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The CRISPR/Cas9 system has been successfully applied in dozens of diverse species; although the screening of successful CRISPR/Cas9 editing events remains particularly laborious, especially for those that occur at relatively low frequency. Recently, a co‐CRISPR strategy was proved to enrich the desired CRISPR events. Here, the co‐CRISPR strategy was developed in the Fall armyworm, Spodoptera frugiperda, with kynurenine 3‐monooxygenase gene (kmo) as a marker. The kmo mosaics induced by single‐guide RNAs (sgRNAs)/Cas9 displayed the darker green color phenotype in larvae, compared with wild type (brown), and mosaic‐eye adults were significantly acquired from the mosaic larvae group. In the kmo knockout strain, no significant difference was observed in larval development and adult reproduction. Acetylcholinesterase 2 (ace2) and Wnt1 were selected as target genes to construct the co‐CRISPR strategy using kmo marker. By co‐injection of kmo and ace2 sgRNAs, the mutant efficiency of ace2 was significantly increased in the kmo mosaic (larvae or adults) groups. Similarly, more malformed pupae with Wnt1 mutations were observed in the darker green larvae group. Taken together, these results demonstrated that kmo was a suitable visible marker gene for the application and extension of co‐CRISPR strategy in Fall armyworm. Using darker green color in larvae or mosaic‐eye in adults from kmo knockout as a marker, the mutant efficiency of a target gene could be enriched in a Fall armyworm group consisting of marked individuals. The co‐CRISPR strategy is helpful for gene function studies by the knockout technique with no or lethal phenotypes. [ABSTRACT FROM AUTHOR]

Simple Summary: Liquid biopsies provide a non-invasive approach to tracing tumor-derived biomarkers in blood, with broad applications in medicine. During the past decade, circulating free DNA (cfDNA) has been turned into an informative resource for cancer management. Mutation-enrichment methods enhance the detection of tumor-derived, low-level mutations in blood. These methods increase the frequency of low-level mutations insofar as they become detectable via routine diagnostic techniques. Enriching mutations prior to PCR (pre-PCR) offers distinctive advantages. Applying the mutation-enrichment process directly to genomic DNA or cfDNA circumvents PCR errors and provides enriched mutation-containing products that different technologies can detect without any required changes in their protocols. In this review, we discuss the recent developments in pre-PCR enrichment methods from the perspective of their applications in liquid biopsies. Liquid biopsy is having a remarkable impact on healthcare- and disease-management in the context of personalized medicine. Circulating free DNA (cfDNA) is one of the most instructive liquid-biopsy-based biomarkers and harbors valuable information for diagnostic, predictive, and prognostic purposes. When it comes to cancer, circulating DNA from the tumor (ctDNA) has a wide range of applications, from early cancer detection to the early detection of relapse or drug resistance, and the tracking of the dynamic genomic make-up of tumor cells. However, the detection of ctDNA remains technically challenging, due, in part, to the low frequency of ctDNA among excessive circulating cfDNA originating from normal tissues. During the past three decades, mutation-enrichment methods have emerged to boost sensitivity and enable facile detection of low-level mutations. Although most developed techniques apply mutation enrichment during or following initial PCR, there are a few techniques that allow mutation selection prior to PCR, which provides advantages. Pre-PCR enrichment techniques can be directly applied to genomic DNA and diminish the influence of PCR errors that can take place during amplification. Moreover, they have the capability for high multiplexity and can be followed by established mutation detection and enrichment technologies without changes to their established procedures. The first approaches for pre-PCR enrichment were developed by employing restriction endonucleases directly on genomic DNA in the early 1990s. However, newly developed pre-PCR enrichment methods provide higher sensitivity and versatility. This review describes the available pre-PCR enrichment methods and focuses on the most recently developed techniques (NaME-PrO, UVME, and DEASH/MAESTRO), emphasizing their applications in liquid biopsies. [ABSTRACT FROM AUTHOR]

Abstract Background CRISPR/Cas9 is widely used for precise genetic editing in various organisms. CRISPR/Cas9 editing may in many plants be hampered by the presence of complex and high ploidy genomes and inefficient or poorly controlled delivery of the CRISPR/Cas9 components to gamete cells or cells with regenerative potential. Optimized strategies and methods to overcome these challenges are therefore in demand. Results In this study we investigated the feasibility of improving CRISPR/Cas9 editing efficiency by Fluorescence Activated Cell Sorting (FACS) of protoplasts. We used Agrobacterium infiltration in leaves of Nicotiana benthamiana for delivery of viral replicons for high level expression of gRNAs designed to target two loci in the genome, NbPDS and NbRRA, together with the Cas9 nuclease in fusion with the 2A self-splicing sequence and GFP (Cas9-2A-GFP). Protoplasts isolated from the infiltrated leaves were then subjected to FACS for selection of GFP enriched protoplast populations. This procedure resulted in a 3–5 fold (from 20 to 30% in unsorted to more than 80% in sorted) increase in mutation frequencies as evidenced by restriction enzyme analysis and the Indel Detection by Amplicon Analysis, which allows for high throughput profiling and quantification of the generated mutations. Conclusions FACS of protoplasts expressing GFP tagged CRISPR/Cas9, delivered through A. tumefaciens leaf infiltration, facilitated clear CRISPR/Cas9 mediated mutation enrichment in selected protoplast populations.

The detection of mutant DNA is critical for precision medicine, but low-frequency DNA mutation is very hard to be determined. CRISPR/Cas9 is a robust tool for in vivo gene editing, and shows the potential for precise in vitro DNA cleavage. Here we developed a DNA mutation detection system based on CRISPR/Cas9 that can detect gene mutation efficiently even in a low-frequency condition. The system of CRISPR/Cas9 cleavage in vitro showed a high accuracy similar to traditional T7 endonuclease I (T7E1) assay in estimating mutant DNA proportion in the condition of normal frequency. The technology was further used for low-frequency mutant DNA detection of EGFR and HBB somatic mutations. To the end, Cas9 was employed to cleave the wild-type (WT) DNA and to enrich the mutant DNA. Using amplified fragment length polymorphism analysis (AFLPA) and Sanger sequencing, we assessed the sensitivity of CRISPR/Cas9 cleavage-based PCR, in which mutations at 1%–10% could be enriched and detected. When combined with blocker PCR, its sensitivity reached up to 0.1%. Our results suggested that this new application of CRISPR/Cas9 system is a robust and potential method for heterogeneous specimens in the clinical diagnosis and treatment management. [ABSTRACT FROM AUTHOR]

Circulating cell-free DNA (ccfDNA) bears great promise as biomarker for personalized medicine, but ccfDNA is present only at low levels in the plasma or serum of cancer patients. E- ice -COLD-PCR is a recently developed enrichment method to detect and identify mutations present at low-abundance in clinical samples. However, recent studies have shown the importance to accurately quantify low-abundance mutations as clinically important decisions will depend on certain mutation thresholds. The possibility for an enrichment method to accurately quantify the mutation levels remains a point of concern and might limit its clinical applicability. In the present study, we compared the quantification of KRAS mutations in ccfDNA from metastatic colorectal cancer patients by E- ice -COLD-PCR with two digital PCR approaches. For the quantification of mutations by E- ice -COLD-PCR, cell lines with known mutations diluted into WT genomic DNA were used for calibration. E- ice -COLD-PCR and the two digital PCR approaches showed the same range of the mutation level and were concordant for mutation levels below the clinical relevant threshold. E- ice -COLD-PCR can accurately detect and quantify low-abundant mutations in ccfDNA and has a shorter time to results making it compatible with the requirements of analyses in a clinical setting without the loss of quantitative accuracy. [ABSTRACT FROM AUTHOR]

It has been a consensus in cancer research that cancer is a disease caused primarily by genomic alterations, especially somatic mutations. However, the mechanism of mutation-induced oncogenesis is not fully understood. Here, we used the mitochondrial apoptotic pathway as a case study and performed a systematic analysis of integrating pathway dynamics with protein interaction kinetics to quantitatively investigate the causal molecular mechanism of mutation-induced oncogenesis. A mathematical model of the regulatory network was constructed to establish the functional role of dynamic bifurcation in the apoptotic process. The oncogenic mutation enrichment of each of the protein functional domains involved was found strongly correlated with the parameter sensitivity of the bifurcation point. We further dissected the causal mechanism underlying this correlation by evaluating the mutational influence on protein interaction kinetics using molecular dynamics simulation. We analyzed 29 matched mutant-wild-type and 16 matched SNP--wild-type protein systems. We found that the binding kinetics changes reflected by the changes of free energy changes induced by protein interaction mutations, which induce variations in the sensitive parameters of the bifurcation point, were a major cause of apoptosis pathway dysfunction, and mutations involved in sensitive interaction domains show high oncogenic potential. Our analysis provided a molecular basis for connecting protein mutations, protein interaction kinetics, network dynamics properties, and physiological function of a regulatory network. These insights provide a framework for coupling mutation genotype to tumorigenesis phenotype and help elucidate the logic of cancer initiation. [ABSTRACT FROM AUTHOR]

In this work, the structure allosteric induced by Endonuclease IV (Endo IV) preference to the specific substrate was ingeniously combined with the asymmetric PCR technique. As a result, it could construct a universal and straightforward strategy for mutant allele enrichment (IVME) to improve the sensitivity of downstream mutation detection methods. At the initial time, both the mutant-type target (MT) and the wild-type target (WT) would hybridize with the probe (FP) containing apurinic/apyrimidinic site to form a flap structure, which could not be used to perform asymmetric PCR. Once Endo IV was added, the substrate formed by FP and MT, not the FP-WT, could be recognized and cleaved due to the high single nucleotide discrimination ability of Endo IV. Subsequently, the flap structure at the 3′ terminal was destroyed and exposed an active 3′-OH, which could act as a primer in the asymmetric PCR. Thus, both the absolute amount and the abundance of MT were enhanced. 1% and 0.01% MT could be directly distinguished by coupling with Sanger sequencing and Endo IV-assisted nucleic probe hybridization-based DNA mutation detection method, respectively. EGFR T790M mutation in the peripheral blood of a non-small cell lung cancer patient was detected by IVME coupled with Sanger sequence. The results were also verified by next-generation sequencing, which demonstrated the potential clinical application of our proposed strategy. • Endo IV and asymmetric PCR were combined to enrich mutant allele for the first time (named IVME). • Various mutation detection techniques are compatible with IVME. • Mutant abundance at 0.01% can be detected by coupling IVME and IVMD. • The proposed strategy is applied for low fraction of EGFR T790M mutation measurement in the peripheral blood of NSCLC patients. [ABSTRACT FROM AUTHOR]

The use of next-generation sequencing (NGS) to profile genomic variation of individual cancer species is revolutionizing the practice of clinical oncology. In liquid biopsy of cancer, sequencing of circulating-free DNA (cfDNA) is gradually applied to all stages of cancer diagnosis and treatment, serving as complement or replacement of tissue biopsies. However, analysis of cfDNA obtained from blood draws still faces technical obstacles due in part to an excess of wild-type DNA originating from normal tissues and hematopoietic cells. The resulting low-level mutation abundance often falls below routine NGS detection sensitivity and limits reliable mutation identification that meets clinical sensitivity and specificity standards. Despite sample preparation advances that reduce sequencing error rates via use of unique molecular identifiers (molecular barcodes) and error-suppression algorithms, excessive amounts of sequencing are still required to detect mutations at allelic frequency levels below 1%. This requirement reduces throughput and increases cost.In this chapter, we describe a sensitive multiplex mutation detection method that enriches mutation-containing DNA during sample preparation, prior to sequencing, thereby increasing signal-to-noise ratios and providing low-level mutation detection without excessive sequencing depth. We couple targeted next-generation sequencing with wild-type DNA removal using Nuclease-assisted Minor-allele Enrichment using Probe Overlap, NaME-PrO, a recently developed method to eliminate wild-type sequences from multiple targets simultaneously. A step by step guide to library preparation and data analysis are provided as well as some precautions during the sample handling., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Genome comparisons provide information on the nature of genetic change, but such comparisons are challenged to differentiate the importance of the actual sequence change processes relative to the role of selection. This problem can be overcome by identifying changes that have not yet had the time to undergo millions of years of natural selection. We describe a strategy to discover accession-specific changes in the rice genome using an abundant resource routinely provided for many genome analyses, resequencing data. The sequence of the fully sequenced rice genome from variety Nipponbare was compared to the pooled (∼114×) resequencing data from 126 japonica rice accessions to discover "Nipponbare-specific" sequences. Analyzing nonrepetitive sequences, 8504 "candidate" Nipponbare-specific changes were detected, of which around two-thirds are true novel sequence changes and the rest are predicted genome sequencing errors. Base substitutions outnumbered indels in this data set by > 28:1, with ∼8:5 bias toward transversions over transitions, and no transposable element insertions or excisions were observed. These results indicate that the strategy employed is effective for finding recent sequence changes, sequencing errors, and rare alleles in any organism that has both a reference genome sequence and a wealth of resequencing data., (Copyright © 2018 by the Genetics Society of America.)

It has been a consensus in cancer research that cancer is a disease caused primarily by genomic alterations, especially somatic mutations. However, the mechanism of mutation-induced oncogenesis is not fully understood. Here, we used the mitochondrial apoptotic pathway as a case study and performed a systematic analysis of integrating pathway dynamics with protein interaction kinetics to quantitatively investigate the causal molecular mechanism of mutation-induced oncogenesis. A mathematical model of the regulatory network was constructed to establish the functional role of dynamic bifurcation in the apoptotic process. The oncogenic mutation enrichment of each of the protein functional domains involved was found strongly correlated with the parameter sensitivity of the bifurcation point. We further dissected the causal mechanism underlying this correlation by evaluating the mutational influence on protein interaction kinetics using molecular dynamics simulation. We analyzed 29 matched mutant-wild-type and 16 matched SNP--wild-type protein systems. We found that the binding kinetics changes reflected by the changes of free energy changes induced by protein interaction mutations, which induce variations in the sensitive parameters of the bifurcation point, were a major cause of apoptosis pathway dysfunction, and mutations involved in sensitive interaction domains show high oncogenic potential. Our analysis provided a molecular basis for connecting protein mutations, protein interaction kinetics, network dynamics properties, and physiological function of a regulatory network. These insights provide a framework for coupling mutation genotype to tumorigenesis phenotype and help elucidate the logic of cancer initiation.