Has anyone been able to reproduce Suzuki et al's results from their HITI paper?

[Luke]

I've been having trouble knocking in a gene into HEK293T cells using the HITI approach.  Has anyone had any luck with this strategy, and if so, would you mind sharing your protocol?

Thanks!

[András Tálas]

I never tried HITI, but we have developed (and used several times) a similar protocol that works pretty good in many cell lines including HEK293T: https://academic.oup.com/dnaresearch/article/doi/10.1093/dnares/dsx029/3904549/A-convenient-method-to-pre-screen-candidate-guide

If you have any questions please do not hesitate to contact me.

Best,

Andras Talas

2017-10-05 6:42 GMT+02:00 Luke <luke...@gmail.com>:

I've been having trouble knocking in a gene into HEK293T cells using the HITI approach.  Has anyone had any luck with this strategy, and if so, would you mind sharing your protocol?

Thanks!

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[crispr.m...@gmail.com]

Hi Andras and Luke,

Andras, I hadn't seen your paper, very interesting.

As a general question of your approach and of course HITI too (they are quite similar in mechanism from what I can tell, HITI using mini-circles of DNA rather than plasmids), do you see random integration of the repair template as well as targeted integration? This is my concern with using 'linear' DNA as a template.

Thanks

Antony

On Saturday, October 7, 2017 at 3:58:17 PM UTC+1, András Tálas wrote:

I never tried HITI, but we have developed (and used several times) a similar protocol that works pretty good in many cell lines including HEK293T: https://academic.oup.com/dnaresearch/article/doi/10.1093/dnares/dsx029/3904549/A-convenient-method-to-pre-screen-candidate-guide

If you have any questions please do not hesitate to contact me.

Best,

Andras Talas

2017-10-05 6:42 GMT+02:00 Luke <luke...@gmail.com>:

I've been having trouble knocking in a gene into HEK293T cells using the HITI approach.  Has anyone had any luck with this strategy, and if so, would you mind sharing your protocol?

Thanks!

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[András Tálas]

Hi,

The short answer is that we have sequenced the integration site (Supp. fig. 4. in our paper) and evaluated the integration but we never checked the random integration events by deep sequencing or WGS because that was not the main goal of our method. When you use any kind of DNA as a repair template (HDR or NHEJ) there is always a chance that you will have random integrations. When we used a control when we linearized the donor plasmid inside the cell but we have not targeted the genome (e.g. Fig3 in our paper) the background random integration was around 3-5% vs. 15-20% targeted integration. When you use a circular donor the random integration was around 2% but the targeted integration was the same (Supp. fig 2. in our paper), so if you have a better random to targeted integration ratio (with linearized plasmids)  the chance that you will have a random integration is much less.

I hope that I answered your question, if you have more please do not hesitate to contact me again,

Best

Andras

---

András Tálas

PhD Candidate

Institute of Enzymology
Research Centre for Natural Sciences

Hungarian Academy of Sciences

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[Neuropath]

Hi Luke,

Have you have any luck with HITI? I have tried it once in human ES cells but it didn't seem to work. The sequence I am trying to insert is a multi-functional cassette with GFP and puromycin selection of about 4.8kb. I got some puro resistant colonies but none of them have integration at the target site. 

~neuropath~

[Neuropath]

Hi Antony,

In the Suzuki paper, they used both mini-circle and plasmids (pCR-blunt II). Mini-circles are only marginally more efficient than plasmids it seems. I haven't seen another publication using HITI the way it was described since that paper.

~neuropath~

[Alex Brown]

I work with HITI frequently (my paper referred to it as NAVI). No matter how you execute your editing (i.e. HDR, NHEJ, NAVI/HITI) it is heavily dependent on the cell type and nuclease/guide-RNA efficiency. If you have a crappy guide, you're going to have a bad time no matter what. As for other, perhaps less obvious, tips that might not be discussed in the paper:

1. Whenever integration is the goal, be sure to include masked regions of the genome during your off-target analysis... you will likely be sorry if you don't.

2. If you linearize your vector and genomic locus by using the same gRNA, you may hurt your efficiency as there is much greater chance for target site regeneration upon integration.

3. If you are using selection, do a kill curve and use the lowest dose possible. Don't just use what your labmates say works best. Your intended outcome is not hundreds of plasmids, each expressing PuroR... It is one or two single alleles, now in a genomic context where expression patterns may not be ideal. This may affect the concentration of selection agent that the cells are able to withstand. I also generally wait about three days before applying selection. Of course, multiple passages help ensure that you are not merely selecting for cells with the original vector, as well.

I know some people remain sceptical, but I find it can really work well for certain applications and it's super easy to try. Good luck!

-Alex