gRNA negative control - lentiCRISPRv2

[Kai]

Hi folks,

As I'm using lentiCRISPRv2-sgRNA to knock out my gene of interest in primary human cells, I'm wondering whether there is an established non-targeting control gRNA sequence that I can use to control for my downstream experiments.

When searching the old posts in the forum, 'Explorer' suggested origene has one (snapshot below). However, when I ran this sequence in crispr.mit.edu, it only scored 84 (snapshot below). I'm wondering if there is a better control out there. Thanks a lot!

[Neville Sanjana]

Hi Kai,

We have 1000 non-targeting controls for human and mouse genomes in the GeCKOv2 libraries. You can retrieve sequences for these guides here:

http://genome-engineering.org/gecko/?page_id=15  (just download the CSV file for the appropriate genome.... both A and B libraries have the same control, non-targeting guides)

Hope that helps,

Neville

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[Kai Wu]

Hi Neville,

Thanks for your quick reply. Yes, I did find a number of really good ones from the library. Do you guys have any preferred ones to use as control? 

Kai

[Neville Sanjana]

That's great to hear.... we don't have any preferred guides. They were designed in the same way, so any should work. (Maybe pick a few more than you need and then settle on a couple based on a downstream assay?)

All the best,

Neville

[Kai Wu]

Thanks Neville!

Kai

[Kai Wu]

Hi Neville,

For lentiCRISPRv2, do you also put an additional G in front of your gRNA (if there is no natural G as the first base) to boost the gRNA expression level from the U6 promoter, as suggested for PX330?

Thanks

Kai

[Neville Sanjana]

Hi Kai.... yes, I do. Of course, if your 20bp sequence already begins with a G, you can omit the extra G. But, in my work, I've always added an extra G for every guide (regardless of the initial base).

Best wishes,

Neville

[Kai Wu]

Hi Neville, 

Thank you for the super-speed reply! I just learned this trick of U6 promoter and realized after your reply that you guys have built the extra G into the cloning protocol as the U6 promoter ends after CACC. I though I need to reorder primers to add the extra G....

The FAQ page explained it well:

I'm wondering how much difference the extra G (with increased gRNA expression level I assume) can make for genome editing using Cas9. Have you done the comparison for a 20nt gRNA (not starting with G) and a 21nt version (G+the 20nt same gRNA) in terms of genome editing capacity/efficiency?

Sorry for the naive quest

Thanks again!

Kai

[Kai Wu]

Hi Neville,

Sorry to keep bugging you. I saw you were using lentiCRISPRv2 transient transfection to get gene KO which avoids the continuous Cas9 expression and thus potential off target effect. After transfection, I'm wondering if you do a puro selection procedure (like using v2 lentivirus) to select the ones that get transfected. Do you use the same concentration of puro for selecting the transiently tranfected ones as the ones transduced by v2 lentivirus?

Thank you!

Kai

[Neville Sanjana]

I have not done a side by side comparison myself but there is previous literature on U6 transcription with G vs. other bases in the initial position. I can't recall the references now, but I believe the difference is quite significant and so I would suggest using the G.

As a caveat, some versions of Cas9, such as the new eSpCas9 which has less off-target activity, may be more sensitive to mismatches and so, in certain cases, you might want to be careful about adding on the extra G.

Best wishes,

Neville

[Neville Sanjana]

Hi Kai,

When I do transient transfection of LCv2, I use the same puro concentation for selection. Just remember to only select for a few days maximum since transiently transfection usually does not result in genomic integration.

Best wishes,

Neville

[Kai Wu]

Thank you very much Neville! It's extremely helpful!

Best,

Kai

[Jixin Ding]

HI, I try to pick some no targeting sequences as control, but Blast the sequence samples in the library and found the sequences were match some genes in the genebank, could someone explain this ?Thanks.

[Robert Norgard]

Hi Neville! 

I used this post serval times to find non-targeting gRNAs. Can you clarify how these were designed? I.e. does the sequence target a place in the genome devoid of a pam sequence or is it just scrambled so much that there is no recognition. It appears that whenever I blast them, they always bind to some sequence (not 100% but still bound which makes me uneasy).

Best,

Bobby 

[fengya...@gmail.com]

Hi Neville, 

I downloaded the data sets for library A and B. I want to know how can I know which sequences are non-targeting controls, is there any tag in the name or ID number? Thanks!

[Geoffrey Siwo]

Hi everyone,

I have generated some non-targeting gRNAs that can be used as negative controls (see link). I generated the sequences using a probabilistic approach based on the frequency of each nucleotide at each position of experimentally validated gRNAs from the paper Kaur et al 2016.  Briefly, each gRNA sequence was generated by sampling each nucleotide at each position using the probability equal to that observed in each position of published gRNAs. You can test these gRNAs using the CRISPT MIT tool or your preferred method to select those that have no matches to your organism of interest and have high scores comparable to your gRNA. 

For example, 'RandomSequence 20' in the fasta file has a quality score of 97 from the CRISPR MIT design tool and its closest off-target site has a score of 0.4 and 4 mismatches in the human genome. To choose a negative control that matches your experiment, you can select a non-targeting gRNA that has a similar score and off-target profile like your 'real' gRNA. 

Let me know if you have any questions or thoughts.

Thanks

Geoffrey