Hi Min,

The good news is that I successfully cloned my sgRNA into the lentiCrispr v2 backbone! The bad news is that it took a lot of troubleshooting and optimization, and I'm still not completely sure how we got it to work. We had trouble with Esp3I too, so we ended up switching back to BsmBI and just trying various digestion conditions: different incubation times and temperatures and plasmid quantities. For example, NEB recommends to digest with BsmBI at 55 degrees, but we found that digesting at 37 degrees helped reduce degradation. Second, we originally did long digestions (even overnight) because we thought it might be difficult to distinguish the large, digested versus full-length, undigested products in a gel, but we ended up having to digest for 1.5 hours (or less) so that it didn't completely degrade. Finally, we switched our gel extraction kit from Qiagen's to Thermo's GeneJET kit, which can be used to purify DNA up to 20 kb in size (unlike Qiagen's standard kit, which only claims to purify up to 10 kb fragments). This combination of changes made our cloning work! Hopefully it works for you too.

-Nicole

Hi, Ashlee.

I think I need some more information. What do you use to elute your DNA when purifyng before digesting? If you use anithing which contains EDTA it may partially chelate your Mg2+ ions in your reaction. Your reagents may also be contaminated with nucleases, have you also tried changing them? Another reason may be that DNA is binding to salts to stabilize itself and decreasing their presence (as DNA is 2 or 3 times higher than usual), which can make your BsmBI go unstable and tending to star activity due to conformation relaxation, which can be of importance since NEBuffer 3.1 is recommended for enzymed that need salt-containing buffers.

I hope this will help!

Can you maybe share your detailed protocol? 

Hi Ashlee, sometimes it is more difficult to find an answer for cloning problems than finding an alternative way to go. I would suggest you to use Esp3I from ThermoFisher. It has the same cutting site as BsmBI but is working at 37°C. We are using it for the LentiCRISPR vector in our lab.

Best

Lena

 Thanks a lot! 

Hi Ashlee,

I've been having the same trouble as you, so I'm going to try Esp3I as well. (Thanks to everyone for their suggestions!) Just as a heads-up, Thermo Scientific recommends adding DTT (dithiothreitol) to the Esp3I digestion, and DTT is not supplied with the enzyme. We didn't have DTT, so we tried digesting without it, and it didn't work. Now we are just waiting for DTT to arrive. Hopefully that does the trick. Good luck!

-Nicole

Hi Ashlee and Nicole

May I know if  Esp3I has worked well with your lentiGuide-Puro vector? I'm really glad I came across your post as I will be using this vector for my project. :)

Please let me know

Thanks

Min

Hi Min,

The good news is that I successfully cloned my sgRNA into the lentiCrispr v2 backbone! The bad news is that it took a lot of troubleshooting and optimization, and I'm still not completely sure how we got it to work. We had trouble with Esp3I too, so we ended up switching back to BsmBI and just trying various digestion conditions: different incubation times and temperatures and plasmid quantities. For example, NEB recommends to digest with BsmBI at 55 degrees, but we found that digesting at 37 degrees helped reduce degradation. Second, we originally did long digestions (even overnight) because we thought it might be difficult to distinguish the large, digested versus full-length, undigested products in a gel, but we ended up having to digest for 1.5 hours (or less) so that it didn't completely degrade. Finally, we switched our gel extraction kit from Qiagen's to Thermo's GeneJET kit, which can be used to purify DNA up to 20 kb in size (unlike Qiagen's standard kit, which only claims to purify up to 10 kb fragments). This combination of changes made our cloning work! Hopefully it works for you too.

-Nicole

Hi Min, I actually didn't follow up with this and I never used Esp3l, but Nicole's answer seems very helpful! Good luck with your research!

Thank you Nicole for your suggestions and opinions! 

Ashlee

Hi Nicole and Ashlee

Thanks very much for your input! I definitely will be trying the digestion at 37C degree instead of 55C degree as suggested by NEB :) Hopefully it works out well

Min

Nicole's suggestion was great. I digested lentiCRISPR v2 with NEB BsmBI at 37 degrees with 30 min and got nice results finally. Thanks a lot!

Hi everyone! While I found this thread helpful to find out others had issues like me... changing from BsmB1 to Esp3I did not help because the issue was more to do with the quality of the extracted DNA that I was trying to digest. After multiple attempts of adding different concentrations of DMSO etc... and being unsuccessful, I sent multiple samples of DNA for Sanger sequencing. What I found was that the samples that I MIDI prepped had many NNNN's though the BsmBI sequence. However, the MINIprep did not. So when I used my MINIprepped samples, all of them beautifully ligated the "stuffer" sequence out and I was able to ligate my oligo sequencing into it in its place as per the LentiCRISPRv2 protocol. Just a little trouble shooting tid bit that might help through what was a very frustrating process for me :)

Min Hui Tan

Hi Min,

Have you made the BsmbI work at last ? I am now getting the same trouble and tried 37 ℃ for 1 hour, but it seems not work at all.

Thanks,

Xiangbin

Hi Xiangbin,

The recommended temperature for digestion with BsmBI is 55C. The isoschizomer Esp3I cuts at 37C. In case of no cutting, I would stick to the recommended temperature.

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