Single lenti vector system with U6-sgRNA-Cas9-GFP or puro with loxP sites or Dox inducible?

[CB]

Hi, 

Does anyone know of a lentiviral plasmid like lentiCRISPRv2 but with loxp sites flanking the U6-sgRNA-Cas9-Puro? I would like to be able to AdCre this lentivirus out after I obtain cells with the KO. I do not want to have long term constitutive expression of sgRNA and Cas9. 

or 

Does anyone know of a Dox inducible lenti plasmid like lentiCRISPRv2? 

In either scenario, I want a single vector system. 

Thanks, 
Carter

[Lu Xiaoyin]

Hi, Carter,

 

Will you considering using an integrase-deficient packaging system? Usually it is changing catalytic D64 to V in packaging vectors like psPAX2. This way the lenti system becomes more like transient expression.

 

The cre-it-out idea seems alright, but I haven’t seen those floxed ones in those “all in one” CRISPR vectors.

 

For Tet-ON system, the concern would be that you also need rtTA for activating the TRE promoter, which actually brings constitutive expression of another protein.

 

Hope that may help,

 

Xiaoyin

在 2016年4月27日星期三 UTC+8下午8:46:42，CB写道：

[Mona Friedrich]

Hi Carter,
did you figure anything out? i would also be very interested in a dox inducible single vector system!
Cheers, Mona

[Carter]

I have the vector in hand. It is the lentiCRISPRv2 converted to dox inducible - LTR<U6-sgRNA-TRE-Cas9-T2A-eGFP-EF-1a-Puro-P2A-rtTA>LTR. It produces good titers and I can see GFP with dox but I am just waiting to get a KO. Just need to validate it. 

[Mona Friedrich]

That sounds awesome! Is your vector deposited on addgene?
So just to be sure I get it right: after transfection, you can do the selection (Puro) before you induce KO, right? Did you select single clones?

[Carter]

No, I am validating it first. It is fully sequenced with NGS and checks out. It produces nice amounts of virus and induces well as monitored with GFP. Yes, the puromycin is driven by a constitutive promoter. Only Cas9-T2A-GFP is inducible. You can transduce cells then select with puro and hit the cells with dox later for the KO. I have not selected single clones yet. I am testing out the KO in a polyclonal population first to see if it cuts and to monitor any background cutting in the -Dox sceanario (just to see if there is leakiness, no GFP is seen in -Dox). 

[Ivan Ivandic]

Hi,

this vector seems an awesome option! Please post here as soon as its possible to get it through any source. Have you tried it on primary cells? If yes, do you have any numbers on transduction efficiency? 

Thx,

Ivan

[Mona Friedrich]

Thanks for the update, Carter! I would also be really thankful if you could let us know in case you'd be willing to share this vector.

[Carter]

Mona, 

The plasmid should be available soon on addgene. Waiting for the QC and MTA before it is released. 

TLCV2 #87360

LentiCRISPR v2 was modified into an all-in-one dox inducible system. The addition of doxycycline induces Cas9-2A-eGFP. The U6 promoter drives constitutive sgRNA expression.

Thanks,

Carter

[CB]

In case anyone is interested. 

TLCV2 

(Plasmid #87360)

https://www.addgene.org/87360/

LentiCRISPR v2 was modified into an all-in-one dox inducible system. The addition of doxycycline induces Cas9-2A-eGFP. The U6 promoter drives constitutive sgRNA expression.

[CB]

It is now available to order

[AJ]

What titers did you achieve ?

[CB]

I perform a functional titer with puromycin selection and I typically end up expanding the well that was transduced with virus that was diluted 1:100 to 1:500 depending on the cell line. It produces titers that are similar to lentiCRISPR v2, at least in my hands. 

[AN]

Hi Carter,

I am very new to the CRISPR technology. I am trying to wrap my head around it. Our group is interested in developing a Lenti-CRISPR/cas9 toolkit for integration of larger DNA fragments to generate a stable mammalian cell line. I came across with this thread while looking for more information regarding the same. I understand that using LV poses a problem of integration and some disadvantages associated with it. On the other hand, IDLV infect cells with high efficiency but are progressively eliminated as the cells divide. I have few questions regarding this dox inducible lenti-CRISPR system. I would really appreciate if you could answer my questions.

1) Since it is a big plasmid already, does the dox-inducible lenti-CRISPR system support multiplexing? 

2) Or would you recommend to clone multiple gRNAs in one vector and cas9 on other? Any other suggestion/advice?

3) If I go with the dox-inducible lenti-CRISPR system then would it be alright to use identical promoter for expression of gRNA (since direct repeats make lentivirals unstable)

I hope my questions don't sound stupid. Thank you for your time.

Regards,

Amu

[GC]

Hi CB,

I was wondering if you were successful in generating KO cells with the TLCV2 plasmid.

Thanks in advance for your input,

Gabriele 

[SJ]

Hi Carter,

I am using TLCV2 plasmid for an inducible KO. Nevertheless, I have an awful titer after virus production. I am afraid that the packaging plasmids that I use are not optimal for TLCV2: pVSVg and p8.91. Could you please give me any advice to get a best titer?

Thank you so much.

Best regards,

Sol

[Monika Davare]

Hi Sol,

Did you ever sort this out? We are gearing up  to use this vector, and preliminary work in our hands also produced extremely low titer virus.

I was just curious if you got an answer or were able to optimize this in any way.

Would really appreciate any further insight.

Thanks,

Monika