Donor HR template transfection while using lenti crispr v2

[Yaiza Núñez]

Hello,

I want to perform a knock in in some difficult to transfect cells, so we have decided to use lenti crispr v2 from addgene (

Plasmid #52961)

 

I want to use ssODN as a donor template to include a tag to my protein but I was wandering if someone has experience with lenti crispr and oligonucleotides. Is it possible to itransduce Cas9 using lentivirus and then the ssODN later by transfection? How much do I have to wait until doing the transfection with the ssODN? Or is better to forget about lentivirus and do electoporation of both elements altogether?  

Thank you very much!

Plasmid #52961

[Neville Sanjana]

Hi Yaiza,

I recommend electroporating lentiCRISPRv2 (which here just drives Cas9 and sgRNA transiently) and the ssODN. This has the advantage of not having constitutively Cas9 expression, which is what would happen if you use virus. 

Of course, if you already have the virus, you can try both ways and see what produces your desired HR outcome more efficiently. Just make sure that the ssODN repair template cannot be cut by the Cas9-sgRNA complex after HR.

Good luck!

Neville

--
You received this message because you are subscribed to the Google Groups "Genome Engineering using CRISPR/Cas Systems" group.
To unsubscribe from this group and stop receiving emails from it, send an email to crispr+un...@googlegroups.com.
For more options, visit https://groups.google.com/d/optout.

[Yaiza Núñez]

Thank you very much for your suggestions Neville.

[Mithil Soni]

Hi Neville,

I have a very basic question. If generate virus using lentiCRISPRv2 it will be integrated and stably expressed all the time. In this case, cas9 and gRNA will be performing gene editing all the time??

And if so then genetic makeup will keep changing all the time? Is that right or I am missing something..

Thanks,
harsh

[Neville Sanjana]

Hi Harsh,

Assuming the sgRNA is well-designed to target a specific site, the Cas9 should only modify that site but, as demonstrated in several papers, Cas9 can cut at a lower frequency at an off-target site. Since lentiviral expression under a constitutive promoter results in continual Cas9 expression, it may modify several others sites as more and more time passes.

For making a clonal cell line that will be used in downstream assays, I think it is better to avoid lentiviral Cas9 and use transient delivery (e.g. transfection of DNA/RNA, protein delivery, microinjection, etc.) Lentiviral Cas9 is better suited to applications like high-throughput screening, where you have a readout that occurs within days to a few weeks after genome modification.

Hope that helps,

Neville

[Mithil]

Thank you Neville for reply.

In order to generate stable cell line, I replaced cas9 gene in lentiCRIPRv2 with cas9n and used the nickase approach.

Since nickase has less off target effects, should I be ok with cas9n and gRNA stable expression. 

Just to make sure, to generate stable cell line Using transient transfection, I can use lentiCRIPRv2 .. Right ? 

Thanks,

Harsh...

[Neville Sanjana]

Hi Harsh,

I don't have any experience with long-term expression of an integrated Cas9 nickase. You may well be correct but it is hard to say for sure.

For a cell line with a particular mutation, you can transiently transfect lentiCRISPRv2  (and even do a transient puromycin selection) and then pick individual cells to create a clonal line. This way Cas9 is not integrated into the genome but modifies the cell during its transient expression. I have done this myself and it works well.

Good luck!

Neville

[Leor Huang]

Hi Yaiza,

Have you successful solved this issue by electroporating lentiCRISPR v2 and ssODNs to the cell? Currently, I also have a problem to determine how to introduce the ssODNs with the lentivirus. Have you try the other possible approach by transfected ssODNs after virus transduction?

If you employ the electroporating, is it necessary to use lentiCRISPR v2 ？ What about using PX335 and ssODNs, deliver to cell by electroporating method.

Thank you,

Leor

[W. P.]

Hi Harsh,

did your lentiCRISPRv2 with cas9n work well? I also tried to use a nickase approach in lentiCRISPRv2. However I couldn't get cas9 mutated due to its lare vector size.

thanks

[hzsh...@sina.com]

Hi Yaiza,

Can you give me one aliquot of lenticrispr v2 plasmid? Because Christmas holiday is near, It will take  long time to deliver, if I order it from addgene.

Thanks in advance.

Op donderdag 27 augustus 2015 00:10:05 UTC+2 schreef Yaiza Núñez:

[hzsh...@sina.com]

Hi Leor,

Can you give me one aliquot of lenticrispr v2 plasmid? Because Christmas holiday is near, It will take  long time to deliver, if I order it from addgene.

Thanks in advance.

Op dinsdag 8 december 2015 06:57:58 UTC+1 schreef Leor Huang:

[Shalini Jain]

Hi Neville,

I want to introduce point mutation in Transferrin gene using SGBS cell line, and want to use lenticrisprv2 and ssODN. We need to do electroporation or we can do transduction of both lentivirusv2 and ssODN to 293T.

Does anybody did that before. Please suggest.

Thanks

On Wednesday, August 26, 2015 at 6:48:42 PM UTC-4, Neville Sanjana wrote:

[Roy Ehling]

Another approach would be to make lentiCRSPR virus but without active guide, leading to constitutive Cas9 production. Then shock ssODN donor as well as crRNA:tracrRNA into your cells.

[CB]

You can try the inducible lentiCRISPR on addgene - TLCV2

Plasmid #87360)

. 

Add Dox when the ssODN is transfected. 

[Shalini Jain]

Thanks for helful information, i will try.

Best,

Shalini