Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 9‐mediated mutagenesis of the multiple edematous wings gene induces muscle weakness and flightlessness in Bactrocera dorsalis (Diptera: Tephritidae)

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 9 (Cas9) system is a versatile, efficient and heritable gene editing tool that can be useful for genome engineering. Bactrocera dorsalis (Hendel) is a major pest of agriculture that causes great economic losses. We used the B. dorsalis multiple edematous wings (Bdmew) gene as the target gene to explore the effectiveness of CRISPR/Cas9 for B. dorsalis genome manipulation. We studied the physiological functions of the Bdmew gene, particularly those related to muscle development. Site‐specific genome editing was feasible using direct microinjection of specific guide RNA and the Cas9‐plasmid into B. dorsalis embryos. Mutation frequencies ranged from 12.1 to 30.2% in the injected generation. Mosaic G0, with the mew mutation, was heritable to the next generation. The G1 displayed a series of defective phenotypes including muscle weakness, flightlessness, failure to eclose, wing folds and unbalanced movement. These results demonstrated that CRISPR/Cas9 can act as a highly specific, efficient, heritable tool for genome manipulation in B. dorsalis and this has significance for gene function research and genetic control of pests. The Bdmew gene possesses key functions in muscle development of B. dorsalis. Bdmew mutations cause a series of serious defects by interfering with muscle development and may provide a means for controlling B. dorsalis via a gene‐based method such as gene drive.     

Methods for the generation of heritable germline mutations in the disease vector Culex quinquefasciatus using clustered regularly interspaced short palindrome repeats‐associated protein 9

Culex quinquefasciatus is a vector of many diseases that adversely impact human and animal health; however, compared to other mosquito vectors limited genome engineering technologies have been characterized for this vector. Clustered regularly interspaced short palindrome repeats‐associated protein 9 (CRISPR‐Cas9) based technologies are a powerful tool for genome engineering and functional genetics and consequently have transformed genetic studies in many organisms. Our objective was to improve upon the limited technologies available for genome editing in C. quinquefasciatus to create a reproducible and straightforward method for CRISPR‐Cas9‐targeted mutagenesis in this vector. Here we describe methods to achieve high embryo survival and mutagenesis rates and we provide details on the injection supplies and procedures, embryo handling and guide RNA (gRNA) target designs. Through these efforts, we achieved embryo survival rates and germline mutagenesis rates that greatly exceed previously reported rates in this vector. This work is also the first to characterize the white gene marker in this species, which is a valuable phenotypic marker for future transgenesis or mutagenesis of this vector. Overall, these tools provide the framework for future functional genetic studies in this important disease vector and may support the development of future gene drive and genetic technologies that can be used to control this vector.     

A novel cell line generated using the CRISPR/Cas9 technology as universal quality control material for KRAS G12V mutation testing

Background

KRAS mutations are the key indicator for EGFR monoclonal antibody‐targeted therapy and acquired drug resistance, and their accurate detection is critical to the clinical decision‐making of colorectal cancer. However, no proper quality control material is available for the current detection methods, particularly next‐generation sequencing (NGS). The ideal quality control material for NGS needs to provide both the tumor mutation gene and the matched background genomic DNA, which is uncataloged in public databases, to accurately distinguish germline polymorphisms and somatic mutations.

Methods

We developed a novel KRAS G12V mutant cell line using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 (Cas9) technique to make up for the deficiencies in existing quality control material and further validated the feasibility of the cell line as quality control material by amplification refractory mutation system (ARMS), Sanger sequencing, digital PCR (dPCR), and NGS.

Results

We verified that the edited cell line specifically had the G12V mutation, and the validation results presented a high consistency among the four methods of detection. The three cell lines screened contained the G12V mutation and the mutation allele fractions of G12V‐1, G12V‐2, and G12V‐3 were 52.01%, 82.06%, and 17.29%, respectively.

Conclusion

The novel KRAS G12V cell line generated using the CRISPR/Cas9 gene editing system is suitable as a quality control material for all current detection methods and provides a new direction in the development of quality control material.

     

Clustered regulatory interspaced short palindromic repeats (CRISPR)‐mediated mutagenesis and phenotype rescue by piggyBac transgenesis in a nonmodel Drosophila species

How behavioural diversity emerged in evolution is an unexplored subject in biology. To tackle this problem, genes and circuits for a behaviour need to be determined in different species for phylogenetic comparisons. The recently developed clustered regulatory interspaced short palindromic repeats/CRISPR associated protein9 (CRISPR/Cas9) system made such a challenge possible by providing the means to induce mutations in a gene of interest in any organism. Aiming at elucidating diversification in genetic and neural networks for courtship behaviour, we attempted to generate a genetic tool kit in Drosophila subobscura, a nonmodel species distantly related to the genetic model Drosophila melanogaster. Here we report the generation of yellow (y) and white mutations with the aid of the CRISPR/Cas9 system, and the rescue of the y mutant phenotype by germline transformation of the newly established y mutant fly line with a y⁺‐marked piggyBac vector. This successful mutagenesis and transformation in D. subobscura open up an avenue for comprehensive genetic analyses of higher functions in this and other nonmodel Drosophila species, representing a key step toward systematic comparisons of genes and circuitries underlying behaviour amongst species.     

Efficient genome editing in the olive fruit fly,Bactrocera oleae

The olive fruit fly, Bactrocera oleae, causes great damage to the quality and quantity of olive production worldwide. Pest management approaches have proved difficult for a variety of reasons, a fact that has brought about a need for alternative tools and approaches. Here we report for the first time in B. oleae the development of the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene editing tool, using the well‐known eye colour marker gene scarlet. Two synthetic guide RNAs targeting the coding region of the scarlet gene were synthesized and shown to work efficiently in vitro. These reagents were then microinjected along with purified Cas9 protein into early‐stage embryos. Successful CRISPR‐induced mutations of both copies of the scarlet gene showed a striking yellow eye phenotype, indicative of gene disruption. Multiple successful CRISPR events were confirmed by PCR and sequencing. The establishment of an efficient CRISPR‐based gene editing tool in B. oleae will enable the study of critical molecular mechanisms in olive fruit fly biology and physiology, including the analysis of insecticide resistance mechanisms and the discovery of novel insecticide targets, as well as facilitate the development of novel biotechnology‐based pest control strategies.     

Site‐specific transgenesis of the Drosophila melanogaster Y‐chromosome using CRISPR/Cas9

Despite the importance of Y‐chromosomes in evolution and sex determination, their heterochromatic, repeat‐rich nature makes them difficult to sequence (due, in part, to ambiguities in sequence alignment and assembly) and to genetically manipulate. Therefore, they generally remain poorly understood. For example, the Drosophila melanogaster Y‐chromosome, one of the most extensively studied Y‐chromosomes, is widely heterochromatic and composed mainly of highly repetitive sequences, with only a handful of expressed genes scattered throughout its length. Efforts to insert transgenes on this chromosome have thus far relied on either random insertion of transposons (sometimes harbouring ‘landing sites’ for subsequent integrations) with limited success or on chromosomal translocations, thereby limiting the types of Y‐chromosome‐related questions that could be explored. Here, we describe a versatile approach to site‐specifically insert transgenes on the Y‐chromosome in D. melanogaster via CRISPR/Cas9‐mediated homology‐directed repair. We demonstrate the ability to insert, and detect expression from, fluorescently marked transgenes at two specific locations on the Y‐chromosome, and we utilize these marked Y‐chromosomes to detect and quantify rare chromosomal nondisjunction effects. Finally, we discuss how this Y‐docking technique could be adapted to other insects to aid in the development of genetic control technologies for the management of insect disease vectors and pests.     

Progress in the application of CRISPR: From gene to base editing

The system of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated endonucleases (Cas) has been utilized for genome editing with great accuracy and high efficiency in generating gene knockout, knockin, and point mutations in eukaryotic genomes. However, traditional CRISPR/Cas9 technology introduces double-stranded DNA breaks (DSBs) at a target locus as the first step to make gene corrections, which easily results in undesired mutations. Thus, it is necessary to develop new methods for correcting the unwanted mutations. In this review, we summarize the recent developments and a new approach to genome and base editing by using CRISPR/Cas9. This methodology renders a conversion of one target base into another, for example, C to T (or G to A), and A to G (or T to C) without producing DSBs, requiring a donor DNA template, or generating excessive insertions and deletions. Furthermore, CRISPR/Cas9-derived base editing also improves efficiency in repairing point mutations in the genome.     

Genomic targeting by recombinase‐mediated cassette exchange in the spotted wing drosophila,Drosophila suzukii

Drosophila suzukii is a significant pest of stone and small fruits. The genome of this species has been sequenced and manipulated by transposon‐mediated transformation and CRISPR/Cas9 gene editing. These technologies open a variety of possibilities for functional genomics and genetic modifications that might improve biologically based population control strategies. Both of these approaches, however, would benefit from genome targeting that would avoid position effects and insertional mutations associated with random transposon vector insertions, and the limited DNA fragment insertion size allowed by gene editing. Here, we describe an efficient recombinase‐mediated cassette exchange (RMCE) system for D. suzukii in which heterospecific lox recombination sites were integrated into the genome by transposon‐mediated transformation and subsequently targeted for double recombination by a donor vector in the presence of Cre recombinase. Three loxN/lox2272 landing site lines have previously been created in D. suzukii, and quantitative PCR determined that polyubiquitin‐regulated enhanced green fluorescent protein expression is least susceptible to position effect suppression in the 443_M26m1 line. We presume that RMCE target sites may also be inserted more specifically into the genome by homology‐directed repair gene editing, thereby avoiding position effects and mutations, while eliminating restrictions on the size of donor constructs for subsequent insertion.     

CRISPR/Cas9-based functional characterization of the pigmentation gene ebony in Plutella xylostella

Body pigmentation is an important character of insects in adapting to biotic and abiotic environmental challenges. Additionally, based on the relative ease of screening, several genes involved in insect melanization have been used in classic genetic studies or as visual markers in constructing transgenic insects. Here, a homologue of the Bombyx mori melanization-inhibiting gene ebony, associated with the conversion of dopamine to N-β-alanyl dopamine, was identified in a global pest, Plutella xylostella. The CRISPR/Cas9 system was applied to generate multiple Pxebony knockout alleles which were crossed to produce a Pxebony knockout strain, showing darker pigmentation in larvae, pupae and adults, compared with wildtype. Interestingly, we observed that Pxebony heterozygotes displayed an intermediate darkened phenotype, indicating partial dominance between the knockout and wildtype alleles. The fitness costs of Pxebony deficiency were also assessed in the mutant strain, indicating that embryo hatchability and larval survival were significantly reduced, while the eclosion rate was not obviously affected. Our work provides a potential target for exploring CRISPR-based genetics-control systems in this economically important pest lepidopteran.     

Knockout of a P‐glycoprotein gene increases susceptibility to abamectin and emamectin benzoate in Spodoptera exigua

P‐glycoprotein [P‐gp or the ATP‐binding cassette transporter B1 (ABCB1)] is an important participant in multidrug resistance of cancer cells, yet the precise function of this arthropod transporter is unknown. The aim of this study was to determine the importance of P‐gp for susceptibility to insecticides in the beet armyworm (Spodoptera exigua) using clustered regularly interspaced short palindromic repeats/CRISPR‐associated 9 (CRISPR/Cas9) gene‐editing technology. We cloned an open reading frame (ORF) encoding the S. exigua P‐gp protein (SeP‐gp) predicted to display structural characteristics common to P‐gp and other insect ABCB1 transporters. A knockout line with a frame shift deletion of four nucleotides in the SeP‐gp ORF was established using the CRISPR/Cas9 gene‐editing system to test its potential role in determining susceptibility to chemical insecticides or insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Results from comparative bioassays demonstrate that knockout of SeP‐gp significantly increases susceptibility of S. exigua by around threefold to abamectin and emamectin benzoate (EB), but not to spinosad, chlorfenapyr, beta‐cypermethrin, carbosulfan indoxacarb, chlorpyrifos, phoxim, diafenthiuron, chlorfluazuron, chlorantraniliprole or two Bt toxins (Cry1Ca and Cry1Fa). Our data support an important role for SeP‐gp in susceptibility of S. exigua to abamectin and EB and imply that overexpression of SeP‐gp may contribute to abamectin and EB resistance in S. exigua.     

Identification and characterization of a Masculinizer homologue in the diamondback moth,Plutella xylostella

Recently, a novel sex‐determination system was identified in the silkworm (Bombyx mori) in which a piwi‐interacting RNA (piRNA) encoded on the female‐specific W chromosome silences a Z‐linked gene (Masculinizer) that would otherwise initiate male sex‐determination and dosage compensation. Masculinizer provides various opportunities for developing improved genetic pest management tools. A pest lepidopteran in which a genetic pest management system has been developed, but which would benefit greatly from such improved designs, is the diamondback moth, Plutella xylostella. However, Masculinizer has not yet been identified in this species. Here, focusing on the previously described ‘masculinizing’ domain of B. mori Masculinizer, we identify P. xylostella Masculinizer (PxyMasc). We show that PxyMasc is Z‐linked, regulates sex‐specific alternative splicing of doublesex and is necessary for male survival. Similar results in B. mori suggest this survival effect is possibly through failure to initiate male dosage compensation. The highly conserved function and location of this gene between these two distantly related lepidopterans suggests a deep role for Masculinizer in the sex‐determination systems of the Lepidoptera.     

Distribution analysis of TRH in Bactrocera dorsalis using a CRISPR/Cas9-mediated reporter knock-in strain

Although the study of many genes and their protein products is limited by the availability of high-quality antibodies, this problem could be solved by fusing a tag/reporter to an endogenous gene using a gene-editing approach. The type II bacterial CRISPR/Cas system has been demonstrated to be an efficient gene-targeting technology for many insects, including the oriental fruit fly Bactrocera dorsalis. However, knocking in, an important editing method of the CRISPR/Cas9 system, has lagged in its application in insects. Here, we describe a highly efficient homology-directed genome editing system for B. dorsalis that incorporates coinjection of embryos with Cas9 protein, guide RNA and a short single-stranded oligodeoxynucleotide donor. This one-step procedure generates flies carrying V5 tag (42 bp) in the BdorTRH gene. In insects, as in other invertebrates and in vertebrates, the neuronal tryptophan hydroxylase (TRH) gene encodes the rate-limiting enzyme for serotonin biosynthesis in the central nervous system. Using V5 monoclonal antibody, the distribution of TRH in B. dorsalis at different developmental stages was uncovered. Our results will facilitate the generation of insects carrying precise DNA inserts in endogenous genes and will lay foundation for the investigation of the neural mechanisms underlying the serotonin-mediated behaviour of B. dorsalis.     

Nonsyndromic X‐linked intellectual deficiency in three brothers with a novel MED12 missense mutation [c.5922G>T (p.Glu1974His)]

X‐linked intellectual deficiency (XLID) is a large group of genetic disorders. MED12 gene causes syndromic and nonsyndromic forms of XLID. Only seven pathological mutations have been identified in this gene. Here, we report a novel mutation segregating with XLID phenotype. This mutation could be in favor of genotype–phenotype correlations.     

Personalized therapeutic strategies for patients with retinitis pigmentosa

Introduction: Retinitis pigmentosa (RP) encompasses many different hereditary retinal degenerations that are caused by a vast array of different gene mutations and have highly variable disease presentations and severities. This heterogeneity poses a significant therapeutic challenge, although an answer may eventually be found through two recent innovations: induced pluripotent stem cells (iPSCs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas genome editing.

Areas covered: This review discusses the wide-ranging applications of iPSCs and CRISPR–including disease modelling, diagnostics and therapeutics – with an ultimate view towards understanding how these two technologies can come together to address disease heterogeneity and orphan genes in a novel personalized medicine platform. An extensive literature search was conducted in PubMed and Google Scholar, with a particular focus on high-impact research published within the last 1 – 2 years and centered broadly on the subjects of retinal gene therapy, iPSC-derived outer retina cells, stem cell transplantation and CRISPR/Cas gene editing.

Expert opinion: For the retinal pigment epithelium, autologous transplantation of gene-corrected grafts derived from iPSCs may well be technically feasible in the near future. Photoreceptor transplantation faces more significant unresolved technical challenges but remains an achievable, if more distant, goal given the rapid pace of advancements in the field.