DOI: 10.5061/dryad.9ghx3fftg
title: Data from: Highly efficient generation of germline mutations using CRISPR/Cas9 in the speckled wood butterfly Pararge aegeria

description: To date, the use of CRISPR/Cas9 technology in ecological-model species for
 validating genotype to phenotype connections has focused primarily on
 visual phenotypes using G0 mutations coupled with analyses of resulting
 mosaic phenotypes. However, studies of physiological phenotypes
 necessitate germline mutations in order to assess non-visible phenotypic
 effects, thus dedicated efforts to developing efficient germline mutations
 in ecological model species are needed. Here we applied the CRISPR/Cas9
 technology to an ecological model species, the speckled wood butterfly
 (Pararge aegeria). We targeted yellow-y, which is required for the
 production of black melanin, as yellow-y loss of function (LOF) mutations
 are not lethal and easy to phenotype, affording efficient assessment of F0
 and germline mutations. To explore what factors may affect efficiency of
 transformation, we employed four alternative treatments, including
 variation in sgRNAs and their concentrations. Color changes in the head
 capsule of first larval instar as well as adult wing color were used as
 indicators of successful knockouts. Individuals with wings that were at
 least 50% transformed were mated, with their F1 offspring assessed for the
 presence of germline mutations. Our CRISPR/Cas9 technique was highly
 efficient at generating LOF mutations in yellow-y. Across all treatments,
 nearly 80% of adults exhibited mosaic LOF phenotypes, of which nearly 30%
 appeared to have 100% LOF phenotypes. Crosses between adults exhibiting at
 least 50% LOF phenotypes resulted in fully transformed offspring,
 revealing high incidence of germline LOF mutations in yellow-y. We provide
 a detailed protocol on how to obtain high germline LOF mutation efficiency
 in order to advance the study of genotype-phenotype connections for
 non-visible physiological traits across natural populations of this and
 other model ecological species.
description: # Data from: Highly efficient generation of germline mutations using
 CRISPR/Cas9 in the speckled wood butterfly Pararge aegeria
 [https://doi.org/10.5061/dryad.9ghx3fftg](https://doi.org/10.5061/dryad.9ghx3fftg) ## Description of the data and file structure ### Files and variables #### File: Larvae\_Yellow\_treatments.txt **Description:** Binary data for testing if the four CRISPR/Cas9 treatments differ in the frequency of individuals appearing as knock-out (excl. controls) based on the color of the head capsule of the first instar larva. ##### Variables * Construct: the treatment the individual was subjected to, y1+3, y1+s4, y1, y2. * Yellow: whether the individuals head capsule appeared dark brown like the wildtype (0) or light brown (1). #### File: Adults\_Yellow\_treatments.txt **Description:** Binary data for testing if the four CRISPR/Cas9 treatments differ in the frequency of individuals appearing as knock-out (excl. controls) based on the wing background color of the adult butterflies. ##### Variables * Construct: the treatment the individual was subjected to, y1+3, y1+s4, y1, y2. * Yellow: whether the individuals background wing color appeared dark brown like the wildtype (0) or mosaic or 100% transformed (1). #### File: Yellow\_treatments\_100\_.txt **Description:** Binary data for testing if the four CRISPR/Cas9 treatments differ in the frequency of adult individuals appearing as fully knock-out. ##### Variables * Construct: the treatment the individual was subjected to, y1+3, y1+s4, y1, y2. * Yellow: whether the individuals background wing color appeared wildtype or mosaic in colors (0) or appeared 100% transformed (1). #### File: Survival\_Adult.txt **Description:** Binary data for testing if the treatments differ in egg survival. ##### Variables * Treatment: the treatment the individual was subjected to, y1+3, y1+s4, y1, y2, C, W. * Survival_Adult: whether the individual survived until eclosion (1) or not (0). #### File: Survival\_Egg.txt **Description:**  ##### Variables * Treatment: the treatment the individual was subjected to, y1+3, y1+s4, y1, y2, C, W. * Survival_Egg: whether the individual survived until hatching (1) or not (0). #### File: Adult\_weight\_G0\_G1.txt **Description:** Data for assessing if knock-out of the *yellow-y* gene has a pleiotropic effect on adult weight. ##### Variables * Generation: the generation the individual belonged to, either G0 (control) or G1 (germline). * Adult weight: the weight of the adult individual in g * Sex: the sex of the individual (M: male, F: female) #### File: Life\_history\_G0\_G1.txt **Description:** Data for assessing if knock-out of the *yellow-y* gene has pleiotropic effects on other life history traits. ##### Variables * Generation: the generation the individual belonged to, either G0 (control) or G1 (germline).  * LDT: the larval development time of the individual in days * PDT: the pupal development time of the individual in days * Pupal weight: the weight of the individual’s pupa in g * Sex: the sex of the individual (M: male, F: female) #### File: Survival\_G0\_G1\_only\_observed\_matings.txt **Description:** Binary data for testing if knock-out of the *yellow-y* gene affects survival to adult eclosion. ##### Variables * Generation: the generation the individual belonged to, either G0 (control) or G1 (germline). * Survival: whether the individual survived until eclosion (1) or not (0). ## Code/software The analyses can be performed in R (version 4.1.2) using the **car** and **emmeans** packages.  G0_G1.R : This R script analyzes whether the knock-out of the *yellow-y* gene affects survival and other life history traits by comparing control (G0) and germline (G1) generations using logistic regression and ANOVA. Treatment_F0.R: This R script evaluates the effect and efficiency of CRISPR/Cas9-mediated *yellow-y* gene knock-out in F0 individuals by comparing treated vs. control groups and different constructs using chi-square tests, logistic regression, ANOVA, and post hoc comparisons. Survival.R: This R script analyzes whether different treatments affect survival at the egg and adult stages using logistic regression, ANOVA, and post hoc comparisons.