anyone has experience cleave human gDNA using crRNA and Cas12a?

[Jing Zhang]

What is the amount of gDNA should be used? Human gDNA is very viscous and it is hard to input >2ug gDNA in 30 ul. 

Let me provide more details about the project:

1. We designed two crRNAs targeting both ends of a cancer gene (50 kb long) to cleave the cancer gene gDNA using crRNA/Cas12. Since human gDNA cannot be sequenced directly, we designed qPCR primers spanning the cleavage site. If the Ct value for the cleaved sample is higher by 1 compared to control samples, we can estimate the cleavage efficiency to be 50%.
2. The second step involves protecting the ends of the cleaved fragment using the dNTPαS Fill-In method .
3. After protection, an exonuclease is used to digest the remaining gDNA. 

Another challenge is that the gDNA and the cleaved fragment are too long to be purified at each step. I tried using an NEB DNA purification column but lost most of the DNA. Each step uses a different buffer/enzyme and should be purified.

Any suggestions?

[Xavier De Deken]

Hi,

To extract gDNA from cells (GeCKO human library) before PCR amplification of the integrated sgRNA cassettes, we successful used the PCI-based protocol in the following paper ( Xu J, Zhou C, Foo KS, et al. Genome-wide CRISPR screen identifies ZIC2 as an essential gene that controls the cell fate of early mesodermal precursors to human heart progenitors. Stem Cells 2020;38(6):741–755; doi: 10.1002/stem.3168.). 

The purified gDNA is of good quality, but usually very concentrated. The stock solution was diluted to 0.5µg/µL to easily pipette the solution for PCR amplification. 

Hope it could help.

Regards,

Xavier DD