A DNA Unwinding Equilibrium Serves as a Checkpoint for CRISPR-Cas12a Target Discrimination.
Jaideep SinghKevin G LiuAleique AllenWei JiangPeter Z QinPublished in: bioRxiv : the preprint server for biology (2023)
CRISPR-associated proteins such as Cas9 and Cas12a are programable RNA-guided nucleases that have emerged as powerful tools for genome manipulation and molecular diagnostics. However, these enzymes are prone to cleaving off-target sequences that contain mismatches between the RNA guide and DNA protospacer. In comparison to Cas9, Cas12a has demonstrated distinct sensitivity to protospacer-adjacent-motif (PAM) distal mismatches, and the molecular basis of Cas12a's enhanced target discrimination is of great interest. In this study, we investigated the mechanism of Cas12a target recognition using a combination of site-directed spin labeling, fluorescent spectroscopy, and enzyme kinetics. With a fully matched RNA guide, the data revealed an inherent equilibrium between a DNA unwound state and a DNA-paired duplex-like state. Experiments with off-target RNA guides and pre-nicked DNA substrates identified the PAM-distal DNA unwinding equilibrium as a mismatch sensing checkpoint prior to the first step of DNA cleavage. The data sheds light on the distinct targeting mechanism of Cas12a and may better inform CRISPR based biotechnology developments.
Keyphrases
- genome editing
- crispr cas
- single molecule
- circulating tumor
- nucleic acid
- cell free
- molecular dynamics
- dna damage
- atomic force microscopy
- living cells
- cell cycle
- circulating tumor cells
- minimally invasive
- genome wide
- electronic health record
- cancer therapy
- big data
- cell proliferation
- aqueous solution
- data analysis
- artificial intelligence
- gene expression
- drug delivery
- machine learning
- transcription factor
- high resolution