In an a recent experiment published in Gene, Li and Handler (2019) utilized readily available study systems and newly emerging genetic technologies and demonstrated both germline and somatic CRISPR/Cas9 induced mutations in Anastrepha ludens. The creation of heritable mutations was […]
Cas9
Heritable Gene Editing in Thermobia domestica
Ohde et al report the first successful development and use of CRISPR/Cas9 in the basally branching insect Thermobia domestica. By co-injecting Cas9 nuclease with sgRNA targeting a T. domestica ortholog of the Drosophila melanogaster white gene (Td-w), the authors created […]
Microinjection of the Sand Fly Lutzomyia longipalpis, a Vector for Leishmaniasis
Two recent papers, Jefferies et al (2018) and Martin-Martin et. al (2018), describe experiments to develop and optimise a sand fly microinjection protocol. There are 2 authors to this post: Alla Madich, Simon Collier, technical constraints on this website allow […]
Cas9 Orthologs with Minimal PAM Requirements
In their recent paper Chatterjee and colleagues (2018) describe a natural protospacer adjacent motif (PAM) plasticity of previously uncharacterized Cas9 endonuclease from Streptococcus canis (ScCas9). Chatterjee et al (2018) report ScCas9’s affinity to minimal 5’-NNG-3’ PAM sequences and describe its accurate editing capabilities, […]
Precision-Guided Sterile Insect Technique
Right after the 2019 New Year, Kandul et al (2019) described a novel CRISPR-based technology which they termed as “precision guided sterile insect technique (pgSIT)”. The idea of traditional SIT was planted in 1930s, which uses radiation to induce sterility […]
A Multiple Gene Drive System
Yan & Finnigan, (2018) recently published a paper in Scientific Reports describing an artificial multi-locus gene drive system by using a single Cas9 and three guide RNA (gRNA) in the budding yeast Saccharomyces cerevisiae. Nuclease-based gene drives do not follow […]
CRISPR to go – a guide to gene editing Tephritid flies at remote facilities
Sim et al. (2018) recently published a paper in Insect Molecular Biology describing CRISPR/Cas 9 methods for gene editing in three genera of Tephritid fruit flies: Anastrepha, Bactrocera, and Ceratitis. CRISPR/Cas9 allows for targeted gene editing through homology driven repair […]
Driving into doublesex
Kyrou et al. (2018) in a recent paper in Nature Biotechnology report identifying a highly conserved target site in the Anopheles gambiae fertility gene doublesex (dsx), that cannot tolerate mutations. They built a gene drive targeting this site in dsx […]
Female Lethality System in Lepidoptera
Zhang et al (2018) in a recent paper in PNAS report their successful integration of transgenes into the W chromosome of Bombyx mori. Initially, they set out to place visible markers on the W. This would enable rapid and accurate […]
Improved HDR-Mediated Genome Editing
Jose Arturo Gutierrez-Triana and colleagues from Heidelberg University report in eLIFE a CRISPR/Cas9 based methodology that simultaneously decreases donor DNA multimerization and increases HDR single-copy integration. A common obstacle that CRISPR/Cas HDR mediated genome editing faces is the accurate and […]
New SpCas9 With Expanded Target Range
Nishimasu et al. (2018) recently reported in Science on their finds related to a new SpCas9 modification referred as SpCas9-NG that has an expanded target range. The authors directionally mutated the wild-type SpCas9 enzyme to achieve its relaxed preferences to the […]
Cas9 Gene Drive, Sex-Conversion and Evolved Resistance
In a recent manuscript, KaramiNejadRanjbar et al. demonstrate the development of a proof of principle Cas9-based suppression gene drive in D. melanogaster that can be applied to pest insects, and discuss the implications of resistance allele formation for practical use […]
Y-chromosome transgenics using CRISPR/Cas9
Buchman et al (2018) from Tata Institute of California University reported in Insect Molecular Biology the use of CRISPR/Cas9-HDR system in order to insert transgenes to specific sites of the Drosophila melanogaster Y chromosome. In addition they investigated its usage […]
Switchable Genome Editing
A recent manuscript by Suzuki et al (2018) demonstrates an effective method to control the expression of CRISPR Cas9 based molecular components with a small molecule. Existing methods for controlling Cas9 suffer from high background activity and/or use compounds […]
An Enhanced CRISPR Repressor
Nan Cher Yeo and colleagues in the Church lab report in Nature Methods the identification of a combination of repressor domains which increases gene repression and could be widely useful. The CRISPR-Cas system is widely used as a powerful gene […]
Mitigating Resistance to Gene Drive
A recent manuscript posted on bioRiv by Hammond et al (2018) report exciting improvements to CRISPR-based suppression gene drives in Anopheles gambiae, with the development of new CRISPR designs that reduce selection of resistance and results in much higher suppression […]
ReMOT control delivery technology holds promise for CRISPR-Cas9 activity independent of embryonic injection
A new report in Nature Communications by Chaverra-Rodriguez et al. details a novel Cas9-delivery approach for insect genetic modification. In their work, Chaverra-Rodriguez et al introduce a technology they term “ReMOT control”, or Receptor-Mediated Ovary Transduction of Cargo. In their […]
Target Site Accessibility and Development of Better Guide RNA Design Tools For CRISPR/Cas9
A recent paper by the Lee et al (2018) has shown that the present CRISPR/Cas9 design tools do not accurately predict the activity of single-gene targeting gRNA sequences. They also report that the non sequence based features like DNA accessibility […]
Comparing Invasiveness of Self-Limiting Gene Drive Systems
A recent modelling study by Dhole et al. in Evolutionary Applications has thus sought to compare the invasiveness of three gene drives previously described as either spatially or temporally self-limiting. Recent discussions on gene drives have begun to shift away […]
Efficient CRISPR/Cas9 Knockout of vermillion in Helicoverpa zea (Boddie)
Perera et al. (2018) recently used CRISPR/Cas gene editing in Helicoverpa zea, a lepidopteran pest of many crops. The authors showed positive correlations between different concentrations of ribonucleoprotein (RNP) complexes and Cas9 protein with mutation percentage of the vermillion […]
Reducing resistance allele formation in CRISPR gene drive
In a recent PNAS article, Champer et al. report variable rates of gene drive-based resistance allele formation. Gene drives have shown early promise as the next big thing in controlling insects both for the benefit of agricultural production and reducing […]
Precise and Efficient Nucleotide Substitution Via Noncanonical Homology-directed Repair.
In a recent paper in Genome Research Nakajima et al. 2018 propose an efficient gene editing strategy by nucleotide substitution that overcomes three general problems associated with current methods. First, indels caused by the traditional Cas9-induced Double Strand Breaks (DSB), […]
Genome Editing without Double-Stranded DNA Breaks
Komor et al. reviewed two strategies for genome editing that do not rely on the Double Strand Breaks (DSB) in a recent issue ACS Chemical Biology. The authors specifically address oligonucleotide-directed mutagenesis and base pairing. Possibly the most harmful type […]
NLS-Fusions to Cas9 Can Improve Editing
In a recent study using zebrafish published in G3: Genes, Genomes, Genetics, Hu et al. (2018) conducted a comprehensive analysis of gene editing efficiency between Cas9 proteins and Cas9 mRNA, each fused with a nuclear-localization signal (NLS) in a different […]
Gene Drive in a Major Agricultural Pest
In a recent report in PNAS, Buchman et al. (2018) describe the construction and development of a MEDEA-based gene drive system in the crop pest, Drosophila suzukii. Buchman et al revisited the MEDEA-based gene drive approach developed in D. melanogaster […]
Optimizing Gene Drive Performance
Roggenkamp et al 2018, in a recent publication in G3:Genes, Genomes, Genetics provide an in-depth analysis of potential ways that gene drive mechanisms can be regulated by targeting four conserved elements which would be found in any CRISPR/Cas9 based gene […]
Forward Genetic Analysis of Mosquito/Parasite Interactions
Dong et al. (2018) in a recent paper in PLOS Pathogens describe the results of the genetic inactivation of a broad-spectrum agonist of the malaria parasite, FREP1 (fibrinogen-related protein 1, member of an immune and pattern recognition gene family called […]
Gene Drives and the Potential Benefits of CRISPR Technology
Marshall and Akbari review a range of different proposed gene drive systems and discuss ways in which CRISPR may be useful in engineering them in a recent issue of ACS Chemical Biology. In the years since translocations were first suggested […]
The Latest on CRISPR/Cas9: A Collection of Papers
A recent issue of ACS Chemical Biology includes some 25 articles on various aspects of CRISPR/Cas systems – mechanisms and applications. While not all of the articles will be of direct interest to insect biologists, many are and anyone working […]
Mosaic Analysis
The Recent review by Germani et al., on “Mosaic Analysis in Drosophila”, which appeared in January edition of Flybook of Genetics (http://www.genetics.org/content/208/2/473) discusses mosaicism, one of the finest and sophisticated techniques, which is not only well-utilized and established in Drosophila model […]
Redkmer – a computational tool to identify X-linked targets for CRISPR-based sex distortion in insects
In the inaugural volume of The CRISPR Journal, Philippos Papathanos and Nikolai Windbichler describe a computational pipeline they call redkmer, which identifies repetitive CRIPSR target sites for X-shredding systems in insects. Back in 2007, Windbichler et al. found that the […]
Standard Operating Procedures for Gene Drive in Mosquitoes and Beyond
In the January 2018 issue of Pathogens and Global Health Adelman et al. seek to establish standard operating procedures (SOPs) for gene drive research in arthropods and ask fundamental questions as to why these procedures are necessary. At a time […]
A Protocol for the Highly Efficient Generation of Stable Mosquito Knockout Lines Using CRISPR/Cas9
In their recent paper in G3: Genes|Genomes|Genetics, Li et al. describe a highly efficient protocol for the generation of stable knockout lines using CRISPR/Cas9. The ability of effectively targeting and disrupting gene function is essential for gene functional analysis studies […]
Socio-Technical Considerations of Gene Drive: Getting From Here to There
The Journal of Responsible Innovation has published a special issue entitled: Roadmap to Gene Drives: Research and Governance Needs in Social, Political, and Ecological Context. This is a collection of 13 papers and is the outcome of workshop held in […]
Recreating tra-2 Alleles in D. suzukii
Li and Handler (2017) have recently reported using CRISPR/Cas9 to make a temperature-sensitive transformer-2 (tra-2) allele in the pest species Drosophila suzukii by mutating the nucleotides orthologous to the Drosophila melanogaster tra-2ts2 allele. This study demonstrates an important proof of […]
Advances in CRISPR/Cas9 Genome Editing in Drosophila Lead the Way for Applications in Other Insects.
In the January edition of FlyBook (Genetics) Bier et al. review the recent advances in CRISPR/Cas technologies applied to the fruit fly, delving into the details of established and cutting-edge methods and their potential applications to future studies in this […]
Scientists Organize to Influence the Fate of Genetic Technologies Research
Synthetic gene drive research is becoming a lightening rod for attention and recently that attention has focused on the efforts of scientist working in or around the field of synthetic gene drive research to voice their opinions at the Convention […]
Principles of Synthetic Gene Drive Research
In a recent issue of Science, supporters and sponsors of synthetic gene drive research respond to the National Academy of Sciences Engineering and Medicine 2016 report on the research and development of synthetic gene drives. . .Some of the sponsors and […]
The CRISPR Journal
In early 2018 Mary Ann Liebert, Inc. will begin publishing The CRISPR Journal. Their publishing interests are broad and manuscripts are being solicited for inclusion in their inaugural issue in early 2018. This is from the journal’s website: “The rampant […]
Anti-CRISPR Proteins
The intense selection pressure that CRISPR/Cas systems placed on viruses has resulted in the evolution of a number of anit-CRISPR proteins meant to silence this bacterial ‘immune system’. Dong et al (2017) report on a database they constructed that aggregates […]
Somatic Mutagenesis as a Powerful Functional Genomics Tool in Insects
Two recent papers published in the Proceedings of the National Academy of Sciences are notable examples of using the CRISPR/Cas9 system to create somatic mutants to probe gene function. Mazo-Varga et. al (2017) and Zhang et al. (2017). Both studies explore […]
Will CRISPR/Cas9 Gene Drives Spread and How Could They Be Prevented From Doing So?
In an interesting paper published in PNAS, Tanaka, Stone and Nelson (2017) explore the potential for spatial spread of CRISPR/Cas9 gene drives. The authors examine a continuous time, spatial generalisation of the CRISPR/Cas9 population genetics model published by Unckless et […]
More Genetically Modified Ants!
Last month, Yan et al. published their work using CRISPR/Cas9 in ants in the journal, Cell. This research was published alongside Trible et al., which was discussed here previously, based on a preprint version available at BioR𝛘iv. Both studies analyzed […]
Strategies for Driving Out Invasive Species
Prowse et al. (2017) present their modelling work in the Proceedings of The Royal Society B on various different CRISPR/Cas9-based gene drives aimed at suppressing populations of exotic vertebrate species on islands (focusing on mice (Mus musculus)). The issues addressed […]
Gene Drive for Agriculture – Alternative Opinions
Not long ago EMBO Reports published the opinions of Courtier-Orgogoza et. al. in which they discuss briefly the potential of synthetic gene drive systems to be used in insects of agricultural importance. Among other things that called for robust discussion […]
Guidance Sought by Gene Drive Researchers
A letter to the editor of Nature Biotechnology authored by several individuals from some 20 organizations and including scientists, regulatory and policy experts make a case for the need to update existing research guidance documents related to research, development and […]
High Knock in/out Rates in Aedes aegypti Expressing Cas9 from a Transgene.
Li et al have published some interesting data on the preprint server bioRΧiv regarding the creation and use of Aedes aegypti lines expressing an SpCas9 transgene in the germline. The report substantial improvements in gRNA-mediated mutagenesis rates. Cas9 mediated […]
Cas9 Expression Patterns Impact on Resistance Evolution in Different Genetic Backgrounds
Gene drive systems are sensitive to the evolution of resistance in the form of polymorphisms in the guide RNA’s target DNA sequence. Champer et al. (2017) have recently reported on their exploration of how Cas9 expression patterns as well as […]
Effects of Life History Traits on Gene Drive.
Modeling gene drive, Drury et al. describe how the presence of pre-existing Cas9 resistance alleles within populations and non-random mating can have significant effects on the extent to which a driving element will successfully increase in frequency. Advances in the […]
Dynamics of Gene Drive Resistance – Lab Cage Trials
In their current pre-publication Hammond et al investigate the emergence of resistance to a homing-based gene drive designed to disrupt a gene essential for female Anopheles gambiae fertility. In recent years, there has been much theoretical design and modeling work […]
Driving Antipathogenic Effector Genes into Vector Populations
Beaghton and colleagues have reported in Genetics their modeling efforts where they examine drive systems intended to modify standing populations of vectors by introgressing genes that affect vector competency. Genetic systems for rapidly increasing the frequency of desired alleles or […]
Trojan Female Technique: Mitochondria-based population suppression.
The ‘Trojan Female Technique” is a genetics-based population suppression strategy. Wolff et al.(2017) report results of empirical tests of the idea using Drosophila melanogaster as a model system and show net population suppression. Genetics-based pest control strategies are becoming increasingly […]
Unintended CRISPR/Cas9 Mutagenesis
In a short correspondence to Nature Methods Schaefer et al. (2017) have reported some results that are echoing around the gene editing community and insect gene editors should be aware of their findings. Off-target effects and second-site mutations have been […]
Should we use gene drive for pest control
“Agricultural pest control with CRISPR-based gene drive: time for public debate ” is a short opinion piece in EMBO Reports by Courtier-Orgogozo et al. that makes a case for increased public discussions regarding the regulation and use of so-called ‘gene […]
Easi-CRISPR: Long ssDNA donors greatly increase efficiency of HR
Quadros et al. (2017) show that injecting long single stranded DNA donors and Cas9-containing ribonucleoproteins results in improved knock-in efficiency in mouse embryos and there is no reason why they would not have the same effect in insect embryos. Injecting […]
RAD51 and Valporic Acid Enhance Cas9-induced HR
Takayama et al (2017) describe in Nucleic Acids Research how they increased the frequency of biallelic editing of genes in human ES/iPS cells and their findings may be useful to insect biologists. The efficiency of Cas9-mediated homologous recombination (HR) can […]
Cas9 Mutagenesis in Houseflies Confirms Male Determining Factor Identification
Sharma et al. (2107) report in a recent issue of Science the identification of a male determining factor in the housefly Musca domestica and provide convincing reverse genetic confirmatory evidence using dsRNA-mediated gene silencing and Cas9-mediated mutagenesis. Alternative splicing of […]
DNA-free genome editing in the agricultural pest Ceratitis capitata
The genome editing revolution creates new opportunities to improve pest management strategies, yet biosafety concerns over off-target effects and the use of foreign DNA present roadblocks to the development of modified organisms destined for field use. In a recent addition […]
Germline Modification of Ants
Trible et al 2017 reported in BioRχiv the first study of genetically modified ants using genome editing. Clonal raider ants (Ooceraea biroi) are potential genetic models to study social complexity and social evolution. It is known that social insects such as […]
Cre/lox-Recombinase-Mediated Cassette Exchange in Aedes aegypti
Häcker et al 2017 reports the use of Cre/lox Recombination-Mediated Cassette Exchange (RMCE) in Aedes aegypti providing a pathway for interrogating specific genomic locations for the use of transgenic constructs in future genetic control methods. Häcker et al 2017 first generated […]
Papilio machaon genome editing: Krazy Glue + microchisel
Editing butterfly genomes, like other insects, can be challenging because of the particulars of the biology of the insects. Li et al. (2017) describe some of the details of genome editing Papilio machaon in a letter to Insect Science and […]
Short-Half Life Cas9 Improves Mutagenesis
Reducing the half-life of Cas9 has some positive and unexpected impacts on the efficiency of mutatgenesis, according Tu et al (2017) in a paper they just published in Scientific Reports. Tu et al were primarily interested in applying Cas9 mutagenesis […]
Rapid Generation of Hypomorphic Mutations
While CRISPR/Cas9 has made null mutations easier to obtained, Arthur et al (2017) describe a general method for creating hypomorphic mutations that should further enable our abilities to tune levels of gene expression to suite particular needs. Eliminating a gene’s […]
Nasonia Genome Editing
Scientists in the Ferree and Akbari laboratories developed a CRISPR/Cas9 protocol for germline editing in Nasonia vitripennis, adding an essential tool for the study of this important insect. They report their results in bioArxiv (Li et. al 2017). With its […]
Modeling Gene Drives in Africa – Seasonal, Spatial and Epidemiological Considerations
Gene drive as a tool for fighting malaria has received much attention because of promising results in the laboratory. Moving these technologies to the field is complicated and Eckoff et al (2016) in the Proceedings of the National Academy of […]
Cas9 Inhibitory Proteins for Modulating Editing Activity
Pawluk et al (2106) describe anti-CRISPR Cas9 proteins that can be used to regulate the activity of type II Cas9s and show that they function in human cells. These inhibitory proteins are likely to become valuable tools for modulating temporal […]
DNA-guided Genome Editing Proves Unrepeatable
Earlier claims this year by Gao et al. (2016) that a new gene editing system unrelated to Cas9 and based on a DNA-guided Argonaute protein from the haloalkaliphilic archaebacterium Natronobacterium gregoryi, NgAgo, appear now to be unrepeatable despite a sizable […]
Call for Sweeping Moratorium on Gene Drive Research and Application at UN Biodiversity Conference
The UN Convention on Biological Diversity is currently holding their Biodiversity Conference in Cancun, Mexico. Monday 160 organizations from six continents released a statement calling for a global moratorium on all work on gene drives, including laboratory research. The […]
Orco Knockout Mutants of Locusta migratoria
A recent report by Li et al. demonstrates that by using a state-of-the-art CRISPR/Cas9 genome editing, it is now possible to study the function of Orco in the locust, Locusta migratoria . The gene odorant receptor co-receptor (Orco) encodes […]
Dominant Maternal Effects Caused by CRISPR/Cas9
Expressing Cas9 and guide RNAs from transgenes integrated into the genome of organisms typically leads to higher rates of mutagenesis of the target gene compared to when those components are provided transiently by microinjection, for example. Lin and Potter describe […]
Precise Bombyx mori Genome Editing
Takasu et al. test the efficiency of long dsDNA plasmids and short ssDNA oligonucleotides for precise genome editing in silkworms. This study follows their previous experiments aimed at inserting GFP into the genome of Bombyx mori using zinc finger nucleases; […]
FlyRNAi.org – Bigger and Better
FlyRNAi.org was initially a website that supported genome-wide RNAi screening in Drosophila cultured cells, beginning in 2003, but has evolved into a functional genomics platform that has much to offer to researchers in communities focused on Drosophila and beyond. Hu […]
Guide-RNA Engineering – Enhancing Activity and Function
The functionality of Cas9 is determined in part by the guide RNA with which it is associated. Norwak et al.(2016) review briefly how guide RNAs can be modified to enhance their function and to functionalize them in novel ways. Norwak […]
Editing Methylation States
Editing DNA nucleotide sequences has become common place but using the programmability of RNA-guided endonucleases to affect other changes to the genome are less developed although their potential for making such changes is quite high. Liu et al. report on […]
Unlimited DNA Editing With Structure-Guided Nucleases
Structure-guided nucleases such as flap endonuclease-1 (FEN-1) bind to particular DNA structures; in the case of FEN-1 the 3′ flap structure arising during lagging strand DNA synthesis and the removal/displacement of RNA primers associated with Okazaki fragments. Xu et al. […]
In Vivo CRISPR Multiplexing In Insects
Producing multiple sgRNAs from a single RNA polymerase II (Pol II) – transcript would enable multiplexed mutagenesis and gene editing as well as the possibility of precisely regulating the expression of sgRNAs in specific temporal and spatial patterns. Port and […]
CRISPR-SD, a programmable Cas9 approach to generating X-shredder lines in insects with XY mating systems
In a recent paper in Scientific Reports, Galizi, Hammond, et al report on the use of CRISPR-Cas9 to develop a sex-distortion system in Anopheles gambiae, which they termed CRISPRSD. Earlier, the group had demonstrated that transgenic An. gambaie expressing the […]
Chromatin’s Impact on Editing
Chen et al (2016) report in Nucleic Acids Research results of a systematic analysis of chromatin conformation on various genome editing tools and observe effects that are not equally shared among the most popular systems. The authors focus on two […]
Gene Editing Spodoptera
Three papers recently described successful CRISPR/Cas9 mutagenesis in two species of Spodoptera. Spodoptera litura and S. littoralis. Bi et al (2016) use Cas9 to create somatic mutations in S. litura in developmental gene abdominal-A, abd-A, and analyzed the resulting larvae […]
Genetic Sources and Consequences of Resistance Alleles to Gene Drive
CRISPR/Cas9 gene drive has garnered much attention for its potential to be used as a biological control tool to mitigate or even eliminate unwanted invasive species but the topic of resistance development in target populations has tend to not be […]
CHOPCHOP v2: Improved online CRISPR/Cas9 target prediction tool
In a paper published in Nucleic Acids Research, Labun et al. (2016), the developers of CHOPCHOP, report on their updated version: CHOPCHOPv2 . The first version of CHOPCHOP was released by a team of researchers from Harvard University a little […]
U.S. National Academy of Science’s Report on Gene Drive Research
The United States National Academy of Sciences published a report entitled Gene Drives on the Horizon: Advancing Science, Navigating Uncertainty, and Aligning Research with Public Values recently. The report was written by the Committee on Gene Drive Research in Non-Human […]
A DNA-Guided Gene Editing System
In Nature Biotechnology Gao et al. (2016) describe a new gene editing system unrelated to Cas9 and based on a DNA-guided Argonaute protein with some interesting characteristics and the potential to become another useful tool. The Cas9 gene editing system […]
In Vivo CRISPR Multiplexing in Insects
Port et al 2016 have posted in bioRχiv a manuscript describing a system and set of vectors for producing multiple CRISPR gRNAs from a single RNA polymerase II or III transcript in Drosophila, resources that will enhance the CRISPR toolbox […]
Brake System for Gene Drive
Wu et al (2016) in February’s issue of Nature describe how they have developed a brake for a Cas9 gene drive system. This is an interesting correspondence reflecting how the field has shifted from proof-of-principle of Cas9 gene drive systems […]
Phosphorothioate oligomers increase knock-in frequency and precision in TALEN- and Cas9-mediated genome editing
In a recent paper by Renaud et al. (2016), the authors investigate methods to increase genome editing knock-in efficiency by modifying the chemistry of the donor oligomers to be resistant to nuclease activity. The modifications they tested included locked nucleic […]
Insect Gene Drives – Reviewed and Compared
Champer et al have published a review on the topic of engineered gene drives to manipulate wild populations in a recent Nature Reviews Genetics. Recent attention on Cas9-based gene drive systems has raised the awareness of these types of genetic […]
Monarch Reverse Genetics
Markert et al. just reported their effective use Cas9 and TALENs to knock out cryptochrome 2 and clock in Danaus plexippus using some strategies and approaches that are generally useful to insect scientists and much more efficient than earlier reports […]
Animal and Insect Transgenics – Parallels
James Murray and Elizabeth Maga recently recounted briefly the development and evolution of animal transgenic technologies in a recent issue of Transgenic Research and the similarities and differences between the development and evolution of insect transgenic technologies, which were ongoing […]
Gene Editing in an Invasive Species
Li and Scott report their successful use of Cas9 to knockout two genes in Drosophila suzukii in a recent issue of Biochemical and Biophysical Research Communications. Drosophila suzukii or the spotted wing Drosophila although a native of Asia has recently […]
Enhancing Homology Repair-Mediated Genome Editing
In a recent paper in Nature Biotechnology, Richardson et al (2016) report on the interaction of target-site bound Cas9 and dCas9 on DNA strand accessibility- and have identified an optimized approach to increase homologous recombination (HR) mediated repair. They found […]
Another High-Fidelity SpCas9 Nuclease
Kleinstiver et al. describe in Nature their successful efforts to create high-fidelity SpCas9 nuclease variants that have high on-target efficiencies and low or no off-target effects. The results are SpCas9-HF1. When using Cas9 or other engineered nuclease systems one hopes […]
An Overview of Gene Drive
Gantz and Bier reflect on and consider the implications of Cas9-based genome editing on genetics in a recent Bioessays that is worth reading if you are trying to keep up on this rapidly evolving part of the genetics ecosystem. Not […]
Homology independent CRISPR Knock-ins
Lackner et al., in an effort to eliminate the need to synthesize multiple, unique homology based templates for Cas9-mediated homologous recombination, report the use of a universal donor template that will insert tags into potentially any genomic site in the human […]
Genetic Manipulation of Insect Cell Lines
Two papers in the December issue of Genetics, Cherbas et al. 2015 and Manivannan et al. 2015, describe an improved method for making Drosophila cell lines with single, targeted mutations. Both works build upon existing recombination-mediated cassette exchange (RMCE) technology […]
Somatic Editing in Parhyale hawaiensis for Loss-of-Function Analysis
In a recent paper in Current Biology, IGTRCN Participant Arnaud Martin and colleagues use Cas9 gene editing technology to study the development of the crustacean Parhyale hawaiensis. In this study by Martin et al., the specific morphology of serially homologous […]
Gene Drive In Anopheles gambiae
Hammond et al.’s just published paper in Nature Biotechnology is the second paper in a month demonstrating effective CRISPR-based gene drive in a mosquito vector of malaria. In this case the main malaria vector in Africa, Anopheles gambiae. Gene drive […]
Modified Cas9 With Reduced Off-Target Activity – Very Useful
Slaymaker et al (2015) describe in Science their successful efforts to modify Cas9 so that it retains its on-target activity but has highly reduced off-target activity. The potential bane of a well-designed and executed gene editing experiment are off-target mutations […]
Cas9-Mediated Gene-Drive In Mosquitoes
Gantz and Bier who demonstrated the fundamental principles of a Cas9-based gene drive system using Drosophila melanogaster in a publication earlier this year have recreated that system in the mosquito Anopheles stephensi with the help of collaborators. Gantz et al. […]
Dopamine receptor knock-outs in crickets
Awata et al. (2015) have used Cas9 to knock-out Dop1 in Gryllus bimaculatus to assess the role of dopamine neurons in mediating reward and punishment. Functional genomics technologies like transposons and first and second generation gene-editing technologies have been available […]
Sawfly and Ladybird Beetle Gene Editing
Hatakeyama et al (2015) have successfully used gene editing technology in Athalia rosae (sawfly) and Harmonia axyridis (ladybird beetle), enlarging the range of insects in which these technologies have been applied. These results are reported in Insect Molecular Biology. Hatakeyama […]
Gene Drive: History and Perspective
Nature Biotechnology has published a News Feature by Laura DeFranceso that tours the intellectual history of gene drive as a tool for manipulating ‘pest’ populations beginning in the 1960’s and various perspectives from some who are working with these systems […]
A New Editing System
As described in a just-published paper in Cell by Zetsche et al (2015), Cpf1 is an endonuclease that is part of a CRISPR system somewhat similar to the Streptococcus pyogenes Cas9-based system that now is the tool of choice for […]
CRISPR/Cas9 Gene Editing in Papilionid Butterflies
Li et al (2015) recently published an exciting paper on butterflies genomics and functional genomics . Li et al have worked towards producing high quality genomes for extremely heterozygous and closely related butterflies P. xuthus and P. machaon. Further, they […]
Cas9-VPR: Activator, Repressor and Nuclease All in One Cell
In a recent communication in Nature Methods Kiani et al investigated the use of Cas9-VPR to serve simultaneously as an DNA endonuclease and a gene activator/repressor. That native Cas9 could be used for both purposes at the same time hinged […]
CASFISH: In Situ Hybridization on Fixed Cells
Deng et al. in an Early Edition of PNAS have reported on the application of dCas9/sgRNA complexes as gene localization probes in situ Fluorescence In Situ Hybridization (FISH) is an important technology for localizing genes and regions of genomes but […]
CRISPRscan – A Tool Aiding Selection of CRISPR-Cas9 gRNAs
Just published online in Nature Methods, Moreno-Mateos et al report testing the activities of >1200 gRNAs in zebrafish embryos and used the results to create an algorithm for predicting gRNA efficacy. The advent of the CRISPR-Cas9 system has revolutionized genome […]
Editing Tribolium’s Genome
Gilles et al (2015) recently published a paper in Development describing CRISPR-mediated gene targeting and gene replacement in the flour beetle Tribolium castaneum. The authors started with the Tribolium line Pig-19, which strongly expresses enhanced green fluorescent protein (EGFP) in […]
Mis-Expressing Insect Genes Using Cas9
Lin et al. have now published a paper that demonstrates overexpression of endogenous genes in Drosophila melanogaster using the CRISPR/Cas system. Over- and miss-expression of genes can be a powerful tool to elucidate gene function and is commonly achieved through […]
Cas9 Gene Drive & Classical Biological Control
Webber et al have published an Opinion essay in PNAS concerning the growing interest in using engineered endonucleases (Cas9 in particular) to create gene drive systems to combat invasive species. Homing endonucleases are well known and well studied site-specific endonucleases […]
Optimized TALEN Application and Screening in Drosophila melanogaster
Lee et al 2015 provide a detailed methodology for gene editing using TALENs in Drosophila melanogaster. While TALEN use is relatively well established in D. melanogaster, fine tuning the injection concentrations of designer nucleases allows for the maximum efficiency in […]
Cas9-Drive Behaves as Expected
Unckless et al (2015) have done some simple modeling of Cas9-based drive systems and show that they behave as expected of ‘gene drive’ systems in general and Akbari et al (2015) make some suggestions concerning safeguards to consider before one […]
Avoiding TALEN Lethality And Getting High Mutation Rates
Naitou et al (2015) have recently published a paper describing their use of TALENs in Daphnia magna and make some interesting observations that could be relevant to those planning on targeted mutagenesis experiments in insects. The water flea Daphnia is […]
Reversible Light-Regulated Cas9
Yuta Nihongaki and colleagues have recently published a paper in Chemistry & Biology where they have utilized a photoinducible dimerization system named Magnets to reversibly regulate Cas9 activity offering researchers a unique system for regulating gene expression. CRISPR-Cas9 system has become […]
More Aedes Editing – More Insights
In a recent PLoS One paper, Dong et al. (2015) describe their successful attempts to edit the genome of Aedes aegypti using CRISPR/Cas9. As proof of principle, they aimed to excise a portion of a transgene that codes for enhanced […]
Modified Guide-RNAs Enhance CRISPR Editing
Hendel et al. (2015) have shown in Nature Biotechnology that certain chemical modifications to guide RNAs can enhance mutagenesis frequencies of the CRISPR/Cas9 system. Clearly, these types of enhancements, should they pan out to be effective beyond human cells, could […]
Microinjection independent genome engineering
Takahashi et al. (2105) describe a microinjection independent genome engineering method for mice that is an interesting innovation that seeks to address technical challenges associated with the creation of transgenic and genetically modified mice that have good analogs in insect […]
Altered PAM Specificities Improves SpCas9 Editing Platform
Kleinstever et al (2015) have just published in Nature the results of their efforts to systematically redesign the PAM specificity of Streptococcus pyogenes Cas9 (SpCAS9)and their results relieve a notable constraint on the S. pyogenes CRISPR/Cas9 platform. As powerful as […]
High throughput screen of genome-wide CRISPR library
Bassett et al (2015) just published a paper in Journal of Genetics & Genomics on constructing a genome-wide CRISPR library for high throughput screening in Drosophila cells. This will be a valuable resource for systematically studying gene functions in fly […]
Reverse Genetic Screening Using CRISPR
Shah et al (2015) just published a paper in Nature Methods on their efforts to use CRISPR/Cas9 as a rapid reverse genetic screening tool. Although they are working with zebrafish, this is a system that has enough similarities to insects […]
Powerful Genetic and Genomic Toolbox of Nasonia Wasps
Jeremy Lynch (2015) has published a nice overview of the genetic/genomic tools currently available for Nasonia species. Insects in the order of Hymenoptera are emerging as new genetic and genomic tools for evolutionary biology, development, and behavioral ecology. Among over […]
Electroporating the CRISPR/Cas9 System
Qin et al. (2015) describe their successful efforts to use electroporation technology to introduce CRISPR/Cas9 system components into mouse embryos and while not exactly insect biology their motivations for pursuing this approach are shared by many insect biologists with an interest […]
CRISPR with Independent Transgenes or Mutagenic Chain Reaction for Creating Homozygotes in Lab?
In fall of 2014, two publications came from George Church’s group (Esvelt et al., 2014; Oye et al., 2014) postulating on the potential for the CRISPR/Cas9 system to be used as an autonomous gene drive for various pest management goals. […]
Cas9 Against RNA Targets In Vivo
Price et al (2015) have recently published a paper in PNAS in which they report that Cas9 can be used as a RNA-targeting system. Clustered, regularly interspersed, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems are a diverse collection of adaptive immune […]
Scr7 Improves Knock-In Rates
Two recent papers, Maruyama et al (2015) and Chu et al. (2015), describe the use of a small molecule inhibitor of non-homologous end-joining known as Scr7 to increase rates of homology directed repair (HDR) triggered by double stranded breaks in DNA by […]
Parallels Between CRISPR-Cas9 and RNAi
CRISPR-Cas9 based genome engineering is gaining ground from its first publication in 2012 (Jinek et al, 2012). The technique is being tested in different model and non-model organisms. The number of publications is increasing exponentially for the last couple of […]
More Mosquito Genome Editing – With Instructions
Kistler et al. have just published the results of their extensive use of CRISPR/Cas9-based knock-out and knock-in technology in Aedes aegypti in Cell Reports and anyone considering using these technologies in insects will find this paper of interest. The authors […]
Clever CRISPR Multiplexing In Vivo
Xie et al, in a recent paper in PNAS, report that multiplex gene editing with CRISPR-Cas9 can be accomplished by producing multiple sgRNAs from a single transcript that is processed by the plant’s endogenous tRNA-processing pathway. One of the elegant […]
CRISPR-Cas9 mediated “Mutagenic Chain Reaction”
A recent paper in Science by Gantz and Bier (2015) investigated the potential of utilizing the CRISPR-Cas9 system as, essentially, a programmable homing endonuclease to generate homozygous loss-of-function (LOF) mutants. Advances in genome editing have allowed for the directed generation of […]
Repurposing CRISPR/Cas9 System to Reprogram Gene Expression
Zalatan et al. in Cell reported a system to generate synthetic transcriptional programs in yeast and human cells based on modified CRISPR-associated RNA scaffolds that could also be used in insects. To fight against invading viruses and plasmids, bacteria developed […]
Exchangeable Cassettes Controlled by Endogenous Genes
Two recent publications, Gnerer et al. and Diao et al., address the issue of recreating the fine transcriptional patterns of endogenous genes with binary expression systems in insects. Binary expression systems are extremely powerful tools for controlled gene manipulation. The […]
Pulling Recent CRISPR/Cas9 Work in Drosophila Together
Xu et al. review the recent history of using CRISPR/Cas9-based technologies in Drosophila melanogaster. Within the last two years there have been numerous publications describing the use of CRISPR/Cas9-based technologies in Drosophila melanogaster. These last two years have been a time […]
Chemically Inducible Cas9 System
The development of a controllable Cas9 system that is chemically inducible would greatly enhance the experimental utility of the CRISPR Cas9 system. In the CRISPR Cas9 system, a guide RNA directs the Cas9 endonuclease to a specific region within the […]
Off- & On-Target Editing Evaluation
Off-target effects and overall efficacy are major considerations in the design of tools for genome editing. In a recent issue of Trends in Biotechnology, Hendel et al review methods used to assess both the efficacy of genome editing technologies as […]
Cascade: A new CRISPR tool for gene silencing
In recent years, the CRISPR-Cas system has gone from an unknown anomaly in bacteria to a technology that is quickly revolutionizing genetics. Found in approximately half of all bacteria, the CRISPR-Cas system provides protection from invading viruses and mobile elements. […]
Step-By-Step CRISPR-Cas9 for Flies
These days it seems like new papers and reviews relevant to CRISPR-Cas9 modification pop up weekly, if not daily, making it hard to keep up. I am admittedly biased—the authors are close colleagues—but that said, a recent chapter in Methods […]
Comparing TALE- and dCas9-Based Designer Transcription Factors
Genome editing with programmable nucleases using transcription activator-like effector nucleases (TALENs) or RNA-guided clustered regularly interspersed short palindromic repeat (CRISPR) associated protein (Cas9) have become popular and important technologies. These systems can be modified to carry other functional protein domains […]
IGTRCN Trainees – Fall 2014!
The IGTRCN is working to diffuse knowledge and technology from those who ‘know’ and ‘have’ to those who do not but need to. The Peer-to-Peer Training Fellowships are intended to facilitate the process by providing modest resources to encourage interactions. […]
Delivering gene-editing proteins in vivo using a new strategy.
Delivering genetic technologies to appropriate cells at the appropriate times is, for many insect systems, the biggest obstacle to the widespread use of these technologies. Microinjecting developing embryos with DNA, RNA or protein remains the dominant mode of delivery in […]
Plasmids for Rapid and Versatile Genome Editing
As is the case for many other organisms, CRISPR has rapidly become the method of choice for targeted genome modification in insects. Much of the method development has been taking place in the model organism Drosophila melanogaster, where, in little […]
CRISPR/Cas9-Mediated Conditional Mutagenesis
A recent paper by Xue et al (2014) has described a novel use of the Gal4 system in conjunction with CRISPR/Cas9 to induce tissue specific gene silencing that may prove be an alternative to more established approaches that utilize RNA […]
Hi, Tide.
The use of sequence-targeting nucleases (TALENs, CRISPR-Cas9, zinc finger nucleases) to introduce small insertions/deletions into the genome of an organism is an effective means of generating specific mutants. Although these technologies are effective, they still require testing and optimization to […]
CRISPR/Cas9 for RNA Cleavage
The CRISPR/Cas9 system is quickly becoming the most versatile method for modifying DNA targets, but researchers seeking equivalent control against RNA transcripts have been left behind. In a new paper by O’Connell et. al, (2014) the authors describe a clever […]
Guide RNA Design & Off-Target Analysis: A Tool for Non-Model Organisms
Modifying genomes using the CRISPR/Cas9 system is becoming popular. Good prior planning can save you much time and effort, and could mean the difference between success and failure. Designing your guide RNAs is your first major challenge. There are multiple […]
“CHOPCHOP”ing with CRISPRs and TALENs: A New Web Resource for Genome Editing Design
With the growing influence and applicability of genome editing technologies, TALENs and more recently CRISPRs, offer genetic capabilities to an ever growing list of organisms. In turn, as more and more researchers become interested in using these technologies, a common […]
Purified Cas9 Protein for Insect Genome Modification with CRISPRs
The interest in CRISPR/Cas9 genome-editing and -modification technologies is quite extensive and there has been a flood of data dealing with optimizing efficiency among other things. In insect systems there are a number […]
Insect Genetic Technologies Service Providers
Insect Genetic Technology Service Providers If you would like to add to this list use the “SUBMISSIONS” button and select “Knowledgebase” For insects other than Drosophila melanogaster Non-Commercial The Institute for Bioscience and Biotechnology Insect Transformation Facility, University of Maryland […]
CRISPR/Cas Web Resources
Web Resources for design and planning of CRISPR/Cas9 studies from Bassett and Liu (2014) CRISPR/Cas9 and Genome Editing in Drosophila. J. Genetics and Genomics 41, 7-19 doi.10.1016/j.gg.2013.12.004 Name Link Brief description Reference Drosophila CRISPR web resources OxfCRISPR (Liu Lab) http://www.oxfcrispr.org/ […]
CRISPR In Drosophila
Comparison of CRISPR genome engineering studies in Drosophila from Bassett and Liu (2014) CRISPR/Cas9 and Genome Editing in Drosophila. J. Genetics and Genomics 41, 7-19 doi.10.1016/j.gg.2013.12.004 Reference Gratz et al., 2013 Bassett et al., 2013 Yu et al., 2013 Kondo and Ueda, 2013 […]
Expanding the CRISPR/Cas Tool Box for Drosophila Engineering
The ability to introduce mutations in the genome of organisms has allowed us to gather information on numerous biological mechanisms. However, there are significant drawbacks in the traditional tools used for mutagenesis – lack of specificity that gives rise to […]
Multigene Mutagenesis of Silk Moth with CRISPRs.
Genome editing using CRISPR/Cas9 is changing the way we think about genome modification. The CRISPR system, because it relies on a small user-provide guide RNA (gRNA) to give the Cas9 endonuclease its sequence specificity, is easy to use, relative to, […]
sgRNA Design & Target Specificity
Specificity of the CRISPR/Cas9 system is determined by the 20-nt guiding sequence of the sgRNA and the presence of a protospacer-adjacent motif (PAM) adjacent to the target sequence in the genome. Some mismatch (3-5 nucleotides) at the PAM-distal end of […]
Vasa Cas9 Experession Yields Good Mutation Rates in Drosophila
Xue and colleagues report efficient knock-out and knock-in manipulations of the Drosophila genome with a Cas9 transgenic system. Others have made similar observations. Xue et al. report the germline mutational rate ranging from 14% to 95%. They also demonstrated the […]