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Genome aware CRISPR gRNA target prediction for parasitic nematodes.
The pace of research towards a genetic model to understand the unique molecular biology of parasitic nematodes has increased recently. This research has developed a diverse suite of genetic tools for a variety of parasitic nematodes. CRISPR/Cas9 technology in particular offers much promise as a game changing tool for researchers studying parasitic nematodes. Unlike RNAi, which depends on diverse nematode effectors to silence gene expression, the effectors for CRISPR/Cas9 mutations are typically supplied by the experimenter, making gene editing via CRISPR/Cas9 ideal for testing on genetically intractable nematode systems. To facilitate the development of CRISPR/Cas9 technology for parasitic nematodes, I here describe a tool for identifying gRNA targets and diagnostic primers to a user supplied sequence. The software attempts to minimize non-specific targets by interrogating the genomes of parasitic nematodes. This software is freely available online and features an intuitive interface to help researchers design effective CRISPR experiments for parasitic nematodes.
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Proxies of CRISPR/Cas9 Activity To Aid in the Identification of Mutagenized Arabidopsis Plants.
CRISPR/Cas9 has become the preferred gene-editing technology to obtain loss-of-function mutants in plants, and hence a valuable tool to study gene function. This is mainly due to the easy reprogramming of Cas9 specificity using customizable small non-coding RNAs, and to the possibility of editing several independent genes simultaneously. Despite these advances, the identification of CRISPR-edited plants remains time and resource-intensive. Here, based on the premise that one editing event in one locus is a good predictor of editing event/s in other locus/loci, we developed a CRISPR co-editing selection strategy that greatly facilitates the identification of CRISPR-mutagenized Arabidopsis thaliana plants. This strategy is based on targeting the gene/s of interest simultaneously with a proxy of CRISPR-Cas9-directed mutagenesis. The proxy is an endogenous gene whose loss-of-function produces an easy-to-detect visible phenotype that is unrelated to the expected phenotype of the gene/s under study. We tested this strategy via assessing the frequency of co-editing of three functionally unrelated proxy genes. We found that each proxy predicted the occurrence of mutations in each surrogate gene with efficiencies ranging from 68 to 100%. The selection strategy laid out here provides a framework to facilitate the identification of multiplex edited plants, thus aiding in the study of gene function when functional redundancy hinders the effort to define gene-function-phenotype links.
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5S rRNA Promoter for Guide RNA Expression Enabled Highly Efficient CRISPR/Cas9 Genome Editing in Aspergillus niger.
The CRISPR/Cas9 system is a revolutionary genome editing tool. However, in eukaryotes, search and optimization of a suitable promoter for guide RNA expression is a significant technical challenge. Here we used the industrially important fungus, Aspergillus niger, to demonstrate that the 5S rRNA gene, which is both highly conserved and efficiently expressed in eukaryotes, can be used as a guide RNA promoter. The gene editing system was established with 100% rates of precision gene modifications among dozens of transformants using short (40-bp) homologous donor DNA. This system was also applicable for generation of designer chromosomes, as evidenced by deletion of a 48 kb gene cluster required for biosynthesis of the mycotoxin fumonisin B1. Moreover, this system also facilitated simultaneous mutagenesis of multiple genes in A. niger. We anticipate that the use of the 5S rRNA gene as guide RNA promoter can broadly be applied for engineering highly efficient eukaryotic CRISPR/Cas9 toolkits. Additionally, the system reported here will enable development of designer chromosomes in model and industrially important fungi.
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Effective Downregulation of BCR-ABL Tumorigenicity by RNA Targeted CRISPR-Cas13a.
AIM: To induce BCR-ABL gene silencing using CRISPR Cas13a. BACKGROUND: CML is a clonal myeloproliferative disorder of pluripotent stem cells driven by a reciprocal translocation between chromosomes 9 and 22 forming a BCR-ABL fusion gene. Tyrosine- kinase inhibitor drugs like imatinib are the mainstay of treatment and cases resistant to these drugs have a poor prognosis in the absence of a compatible stem-cell donor. However with rapid advancements in gene-editing technologies most studies are now focusing on developing a translational model targeting single-gene disorders with a prospective permanent cure. OBJECTIVE: To explore the potential application of the RNA targeting CRISPR-Cas13a system for effective knockdown of BCR-ABL fusion transcript in a CML cell line K562. METHODS: CRISPR Cas13a crRNA was designed specific to the chimeric BCR-ABL gene and the system was transfected as a two-plasmid system into a CML cell line K562. The effects were enumerated by evaluating the expression levels of downstream genes dependent on the expression of the BCR-ABL gene. Also next-generation sequencing was used to ascertain the effects of CRISPR on the gene. RESULTS: The CRISPR system was successfully able to lower the expression of downstream genes [pCRKL and pCRK] dependent on the activated BCR-ABL kinase signal by up-to 4.3 folds. The viability of the CRISPR-treated cells was also significantly lowered by 373.83-fold [p-value= 0.000891196]. The time-dependent kinetics also highlighted the significant in-vitro suppressive activity to last up to 8 weeks [p-value: 0.025]. As per the cDNA sequencing data from the Oxford MinION next-generation sequencer the CRISPR treated cells show 62.37% suspected cleaved reads. CONCLUSION: These preliminary results highlight an excellent potential application of RNA targeting CRISPRs in Haematological neoplasms like CML and should pave the way for further research in this direction.
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Diminished Expression Alleles for Analysis of Virulence Traits and Genetic Interactions in Candida albicans.
Here we present a protocol of interest to those who want to look at the functional consequences of decreasing expression of essential genes constitutively as well as being able to study the regulatory pathways of that essential gene by also deleting one or two additional genes. This allows epistasis relationships to be determined.
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CRISPR GENome and epigenome engineering improves loss-of-function genetic-screening approaches.
CRISPR-Cas9 technology has revolutionized genotype-to-phenotype assignments through large-scale loss-of-function (LOF) screens. However, limitations like editing inefficiencies and unperturbed genes cause significant noise in data collection. To address this, we introduce CRISPR gene and epigenome engineering (CRISPRgenee), which uses two specific single guide RNAs (sgRNAs) to simultaneously repress and cleave the target gene within the same cell, increasing LOF efficiencies and reproducibility. CRISPRgenee outperforms conventional CRISPR knockout (CRISPRko), CRISPR interference (CRISPRi), and CRISPRoff systems in suppressing challenging targets and regulators of cell proliferation. Additionally, it efficiently suppresses modulators of epithelial-to-mesenchymal transition (EMT) and impairs neuronal differentiation in a human induced pluripotent stem cell (iPSC) model. CRISPRgenee exhibits improved depletion efficiency, reduced sgRNA performance variance, and accelerated gene depletion compared to individual CRISPRi or CRISPRko screens, ensuring consistency in phenotypic effects and identifying more significant gene hits. By combining CRISPRko and CRISPRi, CRISPRgenee increases LOF rates without increasing genotoxic stress, facilitating library size reduction for advanced LOF screens.
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Ethical Issues of Using CRISPR Technologies for Research on Military Enhancement.
This paper presents an overview of the key ethical questions of performing gene editing research on military service members. The recent technological advance in gene editing capabilities provided by CRISPR/Cas9 and their path towards first-in-human trials has reinvigorated the debate on human enhancement for non-medical purposes. Human performance optimization has long been a priority of military research in order to close the gap between the advancement of warfare and the limitations of human actors. In spite of this focus on temporary performance improvement, biomedical enhancement is an extension of these endeavours and the ethical issues of such research should be considered. In this paper, we explore possible applications of CRISPR to military human gene editing research and how it could be specifically applied towards protection of service members against biological or chemical weapons. We analyse three normative areas including risk-benefit analysis, informed consent, and inequality of access as it relates to CRISPR applications for military research to help inform and provide considerations for military institutional review boards and policymakers.
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A Living Organism in your CRISPR Toolbox: Caenorhabditis elegans Is a Rapid and Efficient Model for Developing CRISPR-Cas Technologies.
The Cas9 nuclease from Streptococcus pyogenes (SpCas9) is the most popular enzyme for CRISPR technologies. However, considering the wide diversity of microorganisms (discovered and still unknown), a massive number of CRISPR effectors are being and will be identified and characterized in the search of optimal Cas variants for each of the many applications of CRISPR. In this context, a versatile and efficient multicellular system for CRISPR editing such as Caenorhabditis elegans would be of great help in the development of these effectors. Here, we highlight the benefits of using C. elegans for the rapid evaluation of new CRISPR effectors, and for optimizing CRISPR efficiency in animals in several ways such as by modulating the balance between repair pathways, modifying chromatin accessibility, or controlling the expression and activity of nucleases and guide RNAs.
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Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and single-stranded DNA.
The green alga Chlamydomonas reinhardtii is an invaluable reference organism to research fields including algal, plant, and ciliary biology. Accordingly, decades-long standing inefficiencies in targeted nuclear gene editing broadly hinder Chlamydomonas research. Here we report that single-step codelivery of CRISPR/Cpf1 ribonucleoproteins with single-stranded DNA repair templates results in precise and targeted DNA replacement with as much as ∼10% efficiency in C. reinhardtii We demonstrate its use in transgene- and selection-free generation of sequence-specific mutations and epitope tagging at an endogenous locus. As the direct delivery of gene-editing reagents bypasses the use of transgenes, this method is potentially applicable to a wider range of species without the need to develop methods for stable transformation.
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CRISPR Technology: Transforming the Future of Medicine and Diagnostics.
In this report, we examine the extensive research landscape of CRISPR with an emphasis on CRISPR therapeutics and showcase our results from an in-depth analysis of the most up-to-date scientific information consisting of more than 53,000 publications encompassing academic journal articles and patents, spanning nearly three decades, extracted from the CAS Content Collection. Our analysis indicates that cancer and infectious diseases are the most explored in the context of CRISPR. Identified gene targets associated with CRISPR-related publications are led by TP53, c-myc, and hemoglobin beta subunit (HBB). Among the many delivery methods, adeno-associated vectors (AAVs) appear to be highly explored. With >140 CRISPR-based therapeutics in the clinical development pipeline and billions of dollars in investment, the field of CRISPR continues to evolve rapidly. We also briefly discuss the ethical implications of CRISPR technology. While some fundamental challenges persist, the future of CRISPR is undoubtedly bright.
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