Optimization of the donor for HIT-trapping. (A) Diagrams showing strategies to optimize the HIT-trap donor. All of the optimized donors were derived from the same plasmid donor (HIT-trap-pA1C). Donor-P was PCR generated. Donor-X1 was generated by linearization of the plasmid using SbfI or EcoRV. Donor-X2 only contains the insertion part of the plasmid, with the backbone removed in vitro using XbaI. Donor-Ligx2 was produced by self-ligation of Donor X2 via T4 ligase. (B) The frequencies of desired/not desired knock-in (D/ND-KI) patterns by the respective optimized donor. (n) Number of clones checked by PCR. The genotyping data are presented in Supplemental Figure S10.
