lentiCRISPRv2 vs. lentiGuide-Puro

[Yiwen Liu]

Hi,

First of all, thank you for providing such wonderful tools for genome engineering!

I am new to CRISPR technology and lentiviral work, and I have some questions for the lentiCRISPRv1, v2 and lentiGuide-Puro.

I noticed in the description of lentiCRISPRv2 it is stated the titer is 10X higher than v1, and lentiGuide-Puro is 100X higher than v1. What reasons are behind the difference in titers? 

In order to use lentiGuide, target cell line should already contain cas9. Does it mean a stable cell line containing lentiCas9-Blast should be created? What selection method can be used to pick cells with a higher expression level of cas9?

Thank  you so much!

Yiwen

[Neville Sanjana]

The titer difference is because lentiGuide-Puro contains only the sgRNA and puro resistance. There is no Cas9, so the titer is quite high. We have a different vector (Cas9-Blast) that you can use to make a stable cell line with Cas9 and select for it using blasticidin resistance.

hope that helps,

- Neville

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[jian...@gmail.com]

Hi Neville,

Thanks for the kind reply. In the protocol for virus concentration in v1 library, it requires a 300X concentration, so I will assume that for v2 library, we still need a 30X concentration of the virus, right?

Thanks

[Neville Sanjana]

That's partially correct. We found that for our use we needed to concentrate 300X but it is relative. It depends on the efficiency of your virus production, how transducible the cells you are doing the GeCKO screen in are, and other factors. Also, you can compensate for lower titer virus by simply producing more virus. (In our work with lentiCRISPRv1, we compensated for the relatively low titer of the virus by producing it at very large scale.)

What I recommend is that you produced a flask or two of virus using the new lentiCRISPRv2 (or lentiGuide-Puro if your cells already have Cas9 or if you used lentiCas9-Blast to make a Cas9 cell line) and do a functional titering of the virus on *your* specific cell line. This means transducing with different volumes of virus and seeing what percentage of cells are Puro resistant in each case. Please make sure to do a no infection control also (where all cells should die after puro treatment). This way you will know the exact functional titer of your virus and can plan future virus production accordingly.

Good luck!

- Neville

[JP]

Neville,

Do you know why your LTV with Cas9 also had low titers? I made my own versions of CBh-driven Cas9 LTV labeled with GFP or RFP instead of Blast or Puro resistance markers and those titers are also low. Did changing the promoter really make that much difference?

JP

[Neville Sanjana]

Hey JP..... there are only small changes in the promoter from before.... it is essentially still EFS driving Cas9 and Puro. Most of the titer increase came from optimizing the NLS architecture and small changes in positioning of lentiviral elements. We hope to have a manuscript ready soon with full details (stay posted!) but we wanted to get the reagents out to community quickly, so we're making them available *before* publication. 

- Neville

[JP]

I certainly appreciate the context. We have a desperate need of high-titre, color-tagged (not resistance marker-tagged) LTV that delivers Cas9.

[Brad Heller]

Hi JP,

I would be interested in a RFP-expressing Cas9 chimeria in a lentiviral expression vector because I'd prefer to use FACS to select expressors rather than puromycin.  For technical reasons Id prefer to use RFP (or mCherry or tdTomato etc) than EGFP.

Would you be able to share your construct and provide more information to me about it?

Thank you very much,

Brad Heller
UCSF Neurology

[Neville Sanjana]

Have you tried using Lipofectamine 3000? For the first screen I am preforming with GeCKO v1, I used Lipo3000 which seemed to work quite well. I wonder if you have done any comparisons, which might possibly increase titer levels after transfection. Generally our lab has observed higher transfection efficiency with this product.

Haven't tried Lipo3000 myself.

 

By comparing roughly lentiCRISPR v1 and v2 I saw that you inverted some sequences such as the ampicilline resistance gene and modified other sequences. The cas9-puro part and the guide sequence cloning site are still seem the same. Furthermore, you switched from Lucigen E cloni to Endura and grow the bacteria at 32 degrees for library amplification I was wondering whether the modifications of the new lentiCRISPR version and the new protocol reduced recombination problems which scientists experienced in the the first version. How many rounds of library amplification would you suggest doing without losing library representation (and experiencing potential recombination problems)? Do you sequence every time you amplify the library?

Yes, Endura and 32C should increase plasmid stability.

We do sequence every time we amplify the library and recommend that all users do the same. This way you know exactly what is going into any GeCKO screen you perform.

best,

- Neville

[JP]

Brad,

I cloned it from a pX330 derivative into the LTV with my guide sequence in it already -- probably not useful for you. Try Cas9-GFP-ADV from VectorBioLabs -- combine with pLX-sgRNA or any color LTV with the U6-gRNA cloned into it.

JP

[hzsh...@sina.com]

Hi Nevile,

Which cell line did you try? Do you have the lenticripr V2 plasmid? If yes, can you give me one aliquot?

Thanks in advance.

Zongsheng

Op donderdag 15 mei 2014 07:28:40 UTC+2 schreef Neville Sanjana:

[Neville Sanjana]

Hi Zongsheng,

LentiCRISPRv2 is available through Addgene: https://www.addgene.org/52961/  It would be easiest if you can get it from them.

Hope that helps,

Neville

[hzsh...@sina.com]

Hi Nevile,

Thanks, but they do not offer deliver during the holiday until January 4. I cannot wait so long.

I appreciate it if you can offer some for me.

Thanks in advance.

Zongsheng

Op dinsdag 22 december 2015 13:48:38 UTC+1 schreef Neville Sanjana:

[ifen...@gmail.com]

Hi Neville,

Thank you for your explanation. I'm also preparing to use the lentiCRISPRv2 system. I still have some questions regarding the system, I would be grateful if you could provide me with some help.

Based on your protocol, you produced virus in T225 flasks with the final volume of 30ml of virus. Can you recommend an approximated range of volume I should use in the functional titering? How do I decide the optimal volume to use in my future experiments?

Currently I'm in possesion of the lentiCRISPRv2 plasmids (already amplified), from what I've read, it is necessary to perform deep sequencing before and after virus production, correct?

I'm thinking of performing the selection on a cancer cell line, I have some confusion about the number of cells to use for the selection process. In your papers you mentioned using a cell number of approximately 400/sgRNA construct to achieve one sgRNA per cell. If I understood correctly, the GeCKOv2 human library has a total of 122,411 sgRNA constructs, then I would need to perform the infection and selection process with about 50,000,000 cells?

Thanks again!

Young

在 2014年5月16日星期五 UTC+8上午3:02:01，Neville Sanjana写道：

[Neville Sanjana]

Dear Young,

 

Thank you for your explanation. I'm also preparing to use the lentiCRISPRv2 system. I still have some questions regarding the system, I would be grateful if you could provide me with some help.

Based on your protocol, you produced virus in T225 flasks with the final volume of 30ml of virus. Can you recommend an approximated range of volume I should use in the functional titering? How do I decide the optimal volume to use in my future experiments?

Assuming the virus is not concentrated (i.e. 30ml of supernatant virus), I would try using a range between 20 ul to 1 mL in your spinfection (we typically do 3x10^6 cells in 1 well of a 12-well plate for each virus volume to test). You are looking to find the volume of virus where 20-50% of cells survive puro selection post-infection: http://bit.ly/InfectionModel

 

Currently I'm in possesion of the lentiCRISPRv2 plasmids (already amplified), from what I've read, it is necessary to perform deep sequencing before and after virus production, correct?

It is a good idea to deep sequence your amplified plasmid before making virus. We do not sequence the virus itself but we do sequence an "early time point" from the cells shortly after viral transduction.

 

I'm thinking of performing the selection on a cancer cell line, I have some confusion about the number of cells to use for the selection process. In your papers you mentioned using a cell number of approximately 400/sgRNA construct to achieve one sgRNA per cell. If I understood correctly, the GeCKOv2 human library has a total of 122,411 sgRNA constructs, then I would need to perform the infection and selection process with about 50,000,000 cells?

That's correct. For representation of ~400 cells/sgRNA, you would need to have ~5x10^7 cells after puro selection. Also, keep in mind that this means you need to spinfect 2 to 5 times more cells, depending on your puro survival rate (the functional titer discussed above).

Hope that helps,

Neville

[ifen...@gmail.com]

Thank you for replying in such a short time. Your answers really cleared things up for me.

在 2016年1月4日星期一 UTC+8下午8:57:01，Neville Sanjana写道：

[Shuying Sun]

Hi Neville

I am trying to use the 2-vector system for GeCKO library. I have no problem to engineer the cells with Cas9, the virus has very high infection rate. But I have trouble to achieve good infection rate for the library. I am not sure what went wrong... Is it necessary to do spininfection for adherent cells? Will it increase the infection rate? Do you do the spininfection right after trypsinizing the cells or after they attach? Do you always use 3 million cells per 12-well plate, or it varies. How do you decide the cell number/density per well? If not concentrating the virus and just use the supernatant, how much should I expect to add to achieve ~30% infection?

Thank you very much for your help. I really learned a lot from this forum.

Shuying

[Neville Sanjana]

Hi Shuying,

Great to hear about your success with making the Cas9 cells.... it is strange to hear your results for the sgRNA lenti since lentiGuide-Puro should have much higher titer than a Cas9-containing lenti. I recommend trying a restriction digest (and ideally also deep sequencing) to make sure the plasmid is intact.

We typically use for titration 3M per well of a 12-well since this mimics what we use when spinfecting the library in at full representation (e.g. if we need to infect 300M cells, we would do 100 wells of 3M/well). For lentiGuide-Puro, we typically use 20-100 ul of supernatant per well to achieve a 20-50% infection rate. I recommend titrating your virus by trying a few different viral volumes and applying each to 1 well with 3M cells. Spinfection and polybrene both help with increasing transduction efficiency.

Glad to hear the forum is a helpful resource and good luck with optimizing the library transduction,

Neville

[ifen...@gmail.com]

Hi Neville,

I have successfully produced virus with the lentiCRISPRv2 plasmid. I'm preparing to titrate the virus in a few days. In your 2014 Science paper SI, the cells were counted and divided into duplicate wells in 12-well plates the day after spinfection. Is there an optimal cell number per well? Or as long as all the wells share the same number of cell it's ok. I'm a little confused about this step and would be grateful for your advice.

Thank you,

Young

在 2016年1月4日星期一 UTC+8下午8:57:01，Neville Sanjana写道：

Dear Young,

[Davide Eletto]

Hi Neville,
I was wondering if you have already experienced the lenti-guide puro A+B libraries with THP-1 cell line.
If so, do you recommend to use a specific spin-infection protocol?

Thank you

Davide Eletto
(Univ Cambridge)

[Neville Sanjana]

Hi Davide,

Thank you for the question. Unfortunately, I've never used THP-1 cells, so
I have no specific recommendations. If you need a good place to start, I
would use our standard spinfection protocol for lentiviral CRISPR. This is
documented in Shalem, Sanjana et al. (2014) and Chen, Sanjana et al. (2015)
supplementary methods.... good luck!

Neville

[Davide Eletto]

Thanks a lot, Neville.
I will definitely look into it.

I know for a(n empirical) fact that THP-1 are quite easy to trasduce as I have already generated a line stably expressing your flagged-Cas9.

I got another question. This time about concentration of the virus.
Would you mind to specify the parameters (speed in RCF and/or rotor/tube model) used for the following method (as published in Shalem, Sanjana et al. (Science, 2014)
...To achieve 300X concentration of the GeCKO pooled library, the virus was ultracentrifuged (Sorvall) at 24,000 rpm for 2 h at 4°C...

Thank you again.

BW,
Davide

[Neville Sanjana]

No problem. In that paper, I used a Thermo/Sorvall WX 80 Ultra ultracentrifuge with a swinging bucket rotor (AH629 rotor w/ 36ml bucket). I think this should work out to ~125,000 rcf (=24,000 rpm).

Best wishes,

Neville

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[Davide Eletto]

Thank you again for the super-quick reply.

I have already expanded the libraries (A + B in lentiguide-puro), following the protocol that you guys wrote (http://genome-engineering.org/gecko/wp-content/uploads/2013/12/GeCKOv2.0-library-amplification-protocol.pdf).

The protocol worked very well with Lucigen Endura cells. THANKS A LOT!

I have so much DNA (~.75mg/library) but I do not know where to store it.

Addgene delivers/suggests to keep it at 4C..

Do you recommend to store it at 4C or can I put it in -20C?

My maxis are in TE buffer.

Thank you again.

D

[Neville Sanjana]

Hi Davide,

That's great to hear that our protocol was helpful. At the maxiprep concentration, your DNA should be stable at either temperature. For long-term storage, I think most folks prefer -20C but I have had no problems storing maxiprep'd libraries at 4C.

Hope that helps,

Neville