Data from: CRISPR/Cas9 knockout of shell matrix protein 1 in the slipper-snail Crepidula atrasolea
Data files
May 20, 2025 version files 3.23 GB
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Dryad-ICE-Analysis.zip
3.92 MB
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HCR_High_Content_Images_Stitched.zip
671.21 MB
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High-content-imaging-dataset.zip
2.25 GB
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Miseq-raw-reads-June2024.zip
312.38 MB
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README.md
4.23 KB
Abstract
Over the past decade, CRISPR/Cas9 has transformed molluscan research, resulting in the emergence of multiple species tailored for studying molluscan-specific novelties. However, significant barriers persist, preventing CRISPR/Cas9 from being used by more species to address their novelties. Challenges include efficient delivery of reagents to eggs, requiring genomics resources necessary for validating successful gene edits. Shell formation is a novelty that gastropods are well-suited for studying. The goal of the study is to lay the groundwork for using CRISPR/Cas9 to answer biomineralization questions in C. atrasolea. The slipper snail Crepidula atrasolea produces large eggs that are amenable to microinjection of reagents, and gene expression atlases were made for studying shell development. To this end, we established embryo culturing and RNP optimization methods, provided validation of gene editing using sequencing approaches, and demonstrated high-content imaging to validate gene expression phenotypes in crispant embryos. Further, we report the first successful knockout of a shell matrix protein (SMP) in a gastropod mollusc, which we anticipate will lead to more targeted SMP knockouts in the future.
https://doi.org/10.5061/dryad.b2rbnzsq5
Description of the data and file structure
This dataset comprises sequencing and imaging data that were obtained in the present pre-print. Four main datasets are provided, and their directory contents are as follows:
1. MiSeq 2x250 bp sequencing of SMP1 Crispant and Uninjected controls
Miseq-raw-reads-June2024.zip: Raw reads for sequencing of 1, 5, 10, 15, and 30 embryo SMP1 Crispant and uninjected Control amplicons.SMP1-1bro-neg-092223_S8_L001_R1_001.fastq.gz: 1 embryo uninjected left reads.SMP1-1bro-neg-092223_S8_L001_R2_001.fastq.gz: 1 embryo uninjected right reads.SMP1-1bro-pos-092223_S10_L001_R1_001.fastq.gz: 1 embryo crispant left reads.SMP1-1bro-pos-092223_S10_L001_R2_001.fastq.gz: 1 embryo crispant right reads.SMP1-5bro-neg-092223_S7_L001_R1_001.fastq.gz: 5 embryo uninjected left reads.SMP1-5bro-neg-092223_S7_L001_R2_001.fastq.gz: 5 embryo uninjected right readSMP1-5bro-pos-092223_S3_L001_R1_001.fastq.gz: 5 embryo crispant left readsSMP1-5bro-pos-092223_S3_L001_R2_001.fastq.gz: 5 embryo crispant right readsSMP1-10bro-neg-092223_S9_L001_R1_001.fastq.gz: 10 embryo uninjected left readsSMP1-10bro-neg-092223_S9_L001_R2_001.fastq.gz: 10 embryo uninjected right readsSMP1-10bro-pos-092223_S4_L001_R1_001.fastq.gz: 10 embryo crispant left readsSMP1-10bro-pos-092223_S4_L001_R2_001.fastq.gz: 10 embryo crispant right readsSMP1-15bro-neg-092223_S2_L001_R1_001.fastq.gz: 15 embryo uninjected left readsSMP1-15bro-neg-092223_S2_L001_R2_001.fastq.gz: 15 embryo uninjected right readsSMP1-15bro-pos-092223_S6_L001_R1_001.fastq.gz: 15 embryo crispant left readsSMP1-15bro-pos-092223_S6_L001_R2_001.fastq.gz: 15 embryo crispant right readSMP1-30bro-neg-092223_S1_L001_R1_001.fastq.gz: 30 embryo uninjected left readsSMP1-30bro-neg-092223_S1_L001_R2_001.fastq.gz: 30 embryo uninjected right readsSMP1-30bro-pos-092223_S5_L001_R1_001.fastq.gz: 30 embryo crispant left readsSMP1-30bro-pos-092223_S5_L001_R2_001.fastq.gz: 30 embryo crispant right reads
2. Inference of CRISPR Edits (ICE) Analysis for SMP1 Crispant and Controls
Dryad-ICE-Analysis.zip: Sanger trace files for crispant and control amplicons. Inference of CRISPR edits (ICE) was conducted on these sanger trace files.June 2024 - SMP1 vs Bra - 485 - 549 - 234 - T7: Comparison of Multi-guide (g485,g549) crispant SMP1 trace versus Brachyury crispant SMP1 trace. This comparison is to show that Brachyury crispants do not target the *SMP1 locus.June 2024 - WT vs Bra - 485 - 549 - 234 - T7: Comparison of wildtype (uninjected) and Brachyury crispant SMP1 trace files. This ICE analysis comparison is intended to further demonstrate that WT trace and Brachyury trace at the SMP1 locus do not have edits.June 2024 - 549 - 234F - T7: Single guide injection using g549, and compared against a control (uninjected) sample.June 2024 - SMP1 - 485 - 114F - T7: Single guide injection using g485 and compared against a control (uninjected) sample.June 2024 - SMP1 vs WT - 485 - 549 - 234 -T7: Multi-guide (g485, g549) injection comparing Crispant trace with Uninjected control trace. This comparison demonstrates that both guides are effective at introducing cuts in crispant but not control embryos.
3. High-content imaging dataset for Crispant (two injections), Cas9-only injection, Uninjected, and No-probe treatments. Channels for SMP1, SMP3, DAPI.
High-content-imaging-dataset.zip: Directory containing maximum fluorescent intensity tiff images and their metadata.
4. Stitched images of high-content imaging run consisting of Crispant, Cas9-only injection, Uninjected, and No-probe treatments
HCR_High_Content_Images_Stitched.zip: Directory containing maximum fluorescent intensitity images from a separate imaging run on the same dataset as in part 3. Sites from the high-content imaging dataset were stitched together and post-processed. These images are the same as in Figure 4 in the manuscript.