English
 
Help Privacy Policy Disclaimer
  Advanced SearchBrowse
  1. View item / 
  2. Item Summary

Item

ITEM ACTIONSEXPORT
Add to Basket
  Local TagsRelease HistoryDetailsSummary

Released
Journal Article

2.5A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9

MPS-Authors
/persons/resource/persons271658

Gautam,  A       
IMPRS From Molecules to Organisms, Max Planck Institute for Developmental Biology, Max Planck Society;

External Resource
No external resources are shared
Fulltext (restricted access)
There are currently no full texts shared for your IP range.
Fulltext (public)
There are no public fulltexts stored in PuRe
Supplementary Material (public)
There is no public supplementary material available
Citation

Dey, D., Chakravarti, R., Bhattacharjee, O., Majumder, S., Chaudhuri, D., Ahmed, K., et al. (2024). 2.5A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9. Journal of Biological Chemistry, 300(7): 107439. doi:10.1016/j.jbc.2024.107439.


Cite as: https://hdl.handle.net/21.11116/0000-000F-616A-D
Abstract
The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we have focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we have designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation (RMSD) values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability plays a crucial role.