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Molecular safeguarding of CRISPR gene drive experiments.
CRISPR-based homing gene drives have sparked both enthusiasm and deep concerns due to their potential for genetically altering entire species. This raises the question about our ability to prevent the unintended spread of such drives from the laboratory into a natural population. Here, we experimentally demonstrate the suitability of synthetic target site drives as well as split drives as flexible safeguarding strategies for gene drive experiments by showing that their performance closely resembles that of standard homing drives in Drosophila melanogaster. Using our split drive system, we further find that maternal deposition of both Cas9 and gRNA is required to form resistance alleles in the early embryo and that maternally-deposited Cas9 alone can power germline drive conversion in individuals that lack a genomic source of Cas9.
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Evidence for gene essentiality in Leishmania using CRISPR.
The ability to determine the essentiality of a gene in the protozoan parasite Leishmania is important to identify potential targets for intervention and understanding the parasite biology. CRISPR gene editing technology has significantly improved gene targeting efficiency in Leishmania. There are two commonly used CRISPR gene targeting methods in Leishmania; the stable expression of the gRNA and Cas9 using a plasmid containing a Leishmania ribosomal RNA gene promoter (rRNA-P stable protocol) and the T7 RNA polymerase based transient gRNA expression system in promastigotes stably expressing Cas9 (T7 transient protocol). There are distinct advantages with both systems. The T7 transient protocol is excellent for high throughput gene deletions and has been used to successfully delete hundreds of Leishmania genes to study mutant phenotypes and several research labs are now using this protocol to target all the genes in L. mexicana genome. The rRNA-P stable protocol stably expresses the plasmid derived gRNA and has been used to delete or disrupt single and multicopy Leishmania genes, perform single nucleotide changes and provide evidence for gene essentiality by directly observing null mutant promastigotes dying in culture. In this study, the rRNA-P stable protocol was used to target 22 Leishmania genes in which null mutants were not generated using the T7 transient protocol. Notably, the rRNA-P stable protocol was able to generate alive null mutants for 8 of the 22 genes. These results demonstrate the rRNA-P stable protocol could be used alone or in combination with the T7 transient protocol to investigate gene essentiality in Leishmania.
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Rational designs of in vivo CRISPR-Cas delivery systems.
The CRISPR-Cas system initiated a revolution in genome editing when it was, for the first time, demonstrated success in the mammalian cells. Today, scientists are able to readily edit genomes, regulate gene transcription, engineer posttranscriptional events, and image nucleic acids using CRISPR-Cas-based tools. However, to efficiently transport CRISPR-Cas into target tissues/cells remains challenging due to many extra- and intra-cellular barriers, therefore largely limiting the applications of CRISPR-based therapeutics in vivo. In this review, we summarize the features of plasmid-, RNA- and ribonucleoprotein (RNP)-based CRISPR-Cas therapeutics. Then, we survey the current in vivo delivery systems. We specify the requirements for efficient in vivo delivery in clinical settings, and highlight both efficiency and safety for different CRISPR-Cas tools.
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Precise CRISPR/Cpf1 genome editing system in the Deinococcus radiodurans with superior DNA repair mechanisms.
Deinococcus radiodurans, with its high homologous recombination (HR) efficiency of double-stranded DNA breaks (DSBs), is a model organism for studying genome stability maintenance and an attractive microbe for industrial applications. Here, we developed an efficient CRISPR/Cpf1 genome editing system in D. radiodurans by evaluating and optimizing double-plasmid strategies and four Cas effector proteins from various organisms, which can precisely introduce different types of template-dependent mutagenesis without off-target toxicity. Furthermore, the role of DNA repair genes in determining editing efficiency in D. radiodurans was evaluated by introducing the CRISPR/Cpf1 system into 13 mutant strains lacking various DNA damage response and repair factors. In addition to the crucial role of RecA-dependent HR required for CRISPR/Cpf1 editing, D. radiodurans showed higher editing efficiency when lacking DdrB, the single-stranded DNA annealing (SSA) protein involved in the RecA-independent DSB repair pathway. This suggests a possible competition between HR and SSA pathways in the CRISPR editing of D. radiodurans. Moreover, off-target effects were observed during the genome editing of the pprI knockout strain, a master DNA damage response gene in Deinococcus species, which suggested that precise regulation of DNA damage response is critical for a high-fidelity genome editing system.
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Pea early-browning virus-mediated genome editing via the CRISPR/Cas9 system in Nicotiana benthamiana and Arabidopsis.
The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system has enabled efficient genome engineering in diverse plant species. However, delivery of genome engineering reagents, such as the single guide RNA (sgRNA), into plant cells remains challenging. Here, we report the engineering of Tobacco rattle virus (TRV) and Pea early browning virus (PEBV) to deliver one or multiple sgRNAs into Nicotiana benthamiana and Arabidopsis thaliana (Col-0) plants that overexpress a nuclear localization signal containing Cas9. Our data showed that TRV and PEBV can deliver sgRNAs into inoculated and systemic leaves, and this resulted in mutagenesis of the targeted genomic loci. Moreover, in N. benthamiana, PEBV-based sgRNA delivery resulted in more targeted mutations than TRV-based delivery. Our data indicate that TRV and PEBV can facilitate plant genome engineering and can be used to produce targeted mutations for functional analysis and other biotechnological applications across diverse plant species. Key message: Delivery of genome engineering reagents into plant cells is challenging and inefficient and this limit the applications of this technology in many plant species. RNA viruses such as TRV and PEBV provide an efficient tool to systemically deliver sgRNAs for targeted genome modification.
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CRISPR-Associated Transposase System Can Insert Multiple Copies of Donor DNA into the Same Target Locus.
The CRISPR-associated transposase system enables site-specific DNA integration on the genome independent of homologous recombination. Previous studies have demonstrated that the type V-K CRISPR-associated Tn7-like transposase system from Scytonema hofmanni and the type I-F system from Vibrio cholerae have strong target immunity like Tn7, and therefore two or more copies of the donor DNA would not be inserted into the same target location in theory. In this paper, we report that the type I-F system can insert multiple donor copies into one site, which was identified and confirmed by single-strain identification and high-throughput sequencing. This result is beneficial for our application of multicopy chromosomal integration by CRISPR-associated transposases, allowing more donor insertions into the chromosome. This unexpected result shows that the target immunity mechanism of this system has not been fully understood. Attention should be paid to the possibility of multiple insertions and their effects in related research.
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RNA Interference: Story and Mechanisms.
The discovery that gene expression can be silenced by exogenously introduced double-stranded RNAs into cells unveiled a hidden level of gene regulation by a variety of small RNA pathways, which are involved in regulating endogenous gene expression, defending against virus infections, and protecting the genome from invading transposons, both at the posttranscriptional and epigenetic levels. All endogenous RNA interference pathways share a conserved effector complex, which contains at least an argonaute protein and a short single-stranded RNA. Such argonaute-RNA complexes can repress the transcription of genes, target mRNA for site-specific cleavage, or block mRNA translation into proteins. This review outlines the history of RNAi discovery, function, and mechanisms of action. For comparison, it also touches on CRISPR interference.
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* CRISPR-Based Epigenome Editing of Cytokine Receptors for the Promotion of Cell Survival and Tissue Deposition in Inflammatory Environments.
Musculoskeletal diseases have been associated with inflammatory cytokine action, particularly action by TNF-α and IL-1β. These inflammatory cytokines promote apoptosis and senescence of cells in diseased tissue and extracellular matrix breakdown. Stem cell-based therapies are being considered for the treatment of musculoskeletal diseases, but the presence of these inflammatory cytokines will have similar deleterious action on therapeutic cells delivered to these environments. Methods that prevent inflammatory-induced apoptosis and proinflammatory signaling, in cell and pathway-specific manners are needed. In this study we demonstrate the use of clustered regularly interspaced short palindromic repeats (CRISPR)-based epigenome editing to alter cell response to inflammatory environments by repressing inflammatory cytokine cell receptors, specifically TNFR1 and IL1R1. We targeted CRISPR/Cas9-based repressors to TNFR1 and IL1R1 gene regulatory elements in human adipose-derived stem cells (hADSCs) and investigated the functional outcomes of repression of these genes. Efficient signaling regulation was demonstrated in engineered hADSCs, as activity of the downstream transcription factor NF-κB was significantly reduced or maintained at baseline levels in the presence of TNF-α or IL-1β. Pellet culture of undifferentiated hADSCs demonstrated improved survival in engineered hADSCs treated with TNF-α or IL-1β, while having little effect on their immunomodulatory properties. Furthermore, engineered hADSCs demonstrated improved chondrogenic differentiation capacity in the presence of TNF-α or IL-1β, as shown by superior production of glycosaminglycans in this inflammatory environment. Overall this work demonstrates a novel method for modulating cell response to inflammatory signaling that has applications in engineering cells delivered to inflammatory environments, and as a direct gene therapy to protect endogenous cells exposed to chronic inflammation, as observed in a broad spectrum of degenerative musculoskeletal pathology.
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Immunogold Labeling to Detect Streptococcus pyogenes Cas9 in Cell Culture and Tissues by Electron Microscopy.
The CRISPR-Cas9 system is a powerful and yet precise DNA-editing tool in rapid development. By combining immunogold labeling and electron microscopy with the novel CRISPR-Cas9 system, we propose a new method to gain insight into the biology of this tool. In this study, we analyzed different Cas9-induced systems such as HEK293T cell line, murine oligodendrocyte progenitor cells, brain and liver to detect Cas9 expression by immunoelectron microscopy. Our results show that while Cas9 expression could be found in the nuclei and nucleopores of transfected HEK293T cells, in transfected oligodendrocyte precursor cells, Cas9 was found in cytoplasmic vesicles. In Cas9 constitutively expressing oligodendrocyte precursors, the enzyme was located in the cytoplasm of nondividing cells. Finally, while in the liver Cas9 was detected in different cell types, in the brain we found no specifically labeled cells. In conclusion, immunoelectron microscopy opens a new spectrum of opportunities to study the CRISPR-Cas9 system in a more precise manner.
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From designer babies to edited food: comparing public attitudes toward CRISPR technology and gene editing in Japan and the United States.
The perception of bioethics is said to vary based on culture and religion, and research has often been confined to specific topics such as genome-edited babies. In this study, four different scenarios using CRISPR technology and gene editing (designer babies, de novo embryo production, crop improvement, and malaria mosquito control) were presented to measure the attitudes of people in Japan and the United States. The most influential demographic factor was age, with older individuals in both countries expressing greater concern. In the United States, the higher the educational level and annual income, the more people tended to support these scenarios. In both Japan and the United States, the higher the level of knowledge, the more people tended to oppose them. Furthermore, using the ELSI sheet originally developed for AI research to quantify the degree of public concern across ELSI dimensions, it was revealed that Japanese individuals are concerned about the 'L' factor, which corresponds to legal and policy frameworks, while Americans are more concerned about the 'S' factor, representing traditional and religious viewpoints. Therefore, international analysis using the ELSI sheet not only facilitates better dialogue within each country but also proves useful for sharing points to consider when establishing international regulations.
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