023

Front. Pharmacol.

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Complex regions in the human genome such as repeat motifs, pseudogenes and structural (SVs) and copy number variations (CNVs) present ongoing challenges to accurate genetic analysis, particularly for short-read Next-Generation-Sequencing (NGS) technologies. One such region is the highly polymorphic CYP2D loci, containing CYP2D6, a clinically relevant pharmacogene contributing to the metabolism of >20% of common drugs, and two highly similar pseudogenes, CYP2D7 and CYP2D8. Multiple complex SVs, including CYP2D6/CYP2D7-derived hybrid genes are known to occur in different configurations and frequencies across populations and are difficult to detect and characterize accurately. This can lead to incorrect enzyme activity assignment and impact drug dosing recommendations, often disproportionally affecting underrepresented populations. To improve CYP2D6 genotyping accuracy, we developed a PCR-free CRISPR-Cas9 based enrichment method for targeted long-read sequencing that fully characterizes the entire CYP2D6-CYP2D7-CYP2D8 loci. Clinically relevant sample types, including blood, saliva, and liver tissue were sequenced, generating high coverage sets of continuous single molecule reads spanning the entire targeted region of up to 52 kb, regardless of SV present (n = 9). This allowed for fully phased dissection of the entire loci structure, including breakpoints, to accurately resolve complex CYP2D6 diplotypes with a single assay. Additionally, we identified three novel CYP2D6 suballeles, and fully characterized 17 CYP2D7 and 18 CYP2D8 unique haplotypes. This method for CYP2D6 genotyping has the potential to significantly improve accurate clinical phenotyping to inform drug therapy and can be adapted to overcome testing limitations of other clinically challenging genomic regions.

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Section: Discussionmentioning

confidence: 99%

Characterization of complex structural variation in the CYP2D6-CYP2D7-CYP2D8 gene loci using single-molecule long-read sequencing

Turner¹,

Derezinski²,

Gaedigk

et al. 2023

Front. Pharmacol.

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“…The nuclease then cleaves the DNA at the target site, allowing for precise modifications of genome. 141 , 142 …”

Section: Technologies Used In the Detection Of Tumor Biomarkersmentioning

confidence: 99%

“…The nuclease then cleaves the DNA at the target site, allowing for precise modifications of genome. 141,142 By using CRISPR/Cas9 technology to precisely edit cancerrelated genes, researchers have created highly specific molecular probes for the detection of cancer biomarkers in body fluids, such as blood, urine, and saliva. CRISPR/Cas9 system is extensively used for different kinds of cancer biomarkers including virus nucleic acids, ctDNAs (i.e., EGFR mutation), miRNAs (i.e., miR-17, miR-31), proteins (i.e., TGF-β1, CEA, PSA, AFP), and extracellular vesicles.…”

Section: Electron Microscopy Technologymentioning

confidence: 99%

Tumor biomarkers for diagnosis, prognosis and targeted therapy

Zhou,

Tao,

Qiu

et al. 2024

Sig Transduct Target Ther

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Tumor biomarkers, the substances which are produced by tumors or the body’s responses to tumors during tumorigenesis and progression, have been demonstrated to possess critical and encouraging value in screening and early diagnosis, prognosis prediction, recurrence detection, and therapeutic efficacy monitoring of cancers. Over the past decades, continuous progress has been made in exploring and discovering novel, sensitive, specific, and accurate tumor biomarkers, which has significantly promoted personalized medicine and improved the outcomes of cancer patients, especially advances in molecular biology technologies developed for the detection of tumor biomarkers. Herein, we summarize the discovery and development of tumor biomarkers, including the history of tumor biomarkers, the conventional and innovative technologies used for biomarker discovery and detection, the classification of tumor biomarkers based on tissue origins, and the application of tumor biomarkers in clinical cancer management. In particular, we highlight the recent advancements in biomarker-based anticancer-targeted therapies which are emerging as breakthroughs and promising cancer therapeutic strategies. We also discuss limitations and challenges that need to be addressed and provide insights and perspectives to turn challenges into opportunities in this field. Collectively, the discovery and application of multiple tumor biomarkers emphasized in this review may provide guidance on improved precision medicine, broaden horizons in future research directions, and expedite the clinical classification of cancer patients according to their molecular biomarkers rather than organs of origin.

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“…However, these techniques usually rely on PCR amplification to provide highly specific sequencing and are prone to allelic bias and produce non-native DNA entailing the loss of epigenetic information [ 97 ]. In the last few years, alternative tools have been developed, often in combination with the use of Nanopore sequencing, with the aim of expanding the possibilities to investigate nucleic acid features such as the observation of structural variants (SVs) at the haplotype level through long-read sequencing and detection of DNA methylation ( Table 4 ) [ 98 , 99 ]. These tools exploit the CRISPR/Cas system and its two main characteristics: the high specificity of targeting through sgRNA, which also avoids long hybridization times, and the existence of a plethora of Cas proteins with the ability to perform a series of molecular actions on the nucleotide sequence of interest.…”

Section: Crispr/cas As An Enrichment Tool For Next-generation Sequencingmentioning

confidence: 99%

Not Only Editing: A Cas-Cade of CRISPR/Cas-Based Tools for Functional Genomics in Plants and Animals

Devillars,

Magon,

Pirrello

et al. 2024

IJMS

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The advent of CRISPR/Cas9 technology has revolutionized genome editing, enabling the attainment of once-unimaginable goals. CRISPR/Cas’s groundbreaking attributes lie in its simplicity, versatility, universality, and independence from customized DNA-protein systems, erasing the need for specialized expertise and broadening its scope of applications. It is therefore more and more used for genome modification including the generation of mutants. Beyond such editing scopes, the recent development of novel or modified Cas-based systems has spawned an array of additional biotechnological tools, empowering both fundamental and applied research. Precisely targeting DNA or RNA sequences, the CRISPR/Cas system has been harnessed in fields as diverse as gene regulation, deepening insights into gene expression, epigenetic changes, genome spatial organization, and chromatin dynamics. Furthermore, it aids in genome imaging and sequencing, as well as effective identification and countering of viral pathogens in plants and animals. All in all, the non-editing aspect of CRISPR/Cas exhibits tremendous potential across diverse domains, including diagnostics, biotechnology, and fundamental research. This article reviews and critically evaluates the primary CRISPR/Cas-based tools developed for plants and animals, underlining their transformative impact.

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CRISPR-Cas9 Targeted Enrichment and Next-Generation Sequencing for Mutation Detection

Cited by 8 publications

References 71 publications

Characterization of complex structural variation in the CYP2D6-CYP2D7-CYP2D8 gene loci using single-molecule long-read sequencing

Characterization of complex structural variation in the CYP2D6-CYP2D7-CYP2D8 gene loci using single-molecule long-read sequencing

Tumor biomarkers for diagnosis, prognosis and targeted therapy

Not Only Editing: A Cas-Cade of CRISPR/Cas-Based Tools for Functional Genomics in Plants and Animals

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