Re: LentiCRISPR v1 vs v2 - Lipofectamine 3000

[Neville Sanjana]

Very nice genome engineering tools and also many thanks for making available the v2 so soon.

Glad to do so!

 

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 L3000 myself yet. I did not make much of an effort to optimize lenti production since the protocol I was given by a labmate worked quite well from the beginning but I'm sure there's room to improve.

 

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, the changes in the amplification protocol are designed to reduce unwanted recombination. Not sure what you mean by rounds of library amplification.... generally we always re-transform (to amplify more library) from the master stock. I recommend each lab makes an initial master stock (using the aliquot you got from Addgene) and then use that for all future re-transformations. Serial retransformations will result in a loss of representation. I do sequence every time I amplify the library using MiSeq.

best,

- Neville

[jian...@gmail.com]

Hi Neville,

Thanks for the detailed response. As for GeCKO v2 library, I have a couple more questions regarding to the library preparation:

1) In the protocol for v1 library, after electroporation, you used SOC as recovery medium, but in v2 protocol, it says recovery medium (medium provided with the cells). From the company's brochure, it says "Use of SOC or other media will result in lower transfromation efficiencies." Is that the reason you changed to company's recovery medium?

2) In the protocol for v1 library, plasmid was purified by Maxi Kit (cat# 12162). In the supplementary materials of your recent Science paper, it says using Endotoxin-Free Plasmid Maxiprep (Qiagen). I think the cat# for this one will be 12362. Do you think the quality of plasmid prepared from 12162 is enough? I will assume you also use 12162 to prepare the other two plasmids (pVSVg and psPAX2), right?

Thank you very much for your time.

Jack