A Mechanistic Study on the Tolerance of PAM Distal End Mismatch by SpCas9

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2024 Jun 5

PMID 38838774

Authors

Dhritiman Dey

Rudra Chakravarti

Oindrila Bhattacharjee

Satyabrata Majumder

Dwaipayan Chaudhuri

Kazi Tawsif Ahmed

Dipanjan Roy

Bireswar Bhattacharya

Mansi Arya

Anupam Gautam

Rajveer Singh

Rahul Gupta

Velayutham Ravichandiran

Dhrubajyoti Chattopadhyay

Abhrajyoti Ghosh

Kalyan Giri

Syamal Roy

Dipanjan Ghosh

Affiliations

Soon will be listed here.

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 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 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 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 should be taken into account.

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