Megaclear

gRNAs were synthesized and microinjected as described previously [21] (link). Human codon-optimized Cas9 (hCas9) and sgRNA cloning vector a gift from George Church (Addgene plasmid #41815 and #41824, respectively) [20] (link). Essentially, sequences that recognize Dmrt1 and Dmrt3 target sites were introduced into gRNA cloning vector through inverse PCR. gRNA sequences were PCR amplified from the plasmids and served as templates for the in vitro transcription by mMessage mMachine T7 kit (Thermo Fisher Scientific, Waltham, MA, USA). Transcribed gRNAs were purified with Megaclear (Thermo Fisher Scientific) and ethanol precipitation and microinjected into the cytoplasm of the fertilized eggs obtained from intercross of F1 hybrid (C57BL/6×DBA/2) BDF1 (Sankyo Labo Service Corporation). The concentrations of the RNAs are as follows: Dmrt1 gRNA, 166 ng/μl; Dmrt3 gRNA, 166 ng/μl; and hCas9 mRNA, 166 ng/μl. Primer sequences used for the cloning and template amplification of the gRNAs are listed in Supplementary table 1. All animal protocols were approved by the Animal Care and Use Committee of the National Research Institute for Child Health and Development, Tokyo, Japan.

In vitro transcribed RNA (IVT RNA) was generated using the T7 transcription kit (MEGAscript, ThermoFisher) from a PCR-generated amplicon derived from a DNA plasmid (pBluescript III) using cDNA SuperMix (Quanta Biosciences) and T7 promoter and terminator primers (Integrated Technologies). The RVFV L plasmid (provided by Hana Weingartl, National Centre for Foreign Animal Diseases, Canadian Food Inspection Agency, Manitoba, Canada) contains 3482 base pairs of the L segment of RVFV (nucleotides 1–3482 of the ZH501 RVFV strain). IVT RNA was DNAse treated 3×, column purified (MEGAclear, ThermoFisher) and quantitated with spectrophotometry. The copy number was calculated using an online calculator [37 ]. Ten-fold serial dilutions of IVT stock RNA (10⁴–10⁻¹ copies) were utilized to generate a six-point standard curve using six PCR well replicates per dilution using quantitative RVFV real-time RT-PCR [36 (link)]. Copy numbers for samples were mathematically determined using the PCR-determined mean Ct for the L segment (three PCR well replicates) and the slope and intercept of the L segment IVT RNA standard curve. Data are reported as PCR-determined copy number per reaction. Calculated copy numbers less than 15 (equivalent to Ct greater than 35) are considered past the limits of detection for this assay, are classified as equivocal and, thus, are not reported as true positives.