What is the best way to costract a Crispr library

Question

[Assa Yeroslaviz]

We are planning to do a crispr library for several experiments. I would to ask the group here what is the best way to know which genes to take into the library.

Let's say we would like to analyze the RNA Polymerase II activity. Would it be enough to look up all the gene in the related GO category or functional annotations and create the sgRNA-library with these genes? E.g. GO:0001055 (RNA polymerase II activity) orGO:0006366 (transcription by RNA polymerase II) etc.

Or are there any special pre-prepared library one can order/ use?

Do one need to pay attention to any specific restrictions or limitations when such a library is created (ordered)?

I would appreciate any suggestions. thanks

[Ahill]

You might need to provide more details about your experimental goal and methods to get useful feedback on the question. Are you doing a screen to identify modulators of RNA Pol II activity (like a dropout screen), or looking to specifically disrupt function of genes related to RNA Pol II? For the gene selection bit, relying on GO categories only might be a blunt instrument that'll give you both false positives and negatives depending on what your experimental goal is. Are you interested in targeting RNA Pol II regulators or associated genes that might well be in different GO categories than the ones you list? There are commercial providers of sgRNAs and/or sgRNA libraries (e.g. Thermo, Synthego) from which you can order pre-prepared libraries. Different guide sequence selection strategies can apparently lead to better or worse editing performance, so paying attention to the design approach used (for commercial libraries) or selecting the best bioinformatics design approach (for doing it yourself) would also be important. You can of course also check out the literature to see what others have done.

[Assa Yeroslaviz]

Thanks for the reply. This is exactly my question. We are planning do different things and not one specific biological question. We would like to analyze different aspect of the RNA Pol-II and would like to know what kind of library one might use. Does it make sense to create one library for each of the questions or to try and answer all questions with one (maybe bigger) library).