CRISPR-Cas9-guided amplification-free genomic diagnosis for familial hypercholesterolemia using nanopore sequencing
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ABSTRACT:
SUBMITTER: Xu S
PROVIDER: S-EPMC10954175 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:Familial hypercholesterolemia (FH) is considered the genetic cause of coronary heart disease and ischemic stroke. FH is mainly an autosomal codominant pattern-based disorder and is primarily determined by point mutations within the low-density lipoprotein receptor, apolipoprotein B, and proprotein convertase subtilisin/kexin type 9 genes, causing increased low-density lipoprotein cholesterol levels in the serum of untreated individuals. The accumulation will eventually lead to atherosclerotic cardiovascular disease. Although clinical criteria comprising several prognosis scores, such as the Simon Broome, Dutch Lipid Clinic Network, Make Early Diagnosis to Prevent Early Death, and the recently proposed Montreal-FH-SCORE, are the conventional basis of diagnosing FH, the genetic diagnosis made by single nucleotide polymorphism genotyping, multiplex ligation-dependent probe amplification analysis, and sequencing (both Sanger and Next-Generation sequencing) offers unequivocal diagnosis. Given the heterogeneity of known mutations, the genetic diagnosis of FH is often difficult to establish, despite the growing evidence of the causative mutations, as well as the polygenic aspect of this pathology and the importance of cascade screening of the FH patient‚s healthy family members. This review article details different genetic techniques that can be used in FH identification when there is a clinical FH suspicion based on criteria comprised in prognosis scores, knowing that none of these are exhaustive in the diagnosis, yet they efficaciously overlap and complement each other for confirming the disease at the molecular level.
| S-EPMC7222642 | biostudies-literature
Project description:BackgroundOne ongoing concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target activity is challenging. Here, we present SMRT-OTS and Nano-OTS, two novel, amplification-free, long-read sequencing protocols for detection of gRNA-driven digestion of genomic DNA by Cas9 in vitro.ResultsThe methods are assessed using the human cell line HEK293, re-sequenced at 18x coverage using highly accurate HiFi SMRT reads. SMRT-OTS and Nano-OTS are first applied to three different gRNAs targeting HEK293 genomic DNA, resulting in a set of 55 high-confidence gRNA cleavage sites identified by both methods. Twenty-five of these sites are not reported by off-target prediction software, either because they contain four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. Additional experiments reveal that 85% of Cas9 cleavage sites are also found by other in vitro-based methods and that on- and off-target sites are detectable in gene bodies where short-reads fail to uniquely align. Even though SMRT-OTS and Nano-OTS identify several sites with previously validated off-target editing activity in cells, our own CRISPR-Cas9 editing experiments in human fibroblasts do not give rise to detectable off-target mutations at the in vitro-predicted sites. However, indel and structural variation events are enriched at the on-target sites.ConclusionsAmplification-free long-read sequencing reveals Cas9 cleavage sites in vitro that would have been difficult to predict using computational tools, including in dark genomic regions inaccessible by short-read sequencing.
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| S-EPMC6951065 | biostudies-literature
Project description:BackgroundChromosomal translocation has been detected in many human cancers including gliomas and is considered a driving force in tumorigenesis. Co-deletion of chromosome arms 1p and 19q is a hallmark for oligodendrogliomas. On the molecular level, 1p/19q co-deletion results from t(1;19)(q10;p10), which leads to the concomitant formation of a hybrid chromosome containing the 1q and 19p arms. A method to generate 1p/19q co-deletion is lacking, which hinders the investigation of how 1p/19q co-deletion contributes to gliomagenesis.MethodsWe hypothesized that chromosomal translocation, such as t(1;19)(q10;p10) resulting in the 1p/19q co-deletion, may be induced by simultaneously introducing DNA double-strand breaks (DSBs) into chromosomes 1p and 19q using CRISPR/Cas9. We developed a CRISPR/Cas9-based strategy to induce t(1;19)(q10;p10) and droplet digital PCR (ddPCR) assays to detect the hybrid 1q/19p and 1p/19q chromosomes.ResultsAfter translocation induction, we detected both 1p/19q and 1q/19p hybrid chromosomes by PCR amplification of the junction regions in HEK 293T, and U-251 and LN-229 glioblastoma cells. Sequencing analyses of the PCR products confirmed DNA sequences matching both chromosomes 1 and 19. Furthermore, the 1p/19q hybrid chromosome was rapidly lost in all tested cell lines. The 1q/19p hybrid chromosome also become undetectable over time likely due to cell survival disadvantage.ConclusionWe demonstrated that t(1;19)(q10;p10) may be induced by CRISPR/Cas9-mediated genomic editing. This method represents an important step toward engineering the 1p/19q co-deletion to model oligodendrogliomas. This method may also be generalizable to engineering other cancer-relevant translocations, which may facilitate the understanding of translocation roles in cancer progression.
| S-EPMC9547542 | biostudies-literature
Project description:Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods1-4 are limited by the loss of native modifications, short read length, high input requirements, low yield or long protocols. In the present study, we describe nanopore Cas9-targeted sequencing (nCATS), an enrichment strategy that uses targeted cleavage of chromosomal DNA with Cas9 to ligate adapters for nanopore sequencing. We show that nCATS can simultaneously assess haplotype-resolved single-nucleotide variants, structural variations and CpG methylation. We apply nCATS to four cell lines, to a cell-line-derived xenograft, and to normal and paired tumor/normal primary human breast tissue. Median sequencing coverage was 675× using a MinION flow cell and 34× using the smaller Flongle flow cell. The nCATS sequencing requires only ~3??g of genomic DNA and can target a large number of loci in a single reaction. The method will facilitate the use of long-read sequencing in research and in the clinic.
| S-EPMC7145730 | biostudies-literature
Project description:Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA-RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA. Taken together, these findings provide a platform for therapeutic applications, especially for nervous system disease, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 nuclease activity.
| S-EPMC4697396 | biostudies-other
Project description:CHoP-In (CRISPR/Cas9-mediated Homology-independent PCR-product integration) is a fast, non-homologous end-joining based, strategy for genomic editing in mammalian cells. There is no requirement for cloning in generation of the integration donor, instead the desired integration donor is produced as a polymerase chain reaction (PCR) product, flanked by the Cas9 recognition sequences of the target locus. When co-transfected with the cognate Cas9 and guide RNA, double strand breaks are introduced at the target genomic locus and at both ends of the PCR product. This allows incorporation into the genomic locus via hon-homologous end joining. The approach is versatile, allowing N-terminal, C-terminal or internal tag integration and gives predictable genomic integrations, as demonstrated for a selection of well characterised membrane trafficking proteins. The lack of donor vectors offers advantages over existing methods in terms of both speed and hands-on time. As such this approach will be a useful addition to the genome editing toolkit of those working in mammalian cell systems.
| S-EPMC6899835 | biostudies-literature
Project description:We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.
| S-EPMC3730909 | biostudies-other
Project description:Familial hypercholesterolemia (FH) is an autosomal dominant lipid metabolism disorder characterized by severely elevated plasma low-density lipoprotein cholesterol levels. The disease is caused by mutations in 3 genes (LDLR, APOB and PCSK9) while over 90% of the mutations are located within the LDLR gene. Thus, genetic analysis of the LDLR gene is the first step in the genetic diagnosis of FH. However, conventional methods like Sanger and NextGen sequencing are still costly and time-consuming. In contrast, Oxford Nanopore technology sequencing is an emerging third-generation sequencing technology featured by easy operability, low cost, small size and the capability of parallel sample sequencing. Here, we present an easy Nanopore-sequencing-based workflow for the rapid genetic testing of FH taking only 3 days and costing less than $50 per sample without the requirement for deep bioinformatic knowledge. Using our workflow, we were able to identify the underlying pathogenic variants of 10 FH patients including one novel, not yet recorded pathogenic variants. Our workflow allows the rapid evaluation of the pathogenic variants by utilizing detailed variant information from Ensembl. Additionally, our workflow is not restricted to sequencing the LDLR gene alone but can be easily adapted to the other FH-causing genes and more importantly, to any desired gene contributing to any hereditary disease. Therefore, our workflow is an attractive opportunity for every diagnostic laboratory to offer fast and easy in-house genetic diagnostics.
| S-EPMC8864071 | biostudies-literature
Project description:Cas9 is an endonuclease that can be programed to autonomously deliver diverse effectors to specified genetic addresses. High-resolution structures of this protein and its associated CRISPR RNA guide explain the molecular mechanisms of CRISPR-RNA-guided DNA recognition and provide a molecular blueprint that has facilitated structure-guided functional remodeling. Here we retrace events that led from early efforts to understand the central role of Cas9 in CRISPR-mediated adaptive immunity to contemporary efforts aimed at developing and deploying this enzyme for programmable genetic editing.
| S-EPMC7703833 | biostudies-literature