No non-targeting controls within the Zhang SAM library ?!

I am within a project using the Zhang Lab human whole genome SAM sgRNA library for a CRISPRa screening approach. For the analysis I would really like to determine the non-targeting control sgRNA distribution between the different conditions but there are not included in the library, right?

1.) Is there a logical reason why non-template control sgRNAs can not or should not be used in a CRISPRa sceen?

2.) Did you (user of this library) just give up the opportunity to analyze control guide distribution across samples?

3.) Are there separated control sgRNA SAM libraries available?

4.) If I would have to design my own control sgRNA library, is there a rule of thumb how many controls should be included if the targeting library comprises 70.000 guides?

Thank you in advance

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Fuchsia Kelpieabout 2 years ago

To answer your questions:

1. No - this was an oversight when we designed the library.

2. Yes we don't analyze the control guide distribution across amples.

3. No we currently do not have any available.

4. I would suggest including 500-1,000 non-targeting sgRNAs. You can get the target sequences for these from the other libraries, such as GeCKOv2.

Brown Hydraover 1 year ago

I am planning to use this library as well, and have a couple of questions:

1. Can I check if you guys are currently designing or planning to design the non-targeting controls for this library?

2. To clarify, with regards to the previous reply, are you suggesting that we can use the same non-targeting control sequences from the GeCKOv2 lib as controls for the CRISPRa SAM library?

3. If not, perhaps you can provide some tips/advice on how we can design/manipulate them into the existing library?

Pink Trollover 1 year ago

Meanwhile I generated a NTC-CRIPRs-SAM library using 1000 non-targeting guide sequences from the GeCKOv2 library.

I did this strict according to the Joung et al. 2017 protocol and it worked pretty fine.

If you have any questions feel free to ask, I am happy to help.

Fuchsia Kelpieover 1 year ago

happy to hear that this worked out for you.

to answer your questions:

1. No plans for doing this

2. Yes, same non-targeting sequences from the GeCKO for the CRISPRa. I did this for my lncRNA library in Joung Nature 2017 and worked well.

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