How to clone the gRNA in LentiCRISPR V2 (Addgene #52961) ?

[mazed fetta]

Hi all,

I am trying to clone the gRNA into the LenticrisprV2 vector from addgene ( #52961). It's my fourth try and it's still a failure. No bacteria growing in my petri dishes. I am desperate because I have tried several protocols but none have worked.

If someone has already managed to clone in this vector I would be indebted to him for giving me his protocol.

Thank you in advance for the person who will help me and to all the others who took the time to read this post.

Good day to all

Fetta

[Julia Joung]

Hi Fetta,

We have a detailed protocol for cloning gRNAs into the lentiCRISPRv2 vector using golden gate assembly in the validation section of Joung Nature Protocols 2017 that might be helpful for you.

Best,

Julia

--
You received this message because you are subscribed to the Google Groups "Genome Engineering using CRISPR/Cas Systems" group.
To unsubscribe from this group and stop receiving emails from it, send an email to crispr+un...@googlegroups.com.
To view this discussion on the web visit https://groups.google.com/d/msgid/crispr/0da7bf92-9b00-489c-93e0-d71a9c773bc6%40googlegroups.com.

[Robyn]

 We cloned into this vector with no problem. But we didn't follow the instructions they provide on Addgene to phosphorylate the oligos and dephosphorylate the vector. It is unnecessary as the two BsmBI sites in the vector don’t give compatible overhangs when cut, so the vector can't religate. We used highly competent DH5a cells (not Stbl3). Also I am not convinced that you need to gel purify the vector. If you put a high molar excess of annealed oligo into the ligation it should outcompete reinsertion of the stuffer fragment. Just make sure the vector is fully digested and then purify with a cleanup column. This worked for us with both Lenticrispr and px459 (cutting with BpiI for the latter). If all that fails you could try reordering your oligos, sometimes oligo QC is not as good as it should be.

[mazed fetta]

Thanks a lot for your answer! I will come back to you when I have the result

[Anthi Demetriadou]

Dear Robyn,

is it possible to provide a detailed protocol of how you clone sgRNAs into lentiCRISPRv2?

Thank you,

Anthi