CRISPR activation_SAM library

I'm interested in a custom CRISPR activation library using the SAM system and I have some questions:

  1. Which are the flanking sequences for the human SAM library?
  2. Are you aware of a dCas9 activity assay, as the one utilizing a GFP reporter assay for Cas9?, https://www.addgene.org/59702/
  3. Which format is better to use? The 2-vector or the 3-vector system?
  4. I downloaded from Addgene, the file with the Human SAM Target Sequences. The first column represents the target sequence and the fourth column the reverse complement. Which one should I use for extracting the sgRNA sequences?

  5. Which computational tool is the most appropriate for the analysis of CRISPR activation screens? In your Nat Prot 2017, RIGER is suggested. What about MAGeCK?


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Fuchsia Kelpieover 1 year ago

To answer your questions:

1. The flanking sequences are in the Python script for designing targeted libraries in my Nat Prot 2017 paper.

2. I have not used one myself, but that one looks good

3. I use the 3-vector system for easy to work with cell lines, and 2-vector for more finicky cell lines (like human embryonic stem cells).

4. the target sequence

5. I have switched to using MAGeCK for my screens. RIGER is what we used on our screens in the early days.

Purple Ghostover 1 year ago

Thank you very much!

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