Construction of Baculovirus-Inducible CRISPR/Cas9 Antiviral System Targeting BmNPV in Bombyx mori

The silkworm Bombyx mori is an economically important insect. The sericulture industry is seriously affected by pathogen infections. Of these pathogens, Bombyx mori nucleopolyhedrovirus (BmNPV) causes approximately 80% of the total economic losses due to pathogen infections. We previously constructed a BmNPV-specific CRISPR/Cas9 silkworm line with significantly enhanced resistance to BmNPV. In order to optimize the resistance properties and minimize its impact on economic traits, we constructed an inducible CRISPR/Cas9 system for use in transgenic silkworms. We used the 39k promoter, which is induced by viral infection, to express Cas9 and the U6 promoter to express four small guide RNA targeting the genes encoding BmNPV late expression factors 1 and 3 (lef-1 and lef-3, respectively), which are essential for viral DNA replication. The system was rapidly activated when the silkworm was infected and showed considerably higher resistance to BmNPV infection than the wild-type silkworm. The inducible system significantly reduced the development effects due to the constitutive expression of Cas9. No obvious differences in developmental processes or economically important characteristics were observed between the resulting transgenic silkworms and wild-type silkworms. Adoption of this accurate and highly efficient inducible CRISPR/Cas9 system targeting BmNPV DNA replication will result in enhanced antivirus measures during sericulture, and our work also provides insights into the broader application of the CRISPR/Cas9 system in the control of infectious diseases and insect pests.     

Plant Viruses: From Targets to Tools for CRISPR

Plant viruses cause devastating diseases in many agriculture systems, being a serious threat for the provision of adequate nourishment to a continuous growing population. At the present, there are no chemical products that directly target the viruses, and their control rely mainly on preventive sanitary measures to reduce viral infections that, although important, have proved to be far from enough. The current most effective and sustainable solution is the use of virus-resistant varieties, but which require too much work and time to obtain. In the recent years, the versatile gene editing technology known as CRISPR/Cas has simplified the engineering of crops and has successfully been used for the development of viral resistant plants. CRISPR stands for ‘clustered regularly interspaced short palindromic repeats’ and CRISPR-associated (Cas) proteins, and is based on a natural adaptive immune system that most archaeal and some bacterial species present to defend themselves against invading bacteriophages. Plant viral resistance using CRISPR/Cas technology can been achieved either through manipulation of plant genome (plant-mediated resistance), by mutating host factors required for viral infection; or through manipulation of virus genome (virus-mediated resistance), for which CRISPR/Cas systems must specifically target and cleave viral DNA or RNA. Viruses present an efficient machinery and comprehensive genome structure and, in a different, beneficial perspective, they have been used as biotechnological tools in several areas such as medicine, materials industry, and agriculture with several purposes. Due to all this potential, it is not surprising that viruses have also been used as vectors for CRISPR technology; namely, to deliver CRISPR components into plants, a crucial step for the success of CRISPR technology. Here we discuss the basic principles of CRISPR/Cas technology, with a special focus on the advances of CRISPR/Cas to engineer plant resistance against DNA and RNA viruses. We also describe several strategies for the delivery of these systems into plant cells, focusing on the advantages and disadvantages of the use of plant viruses as vectors. We conclude by discussing some of the constrains faced by the application of CRISPR/Cas technology in agriculture and future prospects.     

Upgraded CRISPR/Cas9 tools for tissue-specific mutagenesis in Drosophila

CRISPR/Cas9 has emerged as a powerful technology for tissue-specific mutagenesis. However, tissue-specific CRISPR/Cas9 tools currently available in Drosophila remain deficient in three significant ways. First, many existing gRNAs are inefficient, such that further improvements of gRNA expression constructs are needed for more efficient and predictable mutagenesis in both somatic and germline tissues. Second, it has been difficult to label mutant cells in target tissues with current methods. Lastly, application of tissue-specific mutagenesis at present often relies on Gal4-driven Cas9, which hampers the flexibility and effectiveness of the system. Here, we tackle these deficiencies by building upon our previous CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) tools. First, we significantly improved gRNA efficiency in somatic tissues by optimizing multiplexed gRNA design. Similarly, we also designed efficient dual-gRNA vectors for the germline. Second, we developed methods to positively and negatively label mutant cells in tissue-specific mutagenesis by incorporating co-CRISPR reporters into gRNA expression vectors. Lastly, we generated genetic reagents for convenient conversion of existing Gal4 drivers into tissue-specific Cas9 lines based on homology-assisted CRISPR knock-in. In this way, we expand the choices of Cas9 for CRISPR-TRiM analysis to broader tissues and developmental stages. Overall, our upgraded CRISPR/Cas9 tools make tissue-specific mutagenesis more versatile, reliable, and effective in Drosophila. These improvements may be also applied to other model systems.

The ability to characterize gene function in a tissue-specific manner has been critical for studying developmental and disease mechanisms of essential genes. The CRISPR/Cas9 system has recently provided powerful tools for inducing tissue-specific gene loss of function (LOF). In this system, the endonuclease Cas9 is directed by a small guide RNA (gRNA) to a specific DNA sequence to create double-strand breaks (DSBs) (1). In the absence of homologous repair templates, DSBs are primarily repaired by nonhomologous end joining, an error-prone process that often introduces mutations in the form of insertions or deletions (indels) (2, 3). Because the protospacer adjacent motif required for Cas9 action is ubiquitous in genomes (1, 4), by targeting the expression of Cas9 and gRNAs to specific tissues, mutations can be induced at virtually any gene in a tissue-specific manner. However, current tissue-specific CRISPR/Cas9 tools in Drosophila are still deficient in three areas, limiting the power of CRISPR/Cas9 in analyzing gene functions in broad tissues and biological processes.     

On the Development of Programs in Cancer Research

This paper discusses some problems of the participation of scientists in developing broad, long-range programs of cancer research by national and local medical centers. The 17-year performance of the Scientific Advisory Committee of the National Foundation for Infantile Paralysis in development of polio vaccines is discussed with respect to: (i) the formulation of programs and priorities in the absence of much fundamental knowledge; (ii) the support of basic research, (iii) its attitude on the direction of scientific effort, and (iv) the cooperative activities among numerous capable, strongwilled, independent scientists. This historic effort provides an excellent model for scientific participation in the development of comprehensive programs of cancer research.     

Adeno-Associated Vector-Delivered CRISPR/SaCas9 System Reduces Feline Leukemia Virus Production In Vitro

Feline leukemia virus (FeLV) is a retrovirus of cats worldwide. High viral loads are associated with progressive infection and the death of the host, due to FeLV-associated disease. In contrast, low viral loads, an effective immune response, and a better clinical outcome can be observed in cats with regressive infection. We hypothesize that by lowering viral loads in progressively infected cats, using CRISPR/SaCas9-assisted gene therapy, the cat’s immune system may be permitted to direct the infection towards a regressive outcome. In a step towards this goal, the present study evaluates different adeno-associated vectors (AAVs) for their competence in delivering a gene editing system into feline cells, followed by investigations of the CRISPR/SaCas9 targeting efficiency for different sites within the FeLV provirus. Nine natural AAV serotypes, two AAV hybrid strains, and Anc80L65, an in silico predicted AAV ancestor, were tested for their potential to infect different feline cell lines and feline primary cells. AAV-DJ revealed superior infection efficiency and was thus employed in subsequent transduction experiments. The introduction of double-strand breaks, using the CRISPR/SaCas9 system targeting 12 selected FeLV provirus sites, was confirmed by T7 endonuclease 1 (T7E1), as well as Tracking of Indels by Decomposition (TIDE) analysis. The highest percentage (up to 80%) of nonhomologous end-joining (NHEJ) was found in the highly conserved gag and pol regions. Subsequent transduction experiments, using AAV-DJ, confirmed indel formation and showed a significant reduction in FeLV p27 antigen for some targets. The targeting of the FeLV provirus was efficient when using the CRISPR/SaCas9 approach in vitro. Whether the observed extent of provirus targeting will be sufficient to provide progressively FeLV-infected cats with the means to overcome the infection needs to be further investigated in vivo.     

Disrupting the male germ line to find infertility and contraception targets

Genetically-manipulated mouse models have become indispensible for broadening our understanding of genes and pathways related to male germ cell development. Until suitable in vitro systems for studying spermatogenesis are perfected, in vivo models will remain the gold standard for inquiry into testicular function. Here, we discuss exciting advances that are allowing researchers faster, easier, and more customizable access to their mouse models of interest. Specifically, the trans-NIH Knockout Mouse Project (KOMP) is working to generate knockout mouse models of every gene in the mouse genome. The related Knockout Mouse Phenotyping Program (KOMP2) is performing systematic phenotypic analysis of this genome-wide collection of knockout mice, including fertility screening. Together, these programs will not only uncover new genes involved in male germ cell development but also provide the research community with the mouse models necessary for further investigations. In addition to KOMP/KOMP2, another promising development in the field of mouse models is the advent of CRISPR (clustered regularly interspaced short palindromic repeat)-Cas technology. Utilizing 20 nucleotide guide sequences, CRISPR/Cas has the potential to introduce sequence-specific insertions, deletions, and point mutations to produce null, conditional, activated, or reporter-tagged alleles. CRISPR/Cas can also successfully target multiple genes in a single experimental step, forgoing the multiple generations of breeding traditionally required to produce mouse models with deletions, insertions, or mutations in multiple genes. In addition, CRISPR/Cas can be used to create mouse models carrying variants identical to those identified in infertile human patients, providing the opportunity to explore the effects of such mutations in an in vivo system. Both the KOMP/KOMP2 projects and the CRISPR/Cas system provide powerful, accessible genetic approaches to the study of male germ cell development in the mouse. A more complete understanding of male germ cell biology is critical for the identification of novel targets for potential non-hormonal contraceptive intervention.     

Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system

A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75–99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.     

Efficiency wages in an experimental labor market

There has been recent experimental support for efficiency wage theories of the labor market. This short paper initiates the larger process of evaluating the boundary conditions of the gift- exchange phenomenon. In particular, we will see whether behavior consistent with the fair wage–effort hypothesis can emerge and be sustained under conditions in which there is (i) a nontrivial marginal cost to providing effort and (ii) increased social distance between subject and experimenter.     

Cas9�CcrRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria

Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria and archaea. The silencing of invading nucleic acids is executed by ribonucleoprotein complexes preloaded with small, interfering CRISPR RNAs (crRNAs) that act as guides for targeting and degradation of foreign nucleic acid. Here, we demonstrate that the Cas9–crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system introduces in vitro a double-strand break at a specific site in DNA containing a sequence complementary to crRNA. DNA cleavage is executed by Cas9, which uses two distinct active sites, RuvC and HNH, to generate site-specific nicks on opposite DNA strands. Results demonstrate that the Cas9–crRNA complex functions as an RNA-guided endonuclease with RNA-directed target sequence recognition and protein-mediated DNA cleavage. These findings pave the way for engineering of universal programmable RNA-guided DNA endonucleases.     

From the Cover: Multigeneration analysis reveals the inheritance,specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has emerged as a powerful tool for targeted gene editing in many organisms, including plants. However, all of the reported studies in plants focused on either transient systems or the first generation after the CRISPR/Cas system was stably transformed into plants. In this study we examined several plant generations with seven genes at 12 different target sites to determine the patterns, efficiency, specificity, and heritability of CRISPR/Cas-induced gene mutations or corrections in Arabidopsis. The proportion of plants bearing any mutations (chimeric, heterozygous, biallelic, or homozygous) was 71.2% at T1, 58.3% at T2, and 79.4% at T3 generations. CRISPR/Cas-induced mutations were predominantly 1 bp insertion and short deletions. Gene modifications detected in T1 plants occurred mostly in somatic cells, and consequently there were no T1 plants that were homozygous for a gene modification event. In contrast, ∼22% of T2 plants were found to be homozygous for a modified gene. All homozygotes were stable to the next generation, without any new modifications at the target sites. There was no indication of any off-target mutations by examining the target sites and sequences highly homologous to the target sites and by in-depth whole-genome sequencing. Together our results show that the CRISPR/Cas system is a useful tool for generating versatile and heritable modifications specifically at target genes in plants.Genome engineering tools are important for plant functional genomics research and plant biotechnology. The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been successfully used for efficient genome editing in human cell lines, zebrafish, and mouse (1–3) and recently applied to gene modification in plants (4–10). In this system a short RNA molecule guides the associated endonuclease Cas9 to generate double strand breaks (DSBs) in the target genomic DNA, which lead to sequence mutations as a result of error-prone nonhomologous end-joining (NHEJ) DNA damage repair or to gene correction or replacement as a result of homology-dependent recombination (HR) (11). It was shown that engineered CRISPR/Cas caused mutations in target genes or corrections in transgenes in transient expression assays in plant protoplasts and tobacco leaves (10). Importantly, stable expression of the CRISPR/Cas in transgenic Arabidopsis, tobacco, and rice plants led to mutations (mostly indels) in target genes and correction of a transgene (4–9). However, it was not known whether the gene mutations and corrections occurred in somatic cells only or whether some of the mutations and corrections happened in germ-line cells and thus may be heritable. Additionally, it is unclear how specific the CRISPR/Cas is in plants. Previous studies in human cell lines indicated a high frequency of off-target effect of CRISPR/Cas-induced mutagenesis (12, 13) but a lower off-target effect in mice and zebrafish (14, 15). Here we show that the CRISPR/Cas-induced transgene correction or mutations in endogenous plant genes and transgenes detected in Arabidopsis T1 plants occurred mostly in somatic cells. However, some of the gene modifications were transmitted through the germ line and were heritable in Arabidopsis T2 and T3 plants following the classic Mendelian model. Mutations caused during DSB repair were predominantly 1 bp insertion and short deletions. Furthermore, our deep sequencing and analysis did not detect any off-targets in multiple CRISPR/Cas transgenic Arabidopsis lines, indicating that the mutagenesis effect of CRISPR/Cas is highly specific in plants.     

CRISPR/Cas系统在病原体核酸检测中的应用进展

快速、灵敏、特异的检测方法对于传染病防控至关重要。聚合酶链式反应、等温扩增等体外核酸扩增技术已广泛应用于病原体检测。近年来,基于规则成簇的间隔短回文重复序列及其相关蛋白(CRISPR/Cas)系统的核酸检测方法显示出快速、高灵敏度、高特异性与便携性等优点。本文对CRISPR/Cas系统类型、原理及其在病原体检测中的应用进展进行综述,并对其应用前景进行展望。     

Applications of CRISPR systems in respiratory health: Entering a new ‘red pen’ era in genome editing

Respiratory diseases, such as influenza infection, acute tracheal bronchitis, pneumonia, tuberculosis, chronic obstructive pulmonary disease, asthma, lung cancer and nasopharyngeal carcinoma, continue to significantly impact human health. Diseases of the lung and respiratory tract are influenced by environmental conditions and socio‐economic factors; however, many of these serious respiratory disorders are also rooted in genetic or epigenetic causes. Clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR‐associated (Cas) proteins, isolated from the immune system of prokaryotes, provide a tool to manipulate gene sequences and gene expression with significant implications for respiratory research. CRISPR/Cas systems allow preclinical modelling of causal factors involved in many respiratory diseases, providing new insights into their underlying mechanisms. CRISPR can also be used to screen for genes involved in respiratory processes, development and pathology, identifying novel disease drivers or drug targets. Finally, CRISPR/Cas systems can potentially correct genetic mutations and edit epigenetic marks that contribute to respiratory disorders, providing a form of personalized medicine that could be used in conjunction with other technologies such as stem cell reprogramming and transplantation. CRISPR gene editing is a young field of research, and concerns regarding its specificity, as well as the need for efficient and safe delivery methods, need to be addressed further. However, CRISPR/Cas systems represent a significant step forward for research and therapy in respiratory health, and it is likely we will see the breakthroughs generated from this technology continue.     

Dynamic antibody responses to the Mycobacterium tuberculosis proteome

Considerable effort has been directed toward controlling tuberculosis, which kills almost two million people yearly. High on the research agenda is the discovery of biomarkers of active tuberculosis (TB) for diagnosis and for monitoring treatment outcome. Rational biomarker discovery requires understanding host–pathogen interactions leading to biomarker expression. Here we report a systems immunology approach integrating clinical data and bacterial metabolic and regulatory information with high-throughput detection in human serum of antibodies to the entire Mycobacterium tuberculosis proteome. Sera from worldwide TB suspects recognized approximately 10% of the bacterial proteome. This result defines the M. tuberculosis immunoproteome, which is rich in membrane-associated and extracellular proteins. Additional analyses revealed that during active tuberculosis (i) antibody responses focused on an approximately 0.5% of the proteome enriched for extracellular proteins, (ii) relative target preference varied among patients, and (iii) responses correlated with bacillary burden. These results indicate that the B cell response tracks the evolution of infection and the pathogen burden and replicative state and suggest functions associated with B cell-rich foci seen in tuberculous lung granulomas. Our integrated proteome-scale approach is applicable to other chronic infections characterized by diverse antibody target recognition.     

CRISPR-Associated (CAS) Effectors Delivery via Microfluidic Cell-Deformation Chip

Identifying new and even more precise technologies for modifying and manipulating selectively specific genes has provided a powerful tool for characterizing gene functions in basic research and potential therapeutics for genome regulation. The rapid development of nuclease-based techniques such as CRISPR/Cas systems has revolutionized new genome engineering and medicine possibilities. Additionally, the appropriate delivery procedures regarding CRISPR/Cas systems are critical, and a large number of previous reviews have focused on the CRISPR/Cas9–12 and 13 delivery methods. Still, despite all efforts, the in vivo delivery of the CAS gene systems remains challenging. The transfection of CRISPR components can often be inefficient when applying conventional delivery tools including viral elements and chemical vectors because of the restricted packaging size and incompetency of some cell types. Therefore, physical methods such as microfluidic systems are more applicable for in vitro delivery. This review focuses on the recent advancements of microfluidic systems to deliver CRISPR/Cas systems in clinical and therapy investigations.     

Spectral theory for domains in R of finite measure

Let Ω be a measurable subset of Rⁿ of finite positive Lebesgue measures. The following two problems are considered: (i) Find commuting self-adjoint extensions of the minimal operators -i/x_k, k = 1,..., n (Ω open). (ii) Find a set Λ Rⁿ such that the functions e_λ = exp(iλ₁x₁ +... + iλ_nx_n) for λ Λ form an orthonormal basis for L₂(Ω). The problems are known to be equivalent under mild regularity conditions on Ω, and existence holds in two cases: (i) there is a connected open set Ω′ such that the symmetric difference ΩΔΩ′ is a null set and Ω′ is a fundamental domain for a discrete total subgroup; and (ii)Ω = [unk]_aR (a + [unk]), disjoint union neglecting null sets, in which [unk] is a fundamental domain and R is a “set of representors” for a finite group of translations. Case i is equivalent to a function theoretic condition of Forelli, and case ii is established when the existence of a discrete covariance group is assumed. Generalizations of the geometric results i and ii for spectral sets in arbitrary Lie groups are indicated.     

Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila

The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line–restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25–100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.Experimentally induced mutations in the genomes of model organisms have been the basis of much of our current understanding of biological mechanisms. However, traditional mutagenesis tools have significant drawbacks. Forward genetic approaches such as chemical mutagenesis lack specificity, leading to unwanted mutations at many sites in the genome. Traditional reverse genetic approaches, such as gene targeting by conventional homologous recombination, suffer from low efficiency and therefore are labor intensive. In recent years novel methods have been developed that aim to modify genomes with high precision and high efficiency by introducing double-stand breaks (DSBs) at defined loci (1). DSBs can be repaired by either nonhomologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is an error-prone process that frequently leads to the generation of small, mutagenic insertions and deletions (indels). HDR repairs DSBs by precisely copying sequence from a donor template, allowing specific changes to be introduced into the genome (2).The type II clustered regular interspersed short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has emerged recently as an extraordinarily powerful method for inducing site-specific DSBs in the genomes of a variety of organisms. The method exploits the RNA-guided endonuclease Cas9, which plays a key role in bacterial adaptive immune systems. Target specificity of Cas9 is encoded by a 20-nt spacer sequence in the crisprRNA, which pairs with the transactivating RNA to direct the endonuclease to the complementary target site in the DNA (3). For genome engineering, crisprRNA and transactivating RNA can be combined in a single chimeric guide RNA (gRNA), resulting in a simple two-component system for the creation of DSBs at defined sites (3). Binding of the Cas9/gRNA complex at a genomic target site is constrained only by the requirement for an adjacent short protospacer-adjacent motif (PAM), which for the commonly used Streptococcus pyogenes Cas9 is NGG (4).Several groups recently demonstrated CRISPR/Cas-mediated editing of the genome of Drosophila melanogaster (5–12), a key model organism for biological research. However, the rate of mutagenesis has varied widely both within and among different studies. Differences in the methods used to introduce Cas9 and gRNAs into the fly likely contribute significantly to different experimental outcomes. Kondo and Ueda (8) expressed both Cas9 and gRNA from transgenes stably integrated into the genome, but all other studies have used microinjection of expression plasmids or of in vitro-transcribed RNA into embryos to deliver one or both CRISPR/Cas components (5–7, 9–11). Much of the currently available evidence suggests that transgenic provision of Cas9 increases rates of germ-line transmission substantially (8, 10, 11). However, the influence of different regulatory sequences within cas9 transgenes on the rate of mutagenesis and on the location where mutations are generated within the organism has not been evaluated. The effect of different promoter sequences on the activities of gRNAs also has not been explored systematically. Therefore it is possible that suboptimal tools are being used currently for many CRISPR/Cas experiments in Drosophila.Previous studies in Drosophila have focused on the use of CRISPR/Cas to create heritable mutations in the germ line. In principle, efficient biallelic targeting within somatic cells of Drosophila would represent a powerful system to dissect the functions of genes within an organismal context. However, the feasibility of such an approach has not been explored so far.Here, we present a versatile CRISPR/Cas toolbox for Drosophila genome engineering consisting of a set of systematically evaluated transgenic Cas9 lines and gRNA-expression plasmids. We describe combinations of Cas9 and gRNA sources that can be used to induce, with high efficiency, loss-of-function mutations in nonessential or essential genes and integration of designer sequences by HDR. Finally, we show that our optimized transgenic tools permit efficient biallelic targeting in a variety of somatic tissues of the fly, allowing the characterization of mutant phenotypes directly in Cas9/gRNA-expressing animals.     

CRISPR/Cas9-Mediated Disruption of the lef8 and lef9 to Inhibit Nucleopolyhedrovirus Replication in Silkworms

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes severe disease in silkworms. In a previous study, we demonstrated that by using the CRISPR/Cas9 system to disrupt the BmNPV ie-1 and me53 genes, transgenic silkworms showed resistance to BmNPV infection. Here, we used the same strategy to simultaneously target lef8 and lef9, which are essential for BmNPV replication. A PCR assay confirmed that double-stranded breaks were induced in viral DNA at targeted sequences in BmNPV-infected transgenic silkworms that expressed small guide RNAs (sgRNAs) and Cas9. Bioassays and qPCR showed that replication of BmNPV and mortality were significantly reduced in the transgenic silkworms in comparison with the control groups. Microscopy showed degradation of midgut cells in the BmNPV-infected wild type silkworms, but not in the transgenic silkworms. These results demonstrated that transgenic silkworms using the CRISPR/Cas9 system to disrupt BmNPV lef8 and lef9 genes could successfully prevent BmNPV infection. Our research not only provides more alternative targets for the CRISPR antiviral system, but also aims to provide new ideas for the application of virus infection research and the control of insect pests.     

Metagenomic discovery of CRISPR-associated transposons

CRISPR-associated Tn7 transposons (CASTs) co-opt cas genes for RNA-guided transposition. CASTs are exceedingly rare in genomic databases; recent surveys have reported Tn7-like transposons that co-opt Type I-F, I-B, and V-K CRISPR effectors. Here, we expand the diversity of reported CAST systems via a bioinformatic search of metagenomic databases. We discover architectures for all known CASTs, including arrangements of the Cascade effectors, target homing modalities, and minimal V-K systems. We also describe families of CASTs that have co-opted the Type I-C and Type IV CRISPR-Cas systems. Our search for non-Tn7 CASTs identifies putative candidates that include a nuclease dead Cas12. These systems shed light on how CRISPR systems have coevolved with transposases and expand the programmable gene-editing toolkit.

CRISPR-associated transposons (CASTs) are transposons that have delegated their insertion site selection to a nuclease-deficient CRISPR-Cas system. All currently known CASTs derive from Tn7-like transposons and retain the core transposition genes tnsB and tnsC but dispense with tnsE, and often tnsD, which mediate target selection (1, 2). Tn7 transposons site specifically insert themselves at a single chromosomal locus (the attachment or att site) via the TnsD/TniQ family of DNA-binding proteins while TnsE promotes horizontal gene transfer onto mobile genetic elements. In contrast, Class 1 CASTs replace TnsD and TnsE with a CRISPR RNA (crRNA)–guided TniQ-Cascade effector complex (3–6). These CASTs can use the TniQ-Cascade complexes for both vertical and horizontal gene transfer (5). One notable exception is a family of Type I-B CASTs that retains TnsD for vertical transmission but co-opts TniQ-Cascade for horizontal transmission (7). Similarly, Class 2 CASTs use the Cas12k effector to transpose to the att sites or to mobile genetic elements (8, 9). CASTs also dispense with the spacer acquisition and DNA interference genes found in traditional CRISPR-Cas operons (2). In short, these systems have merged the core transposition activities with crRNA-guided DNA targeting.CASTs are exceedingly rare; only three subfamilies of Tn7-associated CASTs have been reported bioinformatically and experimentally (2, 5, 7, 9, 10). These studies have identified that many, but not all, CASTs encode a homing spacer flanked by atypical (privileged) direct repeats (11). However, the prevalence of such atypical repeats, the diversity of homing strategies, and the molecular mechanisms of why CASTs have evolved these repeats remain unresolved. Moreover, all CASTs that have been identified to date have a minimal CRISPR array with as few as two spacers. These systems are also missing the Cas1–Cas2 adaptation machinery, raising the question of how CASTs target other mobile genetic elements for horizontal gene transfer. Another open question is whether non-Tn7 transposons have adapted CRISPR-Cas systems to mobilize their genetic information.CASTs are also a promising tool for inserting DNA into diverse cells. CASTs have already been used to simultaneously insert large cargos at multiple genomic loci (12–14), build mutant libraries in vivo (15), and edit the genomes of uncultivated members of a bacterial community (16). The characterization of as-yet-undiscovered systems with diverse capabilities may spur additional applications for CASTs in engineering both prokaryotic and eukaryotic cells as had occurred for CRISPR-Cas nucleases. For example, although Cas9 and Cas12a both seemingly catalyze the same reaction—crRNA-guided cleavage of a double-stranded DNA—these enzymes have been harnessed for different biotechnological applications owing to their differing nuclease domain architectures. Cas9 nickases can be readily created by inactivating either the HNH or RuvC nuclease domain, leading to applications such as prime editing (17, 18). Cas12a, in contrast, can cleave nonspecific single-stranded DNA after binding its specific target sequence (19). This activity has been harnessed for a suite of nucleic acid detection technologies (20). We reasoned that an expanded catalog of CASTs may shed light on the many unresolved questions regarding their biological mechanisms and future biotechnological applications.Here, we have systematically surveyed CASTs across metagenomic databases using a custom-built computational pipeline that identifies both Tn7 and non-Tn7 CASTs. Using this pipeline, we have identified unique architectures for Type I-B, I-F, and V CASTs. Type I-F CASTs show the greatest diversity in cas genes, including tniQ-cas8/5 fusions, split cas7s, and even split cas5 genes. Some I-F CASTs likely assemble a Cascade around a short crRNA for homing from a noncanonical spacer. Type I-B CASTs frequently encode two tniQ/tnsD homologs, one of which is used for homing via a crRNA-independent mechanism (7). Remarkably, we have also found I-B systems that encode two tniQ homologs and a homing crRNA, suggesting additional unexplored targeting mechanisms. In addition, we have observed Type I-C and Type IV family Tn7-like CASTs with unique gene architectures. Both of these subfamilies lack canonical CRISPR arrays, suggesting that CASTs use distal CRISPR arrays, perhaps from active CRISPR-Cas systems, for horizontal gene transfer. We have identified multiple self-insertions and gene loss in Type V systems, indicating that target immunity—a mechanism that prevents transposons from multiple self-insertions at an attachment site—is frequently weakened. Finally, we have found a set of Cas12-associated recombination-promoting nuclease/transposase (Rpn) family transposases that may participate in crRNA-guided horizontal gene transfer. We anticipate that these findings will shed additional light on how CASTs have co-opted CRISPR-Cas systems and further expand the CRISPR gene-editing toolbox.