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1. Pax3/7 regulates neural tube closure and patterning in a non-vertebrate chordate

   https://doi.org/10.3389/fcell.2022.999511  

   Kim, Kwantae; Orvis, Jameson; Stolfi, Alberto (September 2022, Frontiers in Cell and Developmental Biology)

   Pax3/7 factors play numerous roles in the development of the dorsal nervous system of vertebrates. From specifying neural crest at the neural plate borders, to regulating neural tube closure and patterning of the resulting neural tube. However, it is unclear which of these roles are conserved in non-vertebrate chordates. Here we investigate the expression and function of Pax3/7 in the model tunicate Ciona. Pax3/7 is expressed in neural plate border cells during neurulation, and in central nervous system progenitors shortly after neural tube closure. We find that separate cis- regulatory elements control the expression in these two distinct lineages. Using CRISPR/Cas9-mediated mutagenesis, we knocked out Pax3/7 in F0 embryos specifically in these two separate territories. Pax3/7 knockout in the neural plate borders resulted in neural tube closure defects, suggesting an ancient role for Pax3/7 in this chordate-specific process. Furthermore, knocking out Pax3/7 in the neural impaired Motor Ganglion neuron specification, confirming a conserved role for this gene in patterning the neural tube as well. Taken together, these results suggests that key functions of Pax3/7 in neural tube development are evolutionarily ancient, dating back at least to the last common ancestor of vertebrates and tunicates. 

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2. Dual CRISPR‐Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica

   https://doi.org/10.1002/biot.201700590  

   Gao, Difeng; Smith, Spencer; Spagnuolo, Michael; Rodriguez, Gabriel; Blenner, Mark (June 2018, Biotechnology Journal)

   CRISPR‐Cas9 technology has been successfully applied inYarrowia lipolyticafor targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest. The targeted regions for excision vary in length from 0.3 kb up to 3.5 kb and contain both non‐coding and coding regions. The majority of the gene excisions are repaired by perfect nonhomologous end‐joining without indel. Based on this dual cleavage system, two targeted markerless integration methods are developed by providing repair templates. While both strategies are effective, homology mediated end joining (HMEJ) based method are twice as efficient as homology recombination (HR) based method. In both cases, dual cleavage leads to similar or improved gene integration efficiencies compared to gene excision without integration. This dual cleavage strategy will be useful for not only generating more predictable and robust gene knockout, but also for efficient targeted markerless integration, and simultaneous knockout and integration inY. lipolytica. 

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3. Generation and validation of a versatile inducible multiplex CRISPRi system to examine bacterial regulation in the Euprymna-Vibrio fischeri symbiosis

   https://doi.org/10.1007/s00203-025-04354-8  

   Pipes, Brian Lynn; Nishiguchi, Michele Kiyoko (May 2025, Archives of Microbiology)

   Abstract TheVibrio fischeri—Euprymna scolopessymbiosis has become a powerful animal—microbe model system to examine the genetic underpinnings of symbiont development and regulation. Although there has been a number of elegant bacterial genetic technologies developed to examine this symbiosis, there is still a need to develop more sophisticated methodologies to better understand complex regulatory pathways that lie within the association. Therefore, we have developed a suite of CRISPR interference (CRISPRi) vectors for inducible repression of specific V. fischeri genes associated with symbiotic competence. The suite utilizes both Tn7-integrating and shuttle vector plasmids that allow for inducible expression of CRISPRi dCas9 protein along with single-guide RNAs (sgRNA) modules. We validated this CRISPRi tool suite by targeting both exogenous (an introduced mRFP reporter) and endogenous genes (luxCin the bioluminescence producingluxoperon, and flrA, the major regulatory gene controlling flagella production). The suite includes shuttle vectors expressing both single and multiple sgRNAs complementary to the non-template strand of multiple targeted genetic loci, which were effective in inducible gene repression, with significant reductions in targeted gene expression levels.V. fischericells harboring a version of this system targeting theluxCgene and suppressing the production of luminescence were used to experimentally validate the hypothesis that continuous luminescence must be produced by the symbiont in order to maintain the symbiosis at time points longer than the known 24-h limit. This robust new CRISPRi genetic toolset has broad utility and will enhance the study of V. fischerigenes, bypassing the need for gene disruptions by standard techniques of allelic knockout-complementation-exchange and the ability to visualize symbiotic regulation in vivo. 

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4. Tomato fruit as a model for tissue-specific gene silencing in crop plants

   https://doi.org/10.1038/s41438-020-00363-4  

   Feder, Ari; Jensen, Sarah; Wang, Anquan; Courtney, Lance; Middleton, Lesley; Van Eck, Joyce; Liu, Yongsheng; Giovannoni, James J. (September 2020, Horticulture Research)

   Abstract Use of CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated 9)-mediated genome editing has proliferated for use in numerous plant species to modify gene function and expression, usually in the context of either transient or stably inherited genetic alternations. While extremely useful in many applications, modification of some loci yields outcomes detrimental to further experimental evaluation or viability of the target organism. Expression of Cas9 under a promoter conferring gene knockouts in a tissue-specific subset of genomes has been demonstrated in insect and animal models, and recently inArabidopsis. We developed an in planta GFP (green fluorescent protein) assay system to demonstrate fruit-specific gene editing in tomato using aphosphoenolpyruvate carboxylase 2gene promoter. We then targeted a SET-domain containing polycomb protein, SlEZ2, previously shown to yield pleiotropic phenotypes when targeted via³⁵S-driven RNA interference and we were able to characterize fruit phenotypes absent additional developmental perturbations. Tissue-specific gene editing will have applications in assessing function of essential genes otherwise difficult to study via germline modifications and will provide routes to edited genomes in tissues that could not otherwise be recovered when their germline modification perturbs their normal development. 

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5. Germline mutagenesis of Nasonia vitripennis through ovarian delivery of CRISPR‐Cas9 ribonucleoprotein

   https://doi.org/10.1111/imb.12663  

   Chaverra‐Rodriguez, D.; Dalla Benetta, E.; Heu, C. C.; Rasgon, J. L.; Ferree, P. M.; Akbari, O. S. (August 2020, Insect Molecular Biology)

   Abstract CRISPR/Cas9 gene editing is a powerful technology to study the genetics of rising model organisms, such as the jewel waspNasonia vitripennis. However, current methods involving embryonic microinjection of CRISPR reagents are challenging. Delivery of Cas9 ribonucleoprotein into female ovaries is an alternative that has only been explored in a small handful of insects, such as mosquitoes, whiteflies and beetles. Here, we developed a simple protocol for germline gene editing by injecting Cas9 ribonucleoprotein in adultN. vitripennisfemales using either ReMOT control (Receptor‐Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules) as ovary delivery methods. For ReMOT Control we used theDrosophila melanogaster‐derived peptide ‘P2C’ fused to EGFP to visualize the ovary delivery, and fused to Cas9 protein for gene editing of thecinnabargene using saponin as an endosomal escape reagent. For BAPC we optimized the concentrations of protein, sgRNA and the transfection reagent. We demonstrate delivery of protein cargo such as EGFP and Cas9 into developing oocytes via P2C peptide and BAPC. Additionally, somatic and germline gene editing were demonstrated. This approach will greatly facilitate CRISPR‐applied genetic manipulation in this and other rising model organisms. 

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