Mouse GeCKO library: Modify the lentiGuide-Puro backbone

For my research project, I would need to add a reporter construct into the lentiGuide-Puro backbone. Before proceeding, I would have few questions:

- Where would be the best place to insert my reporter construct? I can potentially add it upstream (AleI, PpuMI sites) or downstream (PspXI, SmaI sites) of the U6-sgRNA unit.

- Is there a way to add my reporter construct and avoid reconstructing the whole sgRNA library library?

Many thanks for your help!


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Fuchsia Kelpieover 4 years ago

If you do not need the Puromycin, you can replace it with your reporter construct. If you do need it, then downstream of the U6-sgRNA seems like the easiest place to insert it.

I think the cleanest way to do this is probably clone your new plasmid backbone first, PCR amplify the target sequence from the available sgRNA library, and clone into your new plasmid backbone using the library cloning protocol outlined in Joung Nat Protocols 2017. I've suggested this strategy to a few different people, but do not know if it worked well for them yet.

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