HDR knock-in using Lenti CRISPR

[Artur Padzik]

Hi,

Did anyone tried and could recommend vectors/ solution for template based (HDR) knock-in, but using Lentivirus?
Is the only option to transduce CRISPR components and transfect in the same time template?
I would appreciate your input.
Thanks
Artur

[Jahan Dadgar]

hello Artur
I posted essentially the same question?
Have you had any luck finding any information from other sources?

[Artur Padzik]

Hi, Jahan

Unfortunately not. I'm still searching. I'll let you know if I'll find something. Maybe you could drop me a message if you will find anything.
Let's keep each other posted if anything interesting pop up.
Best,
Artur

[JP]

You want to deliver the template as a lenti? Try using integrase-deficient packaging vectors.

[Wolfgang Ruf]

Hi,

where can I get an integrase-deficient packaging system?

Thanks

[JP]

http://www.nature.com/nbt/journal/v25/n11/full/nbt1353.html & refs therein.

If you ask Don Kohn nicely at UCLA by email he might send you some.

[saul kivimae]

Integration deficient lenti system is just changing catalytic D64 in integrase to V in your favorite packaging vector. You can do it in 5 days.

Best
Saul

[WR]

Thanks,

but to be honest it was really hard to mutate D10A lentiCRISPR V2. I think it's because of its size (15kb) and psPAX2 is also large (around 10kb), so I'm not looking forward to do the mutation. I contacted Don Kohn but I haven't got an answer yet.

Best

Wolfgang

[JP]

Try emailing or cc'ing his staff scientist and lab manager Roger Hollis.

Contact info -- http://www.mimg.ucla.edu/faculty/kohn/scientists.html

[WR]

Thanks!

[saul kivimae]

Size of the plasmid doesn't matter.
Quickchange is not the way to go here. Choose 2 closest unique restriction sites either side of the mutation site and overlap-pcr between them with overlap oligos generating the mutation in the middle. Very effective. don't need to pcr the whole plasmid. took me 5 days to make non integrating lenti and retro packaging plasmids. the insert to clone after pcr is short.


Best
Saul

[Richard Maser]

If you can't get the plasmids, I would recommend mutagenesis of your psPax2 (I use pMDLg/pRRE for packaging) with the Q5 mutagenesis protocol (see NEB's website and oligo design tool) - I was able to make D64V, E116N, and E152G individual point mutations (each separately, not together) with Q5 polymerase and it was done in a few hours. You can use their kit or do it yourself with proof-reading polymerase, DpnI, and ligase.

I'd be happy to share my primer design if needed.

Cheers,
Rick

[WR]

Thanks for the helpful hints. I will try to do so. I know the D64V Mutation but what about the others (E116N, and E152G)?

Best

Wolfgang 

[Richard Maser]

They are also mutations that disrupt the integrase to one degree or another. D64V seems to be the most commonly used to package IDLVs. I actually meant to write D116N, not E116N.

It's important to remember that integration is drastically reduced but not non-existent, so make sure you can screen for the correctly targeted mutation.

Good luck!

[Elaine Roby]

This is a great suggestion which I am trying.  Using the IDLV system, the template would then be in the form of an episome, do you suppose the cell would use it in this form for template as opposed to linear DNA?

Thanks

Elaine

[A.N.]

Hello,

Did it work to CRISPR knockin the template via IDLV?

I am aiming for a similar strategy as I would like to introduce a tag sequence at the start or end of a gene. As my cells are impossible to transfect I will be depending on LV-infection. I was wondering whether it is also possible to use an IDLV for the LV plasmid for Cas9 and the gRNA, as I am trying to make a stable cell line and I would not want the Cas9 to have off-target effects after the fluc tag is inserted. 

Best wishes,

Ann

Op woensdag 22 april 2015 11:08:07 UTC+2 schreef Artur Padzik:

[Adrian Cuenca]

Richard,

Thank you for your suggestions. Do you know if there is a good article out there showing how to do an efficient Knock-in using lentivirus in cultured cells?

On Tuesday, May 19, 2015 at 3:39:52 PM UTC-4, Richard Maser wrote:

[nikita modi]

Hey Arthur, 

 Have you had any luck with HDR using lentiviruses?