Only a few steps are required to modify the genome with Cas9-triggered homologous recombination.
Expand the items below to view a protocol for each step, or download our Cloning Guide and Injection Protocols.
Expand the items below to view a protocol for each step, or download our Cloning Guide and Injection Protocols.
▸ Construct design and cloning
▾ Cas9-triggered homologous recombination with unc-119 selection
Our selection protocol is based on, and is highly similar to, the MosSCI protocol developed by Christian Frøkjær-Jensen and colleagues. We are greatly indebted to Dr. Frøkjær-Jensen for developing such an effective procedure and for sharing advice and reagents.
Before the experiment
o 50 ng/μL Cas9-sgRNA construct with your targeting sequence o 10 ng/μL pMA122 (heat-shock driven PEEL-1 negative selection; Addgene #34873) o 10 ng/μL pGH8 (Prab-3::mCherry neuronal co-injection marker; Addgene #19359) o 5 ng/μL pCFJ104 (Pmyo-3::mCherry body wall muscle co-injection marker; Addgene #19328) o 2.5 ng/μL pCFJ90 (Pmyo-2::mCherry pharyngeal co-injection marker; Addgene #19327) DNA should be prepared using Invitrogen's PuroLink mini-prep kit, which gives the highest injection efficiencies. We do not linearize plasmids prior to injection.
Day 7-9: Heat Shock
Day 8-10: Pick Candidate knock-in animals
PCR verification
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