Figure 2.

Figure 2.

(a) GeneWeld reagent components are designed for simultaneous nuclease targeting of genome and donor to reveal short regions of homology. Red arrowheads represent nuclease DSB cut sites. Components include: 1 - Designer nuclease mRNA, either Cas9 to target both the genome and donor, or Cas9 to target the donor and TALEN to cut the genome; 2 - sgRNA for targeting Cas9 to genome; 3 - Universal sgRNA to liberate donor cargo and homologous ends; and 4 - pGTag donor of interest with short homology arms. (b) Stippled and striped boxes represent sticky ends created by Type IIs restriction endonucleases BfuAI and BspQI, allowing digestion and ligation of both homology arms into the donor vector in a single reaction. Homology arm fragments are formed by annealing complementary oligonucleotides to form dsDNA with sticky ends for directional cloning into the vector. XFP = Green or Red Fluorescent Protein. pA = SV40 or β-actin 3’ untranslated region. Red and green fluorescent proteins were cloned into the pGTag vectors, and for each color, subcellular localization sequences for either nuclear localization (NLSs) and membrane localization (CAAX) are provided. (c) Schematic of GeneWeld targeting in vivo. After designer nuclease creates targeted double-strand breaks in the genome and donor, end resection likely precedes homology recognition and strand annealing, leading to integration of the donor without vector backbone.