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1. Proteomic and Transcriptomic Analyses in the Slipper Snail Crepidula fornicata Uncover Shell Matrix Genes Expressed During Adult and Larval Biomineralization

   https://doi.org/10.1093/iob/obac023  

   Batzel, G. O.; Moreno, B. K.; Lopez, L. S.; Nguyen, C. K.; Livingston, B. T.; Joester, D.; Lyons, D. C. (August 2022, Integrative Organismal Biology)

   Synopsis The gastropod shell is a composite composed of minerals and shell matrix proteins (SMPs). SMPs have been identified by proteomics in many molluscs, but few have been studied in detail. Open questions include (1) what gene regulatory networks regulate SMP expression, (2) what roles individual SMPs play in biomineralization, and (3) how the complement of SMPs changes over development. These questions are best addressed in a species in which gene perturbation studies are available; one such species is the slipper snail, Crepidula fornicata. Here, SEM and pXRD analysis demonstrated that the adult shell of C. fornicata exhibits crossed lamellar microstructure and is composed of aragonite. Using high-throughput proteomics we identified 185 SMPs occluded within the adult shell. Over half of the proteins in the shell proteome have known biomineralization domains, while at least 10% have no homologs in public databases. Differential gene expression analysis identified 20 SMP genes that are up-regulated in the shell-producing mantle tissue. Over half of these 20 SMPs are expressed during development with two, CfSMP1 and CfSMP2, expressed exclusively in the shell gland. Together, the description of the shell microstructure and a list of SMPs now sets the stage for studying the consequences of SMP gene knockdowns in molluscs. 

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2. 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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3. The adult shell matrix protein repertoire of the marine snail Crepidula is dominated by conserved genes that are also expressed in larvae

   https://doi.org/10.1186/s12862-024-02237-y  

   Lopez-Anido, Rebecca N; Batzel, Grant O; Ramirez, Gabriela; Wang, Yiqun; Neal, Stephanie; Lesoway, Maryna P; Goodheart, Jessica A; Lyons, Deirdre C (December 2024, BMC Ecology and Evolution)

   Abstract Mollusca is a morphologically diverse phylum, exhibiting an immense variety of calcium carbonate structures. Proteomic studies of adult shells often report high levels of rapidly-evolving, ‘novel’ shell matrix proteins (SMPs), which are hypothesized to drive shell diversification. However, relatively little is known about the phylogenetic distribution of SMPs, or about the function of individual SMPs in shell construction. To understand how SMPs contribute to shell diversification a thorough characterization of SMPs is required. Here, we build tools and a foundational understanding of SMPs in the marine gastropod speciesCrepidula fornicataandCrepidula atrasoleabecause they are genetically-enabled mollusc model organisms. First, we established a staging system of shell development inC. atrasoleafor the first time. Next, we leveraged previous findings inC. fornicatacombined with phylogenomic analyses of 95 metazoan species to determine the evolutionary lineage of its adult SMP repertoire. We found that 55% ofC. fornicata’sSMPs belong to molluscan orthogroups, with 27% restricted to Gastropoda, and only 5% restricted at the species level. The low percentage of species-restricted SMPs underscores the importance of broad-taxon sampling and orthology inference approaches when determining homology of SMPs. From our transcriptome analysis, we found that the majority ofC. fornicataSMPs that were found conserved inC. atrasoleawere expressed in both larval and adult stages. We then selected a subset of SMPs of varying evolutionary ages for spatial-temporal analysis using in situ hybridization chain reaction (HCR) during larval shell development inC. atrasolea. Out of the 18 SMPs analyzed, 12 were detected in the larval shell field. These results suggest overlapping larval vs. adult SMP repertoires. Using multiplexed HCR, we observed five SMP expression patterns and three distinct cell populations within the shell field. These patterns support the idea that modular expression of SMPs could facilitate divergence of shell morphological characteristics. Collectively, these data establish an evolutionary and developmental framework inCrepidulathat enables future comparisons of molluscan biomineralization to reveal mechanisms of shell diversification. 

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4. Efficient CRISPR genome editing and integrative genomic analyses reveal the mosaicism of Cas-induced mutations and pleiotropic effects of scarlet gene in an emerging model system

   https://doi.org/10.1101/2024.01.29.577787  

   Xu, Sen; Neupane, Swatantra; Wang, Hongjun; Pham, Thinh Phu; Snyman, Marelize; Huynh, Trung V; Wang, Li (January 2024, bioRxiv)

   Abstract Despite the revolutionary impacts of CRISPR-Cas gene editing systems, the effective and widespread use of CRISPR technologies in emerging model organisms still faces significant challenges. These include the inefficiency in generating heritable mutations at the organismal level, limited knowledge about the genomic consequences of gene editing, and an inadequate understanding of the inheritance patterns of CRISPR-Cas-induced mutations. This study addresses these issues by 1) developing an efficient microinjection delivery method for CRISPR editing in the microcrustaceanDaphnia pulex; 2) assessing the editing efficiency of Cas9 and Cas12a nucleases, examining mutation inheritance patterns, and analyzing the local and global mutation spectrum in thescarletmutants; and 3) investigating the transcriptomes ofscarletmutants to understand the pleiotropic effects ofscarletunderlying their swimming behavior changes. Our reengineered CRISPR microinjection method results in efficient biallelic editing with both nucleases. While indels are dominant in Cas-induced mutations, a few on-site large deletions (>1kb) are observed, most likely caused by microhomology-mediated end joining repair. Knock-in of a stop codon cassette to thescarletlocus was successful, despite complex induced mutations surrounding the target site. Moreover, extensive germline mosaicism exists in some mutants, which unexpectedly produce different phenotypes/genotypes in their asexual progenies. Lastly, our transcriptomic analyses unveil significant gene expression changes associated with scarlet knock-out and altered swimming behavior in mutants, including several genes (e.g., NMDA1, ABAT, CNTNAP2) involved in human neurodegenerative diseases. This study expands our understanding of the dynamics of gene editing in the tractable model organismDaphniaand highlights its promising potential as a neurological disease model. 

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5. Efficient and modular CRISPR‐Cas9 vector system for Physcomitrella patens

   https://doi.org/10.1002/pld3.168  

   Mallett, Darren R.; Chang, Mingqin; Cheng, Xiaohang; Bezanilla, Magdalena (September 2019, Plant Direct)

   Abstract CRISPR‐Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA‐guided nuclease to initiate double‐strand breaks. Until recently, classical RAD51‐mediated homologous recombination has been a powerful tool for gene targeting in the mossPhyscomitrella patens. However, CRISPR‐Cas9‐mediated genome editing inP. patenswas shown to be more efficient than traditional homologous recombination (Plant Biotechnology Journal, 15, 2017, 122). CRISPR‐Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR‐Cas9 genome editing inP. patens, here we describe the generation and use of a flexible and modular CRISPR‐Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology‐directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5′ and 3′ ends of gene coding regions. With regard to homology‐directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a “stop codon cassette” with downstream near‐effortless genotyping. 

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