Restriction site BsmBI

[Rolf Warta]

Hello Zhang's lab,
I am new to the field of CRISPR and have first some probably stupid questions about the cloning of the sgRNA into the vector. I ordered the LentiCRISPR vector and want to insert a specific sgRNA. I designed it accordingly to your protocol with an CACCG-NNN… 5’ overhang for oligo1 and AAAC-NNN …5’ overhang for oligo2. However, if I digest the LentiCRISPR vector with BsmBI non of the two sequences will be produced. How does the ligation of sgRNA into the vector work?

Another question would be if the G-C basepair in front of the 20mer genomic sequence is optional if the specifc sequence will not start with a G?

Thanks for your help.

Rolf

[Neville Sanjana]

Hi Rolf,

The overhangs are produced after BsmBI digestion (and gel purification of the backbone piece). Please see the vector map and exact sequence: http://bit.ly/pLentiCRISPR

And, yes, for the initial G, you need to basepair it on the bottom strand with a C. If you choose not to use it, please keep in mind that U6 transcription is less efficient without the initial G.

hope that helps,

- Neville

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[Rolf Warta]

Hi Neville,
thank you for your explanations. I have already recognized the restriction site in the vector backbone. But it is still not clear to me in which way the 5’ overhangs of the sgRNA can interact with the sticky ends produced by BsmBI. Maybe I miss some very easy fundamental things.

Thanks for the information of the leading G. Would you also insert this G if the target sequence already starts with GGG.

Thanks

Rolf

[Neville Sanjana]

Hi Rolf,

If your 20bp targeting sequence starts with a G, it is fine to omit the extra G and just place the 20bp sequence after the CACC at the end of the U6 promoter.

Good luck!

- Neville

[troym...@gmail.com]

Rolf,

The beauty of BsmBI is that it cuts *outside* its recognition sequence.  See the NEB website.  I think this is where you're getting hung up.

Good luck,

Troy

[Rolf Warta]

Hi Troy,
indeed this is the answer to my question.
Thanks for the explanation.