Q1: Does the backbone sequence of the donor vector insert into the target genome during knock-in?
A1: Yes, it will insert.

Q2: Is there a directionality bias in the orientation of the inserted vector?
A2: Both forward and reverse orientations have been observed to occur at the same frequency (Sawatsubashi et al. 2018, Fig. 3).

Q3: Does in/del occur on the cut site?
A3: This can happen. However, an investigation of 81 fragments found that in/del did not occur in about 75% of cases.
For details, please refer to Supplemental Tables 2 and 3 of Sawatsubashi et al. 2018.

Q4: What would happen if you linearized the donor vector in advance?
A4: The knock-in success rate would greatly decrease. 
For NHEJ repair-dependent knock-in, the donor vector needs to be cleaved within cells (Cristea et al. 2013; Sawatsubashi et al. 2018, Supplementary Fig. S1).

Q5: Positive selection is performed using a resistance gene expression cassette in the donor vector, but is there a method to isolate knock-in cells without positive selection?
A5: This is not currently possible, but could occur if the limiting dilution method was applied.

Q6: Can you knock-in different donors to multiple loci at the same time?
A6: We are currently attempting to do this. Successful introduction of multiple different cassettes at the same locus has already been achieved (Sawatsubashi et al. 2018, Supplementary Fig. 3).

Q7: Is it possible to apply the VIKING method in mouse and rat zygotes?
A7: It is theoretically possible, but we have not yet attempted this.

Q8: Which vector series are available as donors?
A8: pUC, pcDNA3, pENTR, and pBluescript II can be used.

Q9: Is it possible to entrust the generation of knock-in cells by this method?
A9: This is dependent on collaborative research or good commercial entrustment.
Please contact vikingknockin.contact@gmail.com for further information.