DOI: 10.5061/dryad.b2rbnzsq5
title: Data from: CRISPR/Cas9 knockout of shell matrix protein 1 in the slipper-snail Crepidula atrasolea

description: 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.
description: # CRISPR/Cas9 knockout of shell matrix protein 1 in the slipper-snail
 Crepidula atrasolea
 [https://doi.org/10.5061/dryad.b2rbnzsq5](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 read   * `SMP1-5bro-pos-092223_S3_L001_R1_001.fastq.gz`: 5 embryo crispant left reads * `SMP1-5bro-pos-092223_S3_L001_R2_001.fastq.gz`: 5 embryo crispant right reads * `SMP1-10bro-neg-092223_S9_L001_R1_001.fastq.gz`: 10 embryo uninjected left reads * `SMP1-10bro-neg-092223_S9_L001_R2_001.fastq.gz`: 10 embryo uninjected right reads * `SMP1-10bro-pos-092223_S4_L001_R1_001.fastq.gz`: 10 embryo crispant left reads * `SMP1-10bro-pos-092223_S4_L001_R2_001.fastq.gz`: 10 embryo crispant right reads * `SMP1-15bro-neg-092223_S2_L001_R1_001.fastq.gz`: 15 embryo uninjected left reads * `SMP1-15bro-neg-092223_S2_L001_R2_001.fastq.gz`: 15 embryo uninjected right reads * `SMP1-15bro-pos-092223_S6_L001_R1_001.fastq.gz`: 15 embryo crispant left reads * `SMP1-15bro-pos-092223_S6_L001_R2_001.fastq.gz`: 15 embryo crispant right read * `SMP1-30bro-neg-092223_S1_L001_R1_001.fastq.gz`: 30 embryo uninjected left reads * `SMP1-30bro-neg-092223_S1_L001_R2_001.fastq.gz`: 30 embryo uninjected right reads * `SMP1-30bro-pos-092223_S5_L001_R1_001.fastq.gz`: 30 embryo crispant left reads * `SMP1-30bro-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.