Protocols: Injection mix

Injection Mix

All plasmids intended for injection should be prepared using either a midiprep or maxiprep kit to generate high-quality DNA. Dilute all plasmids and/or single-stranded donors in dH20 to the following concentrations and inject following standard embryo injection protocols. To increase survival we try to keep the total concentration of the injection mixture 1,000 ng/µL or less.

 

Cas9

C​as9 can either be introduced through coinjection of hsp70-Cas9 or it can provided through germline expression by injecting into vasa-Cas9 flies. While both methods work reliably, vasa-Cas9 expression leads to the higher targeting efficiency. We inject hsp70-Cas9 at concentrations of 250 ng/µL to 500 ng/µL. When injecting into vasa-Cas9, you do not need to add any hsp70-Cas9 to the injection mix.

gRNAs

Depending on the intended modification, one or two guideRNAs (gRNAs) may be used. For generating mutations via imperfectly repaired DSBs a single gRNA is used. Larger deletions can  generated by using two gRNAs that define the limits of the intended deleiton. For homology-directed repair we also used two gRNAs injected with a homology containing donor. We inject gRNAs at concentrations of 100 ng/µL to 500 ng/µL depending on the application.

Donors

For homology-directed repair we have used both small single-stranded donors (ssODNs) and larger dsDNA donors. We inject ssODNs at a concentration of 100 ng/µL. For dsDNA donor vectors, like pHD-DsRed-attP, we typically inject at a concentration of 500 ng/µL.

 

Typical Hsp70-Cas9 Injection Mixtures

  • Indels: phsp70-Cas9 (500 ng/µL), pU6-BbsI-chiRNA (500 ng/µL)
  • Deletions: phsp70-Cas9 (500 ng/µL), pU6-BbsI-chiRNA (250 ng/µL each)
  • HDR with ssODNs: phsp70-Cas9 (500 ng/µL), pU6-BbsI-chiRNA (250 ng/µL each), ssODN (100 ng/µL)
  • HDR with dsDNA: phsp70-Cas9 (250 ng/µL), pU6-BbsI-chiRNA (100 ng/µL each), pHD-DsRed-attP donor (500 ng/µL)

Typical vasa-Cas9 Injection Mixtures

  • Indels: pU6-BbsI-chiRNA (250 ng/µL)
  • HDR with dsDNA: pU6-BbsI-chiRNA (100 ng/µL each), pHD-DsRed-attP donor (500 ng/µL).