Sanger sequenced

KI of donor plasmids was attempted using laser-assisted cytoplasmic injection^{20 (link)} of in vitro matured oocytes after 18 h of maturation and in vitro fertilized embryos 6 hpi with 6 pL of a solution containing 67 ng/μL of in vitro transcribed gRNA, 167 ng/μL of Cas9 protein (PNA Bio) and 133 ng/μL of donor plasmid. Injected MII oocytes were subsequently co-cultured with cumulus-oocyte-complexes (COCs) and in vitro fertilized following procedures previously described for sheep^{26 (link)}. Embryos were scored for developmental stage reached at day 7–8. Embryos that reached blastocyst stage were lysed as described above and underwent whole-genome amplification using the Repli-G Mini kit (Qiagen). Target regions were amplified using primers developed using Primer3 (Supplementary Information, Fig. S1 and Table S4)^{39 (link),40 (link)}. PCR was performed on a thermal cycler with 12.5 μL LongAmp Taq 2X Master Mix (NEB), 9.5 μL of H₂O, 1 μL of each primer at 10 mM and 1 μL of DNA for 5 min at 94 °C, 35 cycles of 30 s at 94 °C, 30 s at anneal temp (Supplementary Information, Table S4) and 4 min at 65 °C, followed by 15 min at 65 °C. Products were visualized on a 1% agarose gel using a gel imager, purified using the QIAquick Gel Extraction Kit (Qiagen) and Sanger sequenced (GeneWiz).

Mosquito saliva was collected as described above (Mosquito infection—force salivation). The DMEM plus saliva mixture was mixed with TRIzol^TM reagent, RNA extracted, and cDNA synthesized as described above (filter paper assay). We amplified the structural region of the CHIKV genome using two overlapping PCR products (Fragment 1 and Fragment 2—primer sequences found in Table S1, Supplementary Materials). Resulting PCR products of the correct size were purified using a PCR cleanup kit (Macherey–Nagel, Bethlehem, PA, USA) and Sanger sequenced (Genewiz, South Plainfield, NJ, USA) (sequencing primer sequences can be found in Table S1, Supplementary Materials).