GECKO library 2nd or 3rd generation lenti?

[JP]

Does the lenti-CRISPR use 2nd or 3rd generation lentivirus, i.e. is there a self-inactivating LTR?

[Feng Zhang]

Second generation.

On Tuesday, January 14, 2014, JP wrote:

Does the lenti-CRISPR use 2nd or 3rd generation lentivirus, i.e. is there a self-inactivating LTR?

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[Neville Sanjana]

Hi JP,

I just wanted to clarify what Feng said about the lentiCRISPR vector. The generation terminology can be a bit confusing since many different lentiviral systems have been referred to over the years as different generations. Using Addgene's terminology (http://www.addgene.org/lentiviral/packaging/), our vector is a 3rd generation transfer plasmid, which means you can package it using either 2nd generation (2 plasmids, not including transfer vector) or 3rd generation (3 plasmids, not including transfer vector) packaging plasmids.

It does have a 3' SIN LTR. This is an important biosafety feature since the 3’ LTR primes the transcription before integration, resulting in a short inactivated 5’ and 3’ LTR after integration. As mentioned on the Addgene site, the defining feature for 3rd generation plasmids is that Tat (a transcriptional activator) is not required for transcription of the transfer vector; instead, in lentiCRISPR, a RSV promoter drives transcription from the 5' LTR. 

As I said, you can use either 2nd or 3rd generation packaging plasmids to package lentiCRISPR (again, refer to http://www.addgene.org/lentiviral/packaging/ if you do not already have these in your lab). For the original GeCKO work, I used 2nd generation packaging plasmids (both the Weinberg and the Trono ones at different times) and both of these 2nd generation plasmids work well for lentiCRISPR. The consensus in the field is that pVSVg and psPAX2 are the optimal 2nd generation packaging plasmids and these are what I would recommend using with lentiCRISPR. 

Hope that helps,

- Neville

On Tuesday, January 14, 2014 5:33:26 PM UTC-5, Feng Zhang wrote:

Second generation.

On Tuesday, January 14, 2014, JP wrote:

Does the lenti-CRISPR use 2nd or 3rd generation lentivirus, i.e. is there a self-inactivating LTR?

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[Daniel Ortiz Velez]

Hello,

Is this still true for lentiCRISPR-V2?

Thank you!

[Neville Sanjana]

Yes it is. You can use either the 2 packaging plasmid or 3 packaging plasmid system for making virus from LCv2.

Best,

Neville