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CRISPR/Cas9-mediated efficient white genome editing in the black soldier fly Hermetia illucens.
The CRISPR/Cas9 system is the most straightforward genome-editing technology to date, enabling genetic engineering in many insects, including the black soldier fly, Hermetia illucens. The white gene plays a significant role in the multifarious life activities of insects, especially the pigmentation of the eyes. In this study, the white gene of H. illucens (Hiwhite) was cloned, identified, and bioinformatically analysed for the first time. Using quantitative real-time polymerase chain reaction (qPCR), we found that the white gene was expressed in the whole body of the adult flies, particularly in Malpighian tubules and compound eyes. Furthermore, we utilised CRISPR/Cas9-mediated genome-editing technology to successfully generate heritable Hiwhite mutants using two single guide RNAs. During Hiwhite genome editing, we determined the timing, method, and needle-pulling parameters for embryo microinjection by observing early embryonic developmental features. We used the CasOT program to obtain highly specific guide RNAs (gRNAs) at the genome-wide level. According to the phenotypes of Hiwhite knockout strains, the pigmentation of larval stemmata, imaginal compound eyes, and ocelli differed from those of the wild type. These phenotypes were similar to those observed in other insects harbouring white gene mutations. In conclusion, our results described a detailed white genome editing process in black soldier flies, which lays a solid foundation for intensive research on the pigmentation pathway of the eyes and provides a methodological basis for further genome engineering applications in black soldier flies.
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CRISPR-Cas9 Editing Induces Loss of Heterozygosity in the Pathogenic Yeast Candida parapsilosis.
Genetic manipulation is often used to study gene function. However, unplanned genome changes (including single nucleotide polymorphisms [SNPs], aneuploidy, and loss of heterozygosity [LOH]) can affect the phenotypic traits of the engineered strains. Here, we compared the effect of classical deletion methods (replacing target alleles with selectable markers by homologous recombination) with CRISPR-Cas9 editing in the diploid human-pathogenic yeast Candida parapsilosis. We sequenced the genomes of 9 isolates that were modified using classic recombination methods and 12 that were edited using CRISPR-Cas9. As a control, the genomes of eight isolates that were transformed with a Cas9-expressing plasmid in the absence of a guide RNA were also sequenced. Following gene manipulation using classic homologous recombination, only one strain exhibited extensive LOH near the targeted gene (8.9 kb), whereas another contained multiple LOH events not associated with the intended modification. In contrast, large regions of LOH (up to >1,100 kb) were observed in most CRISPR-Cas9-edited strains. LOH most commonly occurred adjacent to the Cas9 cut site and extended to the telomere in four isolates. In two isolates, we observed LOH on chromosomes that were not targeted by CRISPR-Cas9. Among the CRISPR-edited isolates, two exhibited cysteine and methionine auxotrophy caused by LOH at a heterozygous site in MET10, approximately 11 and 157 kb downstream from the Cas9 target site, respectively. C. parapsilosis isolates have relatively low levels of heterozygosity. However, our results show that mutation complementation to confirm observed phenotypes is required when using CRISPR-Cas9. IMPORTANCE CRISPR-Cas9 has greatly streamlined gene editing and is now the gold standard and first choice for genetic engineering. However, we show that in diploid species, extra care should be taken in confirming the cause of any phenotypic changes observed. We show that the Cas9-induced double-strand break is often associated with loss of heterozygosity in the asexual diploid human fungal pathogen Candida parapsilosis. This can result in deleterious heterozygous variants (e.g., stop gain in one allele) becoming homozygous, resulting in unplanned phenotypic changes. Our results stress the importance of mutation complementation even when using CRISPR-Cas9.
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Precision BioSciences, Novartis Launch Up-to-$1.5B In Vivo Genome Editing Collaboration.
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CRISPR/Cas-Based Techniques for Live-Cell Imaging and Bioanalysis.
CRISPR/Cas systems have found widespread applications in gene editing due to their high accuracy, high programmability, ease of use, and affordability. Benefiting from the cleavage properties (trans- or cis-) of Cas enzymes, the scope of CRISPR/Cas systems has expanded beyond gene editing and they have been utilized in various fields, particularly in live-cell imaging and bioanalysis. In this review, we summarize some fundamental working mechanisms and concepts of the CRISPR/Cas systems, describe the recent advances and design principles of CRISPR/Cas mediated techniques employed in live-cell imaging and bioanalysis, highlight the main applications in the imaging and biosensing of a wide range of molecular targets, and discuss the challenges and prospects of CRISPR/Cas systems in live-cell imaging and biosensing. By illustrating the imaging and bio-sensing processes, we hope this review will guide the best use of the CRISPR/Cas in imaging and quantifying biological and clinical elements and inspire new ideas for better tool design in live-cell imaging and bioanalysis.
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Modulation of Equid Herpesvirus-1 Replication Dynamics In Vitro Using CRISPR/Cas9-Assisted Genome Editing.
(1) Background: equid alphaherpesvirus-1 (EHV-1) is a highly contagious viral pathogen prevalent in most horse populations worldwide. Genome-editing technologies such as CRISPR/Cas9 have become powerful tools for precise RNA-guided genome modifications; (2) Methods: we designed single guide RNAs (sgRNA) to target three essential (ORF30, ORF31, and ORF7) and one non-essential (ORF74) EHV-1 genes and determine their effect on viral replication dynamics in vitro; (3) Results: we demonstrated that sgRNAs targeting essential lytic genes reduced EHV-1 replication, whereas those targeting ORF74 had a negligible effect. The sgRNAs targeting ORF30 showed the strongest effect on the suppression of EHV-1 replication, with a reduction in viral genomic copy numbers and infectious progeny virus output. Next-generation sequencing identified variants with deletions in the specific cleavage site of selective sgRNAs. Moreover, we evaluated the combination between different sgRNAs and found that the dual combination of sgRNAs targeting ORF30 and ORF7 significantly suppressed viral replication to lower levels compared to the use of a single sgRNA, suggesting a synergic effect; (4) Conclusion: data demonstrate that sgRNA-guided CRISPR/Cas9 can be used to inhibit EHV-1 replication in vitro, indicating that this programmable technique can be used to develop a novel, safe, and efficacious therapeutic and prophylactic approach against EHV-1.
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Crops exposed to extreme conditions: perspectives of gene editing to improve stress tolerance.
Extreme environmental conditions, such as drought, soil salinity, and extreme temperatures, seriously limit agricultural production. Traditional breeding can produce more tolerant cultivars, but has limitations due to the multigene-encoded traits and long time needed to generate improved varieties. Gene editing can introduce single or multiple point mutations, generate microinsertion/deletions (InDel), modify selected bases or DNA fragments, and modulate gene expression, producing novel alleles resembling natural variability. In this review, we cover the strategies and perspectives for editing nuclear or organelle-encoded genes to enhance tolerance to extreme environmental conditions, such as drought, salinity, cold, and heavy metals. A list of 73 arabidopsis genes is assembled, whose function in abiotic stress responses has been demonstrated with insertion or gene-edited mutants. A collection of 97 gene-edited mutants in crops with tolerance to one or more abiotic stresses was compiled. The power of multiplex editing is demonstrated to target multigene-encoded traits related to stress tolerance. Special attention is paid to encouraging results of 20 gene editing programs in various crops, which confirmed improved tolerance in field conditions, demonstrating the feasibility to incorporate this powerful technology into plant breeding. As gene editing generates molecular changes similar to natural polymorphism, legal conditions adopting NGT-friendly frameworks are expected to facilitate commercialization of such engineered varieties in most countries.
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Not Only Editing: A Cas-Cade of CRISPR/Cas-Based Tools for Functional Genomics in Plants and Animals.
The advent of CRISPR/Cas9 technology has revolutionized genome editing, enabling the attainment of once-unimaginable goals. CRISPR/Cas's groundbreaking attributes lie in its simplicity, versatility, universality, and independence from customized DNA-protein systems, erasing the need for specialized expertise and broadening its scope of applications. It is therefore more and more used for genome modification including the generation of mutants. Beyond such editing scopes, the recent development of novel or modified Cas-based systems has spawned an array of additional biotechnological tools, empowering both fundamental and applied research. Precisely targeting DNA or RNA sequences, the CRISPR/Cas system has been harnessed in fields as diverse as gene regulation, deepening insights into gene expression, epigenetic changes, genome spatial organization, and chromatin dynamics. Furthermore, it aids in genome imaging and sequencing, as well as effective identification and countering of viral pathogens in plants and animals. All in all, the non-editing aspect of CRISPR/Cas exhibits tremendous potential across diverse domains, including diagnostics, biotechnology, and fundamental research. This article reviews and critically evaluates the primary CRISPR/Cas-based tools developed for plants and animals, underlining their transformative impact.
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Intrinsic RNA-targeting activity limits the applicability of CRISPR-Cas13.
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Precision genome editing.
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Gene editing to enhance pesticide resistance in a beneficial predatory mite.
Successful integrated pest management (IPM) often depends on a suite of control strategies that are compatible with one another. The predatory mite Neoseiulus californicus (Acari: Phytoseiidae) is a key biological control agent of spider mites (especially Tetranychus spp.) and other diminutive, yet important arthropod pests of agriculture. However, like many natural enemies, N. californicus is highly sensitive to chemical pesticides, limiting its overall effectiveness in the field. Here, we used CRISPR/Cas9 gene editing and Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) to create N. californicus harboring loss-of-function mutations in the nicotinic acetylcholine receptor α6 subunit (nAChRα6), the target of the pesticide, spinetoram. The resulting knockout strain (FZ-α6KO) exhibited a 23-fold increase in resistance to spinetoram compared to its parental strain. Inheritance of resistance to spinetoram in FZ-α6KO was autosomal, recessive, and tightly linked with the nAChRα6 gene. We demonstrate that by pairing gene editing with ReMOT Control, extremely small and fragile beneficial arthropods can be genetically manipulated. These results suggest that genetic modification to enhance pesticide resistance in beneficial predatory mites could improve the compatibility between the use of pesticides and biological control organisms.
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