Optimal method to determine the lentiviral titer of GeCKO Pooled 1 vector Library

[ct...@lji.org]

Hello,

I am relatively new to CRISPR-Cas9 and the GeCKO system and I'm hoping to get some suggestion for the problems I am experiencing. I have been working to generate GeCKO 1 vector system lentivirus to transduce my cell line for a functional screening. I followed the GeCKO guidelines and successfully amplified the 1 vector pooled library purchased from Addgene. I've then transfected the purified plasmids with 2nd generation lentivirus packaging system into HEK293T using FuGene 6 (using the protocol and plasmid ratio suggested by the Broad Institute). I collected, pooled, and filtered the viral supernatant.

For the screening, we want to transduce the target cells with a low MOI, and therefore determining an accurate titer for the viral supernatant became important. I wanted to bypass the cell culture to save time and found a ELISA titer kit from Clontech that measures the level of p24 (capsid protein made by the HIV-1 Gag gene). To verify the method, I performed transduction with various titer dilution of a GFP lentivirus (made with the plasmid pCDH-EF1-MCS-T2A-copGFP) in NIH-3T3 and ran FACS (after 48Hrs of culture) to determine the transduction efficiency. The titer calculated from ELISA is overestimated compared to the one FACS calculated (~10^8 vs. 10^7), but the difference in the calculated titer is reproducible (done using the GFP lentivirus). 

I then ran the same transduction on NIH-3T3 for my pooled library virus and performed FACS via intracellular staining for the FLAG tag located on the Cas9. I expected to see similar magnitude of drop in titer compared to the ELISA, but after 48Hrs, barely any cells turned up FLAG+ (~0.3% transduction efficiency, which will lead to a significant difference in the 2 calculated titer: 10^8 vs 10^4). The intracellular staining method works since I had positive staining (> 50% FLAG+) with the GeCKO virus transduced cells selected using puromycin.

Does this mean the ELISA assay is not optimal for determining the titer for the GeCKO pooled library virus? Or should I be performing my cell culture a bit different for titering the pooled library (longer incubation time: >48Hrs, shorter incubation time: <24Hrs, etc.)?

I would also like to know what method/assay everyone uses to titer for the GeCKO virus.

Any suggestions/recommendations are welcome

Thank you

Chia-Hung (Jay) Tai

[Neville Sanjana]

Hi Jay,

I recommend you skip the titering kit and instead do a functional titer by measuring % puro resistant cells after transducing with the 1-vector GeCKO library pooled virus. This will directly allow you to calculate MOI for library virus without the intermediate steps of ELISA, etc.

Hope that helps,

Neville

[ct...@lji.org]

Thank you Neville, I will give that a try

I have another quick question: The reason why I have been focusing on the viral titer is because we want to transduce our cells with a MOI of 0.3 as stated in your paper "Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells". We want to transduce HL-60 (neutrophil-like cells) using the GeCKO pool library lentivirus, but the cell-line is not so easily transfectable. I'm curious to find out what your typical transduction efficiency is with A375 and HUES62 at the low MOI so I can determine whether it's feasible to perform the screening using our cell line, which has 5-20% transfection efficiency through other methods such as electroporation. 

I understand that we will be selecting the cells under puromycin, but selection with such low efficiency can be potentially difficult. The MOI is kept low to prevent potential mult-gene knockout in a single cell, but is there a range of MOI that can be used (to give room for error in the titer calculation)? Would you recommend using a slightly higher MOI to boost the transduction efficiency, or are there other methods to boost the transduction without increase the amount of virus added. 

Much appreciated for all the suggestions

Warm regards,

Chia-Hung (Jay)

[Trent Ferguson]

Hi Neville,

Thanks for answering all the questions coming up regarding CRISPR screening. We're trying to determine the best way to assess titer in our lab and so far haven't had great success with staining for Flag, nor doing gPCR (though we're trying to optimize).

Regarding doing the puro kill curve, are you quanitfying by simply checking for cell viability after a day or two? It seems the problem with this approach is that the resistant clones will grow over two days and this will confound the results. Do you simply factor in that the cells  double a certain amount and then back calculate the titer? Also, regarding cell viability, do you you check simply by using a cell counter or do you FACS with a viability stain?

Any help/advice you can provide would be greatly appreciated. Thanks in advance!

Best,
Trent

[Neville Sanjana]

Hi Jay,

Yes, there is a range. As long as your % survival after puro selection is in a reasonable range (20-50%), you can be fairly certain that most of the surviving cells are single infection events. As for boosting transduction efficiency, I think it is generally accepted in the field to go up to 50% puro survival. Beyond that, you will have a high probability of multiple infection events.

If you are interested in how to convert between MOI, % puro survival and single infection percentage (SIP), you can check out the Supplementary methods of our 2015 paper (A genome-wide CRISPR screen in a mouse model of tumor growth and metastasis). Wikipedia also has a nice explanation and Poisson model computation: https://en.wikipedia.org/wiki/Multiplicity_of_infection

Hope that helps,

Neville

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[Neville Sanjana]

Hi Trent,

I think it will be difficult to accurately quantify viral titer with FLAG-Western or qPCR methods, as these methods don’t factor in specifics of transduction. What we do is transduce cells with different volumes of virus, wait 24 hours and then split each well (each viral volume) into 2 wells (+ puro and - puro). When you do this split, make sure to plate at relatively low density (enough that cells will not be near confluence after 48 hours, which is how long to wait before counting). In parallel to the CRISPR virus transduction, make sure to have a no infection control or infection with a virus like CMV-EGFP that does not confer puro resistance. 

Usually, after 2 days, all cells are dead in the control well (+ puro). Then, you can compute the percent survival for each viral volume by taking the ratio of the + puro to the - puro well for each viral volume. Typically, we titer using the exact same number of cells per well that we’d use in the real screen. We select the viral volume that is closest to 20-50% puro survival. 

For cell counts, we have used Alamar Blue, fancy cell counters (Nexcelom Cellometer or Coulter counter, no stain), and a regular hemocytometer with manual count (no stain). All methods work equally well, so feel free to use whatever is most convenient for you.

Hope that helps,

Neville

[Trent Ferguson]

Hi Neville,

Thanks for the detailed reply! Very helpful and we'll switch to the puro resistance method.