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 […]
editing
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 […]
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 […]
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), […]
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 […]
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 […]
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 […]
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 […]
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 […]
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 […]
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 […]
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 […]
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 […]
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 […]
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 […]
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 […]
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 […]
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 […]
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 […]
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 […]
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 […]
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 […]
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 […]
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 […]
Trending to the Synthetic
While relatively few insects are highly accessible to the most sophisticated and advanced genome manipulation and modification technologies one gets the feeling the this is going to change very soon. The technologies available now are beginning to allow for the […]
2015 Technical Course
2015 IGTRCN Short Course on Insect Genetic Technologies: Theory and Practice August 17 – 21, 2015 The Insect Transformation Facility Institute for Bioscience and Biotechnology Research University of Maryland College Park Applications Required (25 seats available) by May 29, 2015 […]
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 […]
BuD: Another Tunable DNA-Binding Domain for Genome Modification
Tunable DNA-binding domains with high specificity remain important tools for constructing genome modifications even with the current popularity of the CRISPR/Cas9 system. Stella at al (2014) describe their analysis of a DNA-binding protein, BurrH, from the symbiotic bacterium Burkholderia rhizoxinica […]
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, […]
Delivering Reprogramed Meganucleases With Lentiviruses- Is this Applicable to “Difficult” Insects?
Delivering biologically relevant nucleic acids and proteins to appropriate cells of multicellular organisms for the purposes of genetically modifying either somatic or germline genomes is a central problem for not only insect biologists but for those working on other organisms […]
Genetically Modified Annelids: Platynerieis dumerilii. Not Unlike Modifying Insects
The bristle worm, Platynerieis dumerilii, is a marine annelid with a biology that makes it of interest because of the development of its nervous system, reproductive cycles that are synchronized with lunar phases and its neuroendocrinology among other things. Even […]
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 […]
Development, Growth & Differentiation Devote Issue to Genome Editing
Genome editing with engineered nucleases is an emerging technology that enables manipulation of targeted genes in many organisms and cell lines. Development, Growth & Differentiation has devoted an entire issue to genome editing. The paper describing the successful use of […]