4. PCR primer sets (10 μM) (Figs. 4 and 5, see Table 1).
5. Thermocycler.
6. Agarose, loading buffer, and TAE.
7. Agarose gel electrophoresis tank and power supply.
Fig. 4 Knock-in donor construct for gene targeting. (a) Schematic representation
endogenous ROS1 genomic sequence structure, showing ROS1-GFP donor and
endogenous ROS1 knock-in site. Red letters in sgRNA sequence indicate PAM,
and green letters in ROS1 partial sequence indicate stop codon. (b) A flow chart
for knock-in donor vector construction. First round of PCR involves amplification
of homology arms and knock-in sequence with specific primers, followed by
second round of PCR to fuse them. (c) Knock-in donor vector construct harbors
AtU6-sgRNA cassette, knock-in donor, and selection marker (bar) gene
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