Dmc1−/− and Spo11−/− mice were previously reported and purchased from the Jackson Laboratory (3, 4). Null allele for Spo16 was generated using CRISPR-Cas9 technology according to standard protocols (34). A guide sequence of 5′-CCTAGAAAATCGAAGCCACA-3′ with a protospacer adjacent motif sequence was selected on the basis of the website prediction results (https://zlab.bio/guide-design-resources?). The guide sequence was cloned to the pUC57-sgRNA vector. Following linearization, sgRNA and Cas9 mRNA were in vitro transcribed and purified according to the manufacturer’s instructions (AM1345, AM1354, and AM1908, Ambion; and 74104, QIAGEN). Mouse zygotes were obtained by superovulation of 7- to 8-week-old females mating with males of the same strain. A mixture of Cas9 mRNA (40 ng/μl) and sgRNA (40 ng/μl) was injected into zygotes using the Eppendorf TransferMan NK2. Injected zygotes were transferred into pseudopregnant ICR (Institute of Cancer Research) female mice (15 to 25 zygotes per mouse) after 2-hour recovery culture in KSOM (potassium-supplemented simplex optimized medium).

All mice were maintained under specific pathogen–free conditions in a controlled environment of 20° to 22°C, with a 12-hour light/12-hour dark cycle, 50 to 70% humidity, and food and water provided ad libitum. Animal care and experimental procedures were conducted in accordance with the University of Gothenburg, Sweden. All mutant mouse strains had a C57BL/6 background. Genotyping primers used are listed in table S2. For each mouse experiment, at least three mice were analyzed, otherwise stated.

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