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General results screen for gene knock-out using the gene elof1 in the Danio rerio genome danRer11 as an example. On the top is the genome view of the gene with identified gRNAs. This view can be toggled left/right and zoomed in/out. The gene model is drawn with the name of each isoform. Thick blue boxes represent exons, thin blue boxes represent untranslated regions, and green lines indicates in-frame start codons. gRNAs are displayed as arrows, with colors matching the scoring. Directionality is indicated by black arrowheads. Immediately below the genome view are options to download all identified gRNA targets (left) or view the results in the UCSC Genome Browser (right). On the bottom is a ranked list of all gRNA targets found, with all features necessary to make an informed decision. Clicking on a gRNA in the list or the graphical representation opens a detailed results view (Fig. 4).
Source publication
The design of optimal guide RNA (gRNA) sequences for CRISPR systems is challenged by the need to achieve highly efficient editing at the desired location (on‐target editing) with minimal editing at unintended locations (off‐target editing). Although laboratory validation should ideally be used to detect off‐target activity, computational prediction...
Contexts in source publication
Context 1
... In the genome locus view of the results (Fig. 3, top), identify a gRNA that fits your ...
Context 2
... Protocols Figure 4 Detailed results view for gRNA rank 4 from Figure 3. The top-left corner summarizes details about the specific gRNA hit. ...
Context 4
... Download the complete list of proposed targets by choosing a preferred format in the "Download results" box ( Fig. 3, 14. Inspect the detailed view (Fig. 4) for important ...
Context 5
... In the genome locus view of the results (Fig. 3, as in Basic Protocol 1), identify a gRNA that fits your ...
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Citations
... First identified as an immune protection system in prokaryotes, the CRISPR system has recently gained widespread attraction for its potential in gene regulation and editing and has thus proved to be a competitive technology in disease diagnostics [42]. CRISPR consists of a Cas protein and a guide RNA (gRNA) which directs Cas to the target site [43]. Researchers can target any gene of interest by changing the gRNA sequence to identify a specific site of interest when configuring the Cas protein to target that specific sequence. ...
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... We performed reciprocal BLAST of human SPOUT1/CENP-32 (NP_057474.2) against the translated zebrafish genome and identified a single spout1/cenp-32 zebrafish ortholog (Ensembl ID: GRCz11: ENSRDARG00000019707) with 76% identity and 88% similarity. To identify single guide (sg) RNA sites targeting spout1/cenp-32, we used CHOPCHOP [77][78][79] (Supplementary Table S1). Using synthetic oligos as template, we synthesized sgRNAs targeting exon 5 and exon 7 of ENSDART00000099535.5 using the GeneArt Precision gRNA Synthesis Kit (ThermoFisher Scientific), according to the manufacturer's guidelines. ...
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... This recognition allows for precise cutting of the target (cis activity) and nearby nucleic acids (trans activity), enabling accurate sequence-specific editing and detection. Tools for accelerated design of crRNAs-such as PrimeSHERLOCK [47], CHOPCHOP [48], CRISPRdirect [49], CaSilico [50], DeepCRISPR [51], GuideMaker [52], CRISPRscan [53], CRISPR-DT [54], benchling [55] and pathoGD [56]-are now readily available. PathoGD, in particular, has begun incorporating genomic databases into its target design. ...
Purpose of Review
Bacterial infections and antibiotic resistance contribute to global mortality. Despite many infections being preventable and treatable, the lack of reliable and accessible diagnostic tools exacerbates these issues. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based diagnostics has emerged as a promising solution. However, the development of CRISPR diagnostics has often occurred in isolation, with limited integration of genomic data to guide target selection. In this review, we explore the synergy between bacterial genomics and CRISPR-based point-of-care tests (POCT), highlighting how genomic insights can inform target selection and enhance diagnostic accuracy.
Recent Findings
We review recent advances in CRISPR-based technologies, focusing on the critical role of target sequence selection in improving the sensitivity of CRISPR-based diagnostics. Additionally, we examine the implementation of these technologies in resource-limited settings across Asia and Africa, presenting successful case studies that demonstrate their potential.
Summary
The integration of bacterial genomics with CRISPR technology offers significant promise for the development of effective point-of-care diagnostics.
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... We designed pairs of single guide RNA (sgRNA) 34 to target MIR155HG. Schema of MIR155HG loci identifying the pairs of guide RNAs flanking regions of interest are shown in Figure 1A and genomic PCR primers used to confirm deletion are listed in Supplemental Table 1. ...
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... This tool offers precise localization of various subsections, including coding regions, UTRs, splice sites, and specific exons, spanning both protein-coding and noncoding genes. It adeptly identifies potential off-target sites for all sgRNAs, automatically generates primers for target sites, and presents all relevant elements through a [52]. ...
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... uib.no), an online tool that offers a wide range of inputs and alignments to minimize search times. This tool is the most authentic and reliable for designing gRNA (Labun et al., 2021). ...
... Upon clicking the "Find Target" button, the tool provides a list of all gRNA sequences present in the selected gene. The user can then select the desired gRNA sequence for their experiment (Labun et al., 2021). ...
Amyotrophic lateral sclerosis (ALS) is a progressive neu-rodegenerative disorder that leads to the degenera-tion of motor neurons and the weakening of muscles. Despite extensive research efforts, there is currently no cure for ALS and existing treatments only address its symptoms. To address this unmet medical need, ge-nome editing technologies, such as CRISPR-Cas9, have emerged as a promising solution for the development of new treatments for ALS. Studies have shown that CRISPR-Cas9-guided RNAs have the potential to provide accurate and effective silencing in the genetic disease of ALS. Results have demonstrated a 67% on-target score and a 98% off-target score with GC content within the range of 40-60%. This is further validated by the correlation between the gRNA's structural accuracy and the minimum free energy. The use of CRISPR-Cas9 provides a unique opportunity to target this disease at the molecular level, offering hope for the development of a more effective treatment. In silico and computational therapeutic approaches for ALS suggest that the CRISPR-Cas9 protein holds promise as a future treatment candidate. The CRISPR mechanism and the specificity of gRNA provide a novel therapeutic approach for this genetic disease , offering new hope to those affected by ALS. This study highlights the potential of CRISPR-Cas9 as a promising solution for the development of new treatments for ALS. Further research is required to validate these findings in preclinical and clinical trials and to establish the safety and efficacy of this approach in the treatment of ALS.
... Embryos were injected with 500 to 750 pg of Cas9, with 500-750 pg of gRNAs (Synthego Inc), or with a combination of 500 pg of Cas9 protein and 500 pg of gRNAs for the AK7 KO condition. Single gRNAs were designed using CHOPCHOP (Labun et al., 2021). Two gRNAs were used in concert to increase the likelihood of editing. ...
The skin of Xenopus embryos contains numerous multiciliated cells (MCCs), which collectively generate a directed fluid flow across the epithelial surface essential for distributing the overlaying mucous. MCCs develop into highly specialized cells to generate this flow, containing approximately 150 evenly spaced centrioles that give rise to motile cilia. MCC-driven fluid flow can be impaired when ciliary dysfunction occurs, resulting in primary ciliary dyskinesia (PCD) in humans. Mutations in a large number of genes (~50) have been found to be causative to PCD. Recently, studies have linked low levels of Adenylate Kinase 7 (AK7) gene expression to patients with PCD; however, the mechanism for this link remains unclear. Additionally, AK7 mutations have been linked to multiple PCD patients. Adenylate kinases modulate ATP production and consumption, with AK7 explicitly associated with motile cilia. Here we reproduce an AK7 PCD-like phenotype in Xenopus and describe the cellular consequences that occur with manipulation of AK7 levels. We show that AK7 localizes throughout the cilia in a DPY30 domain-dependent manner, suggesting a ciliary function. Additionally, we find that AK7 overexpression increases centriole number, suggesting a role in regulating centriole biogenesis. We find that in AK7-depleted embryos, cilia number, length, and beat frequency are all reduced, which in turn, significantly decreases the tissue-wide mucociliary flow. Additionally, we find a decrease in centriole number and an increase in sub-apical centrioles, implying that AK7 influences both centriole biogenesis and docking, which we propose underlie its defect in ciliogenesis. We propose that AK7 plays a role in PCD by impacting centriole biogenesis and apical docking, ultimately leading to ciliogenesis defects that impair mucociliary clearance.
