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Updated: Jun 28, 2025
Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
Published on: February 26, 2019
Live-cell imaging reveals the trade-off between target search flexibility and efficiency for Cas9 and Cas12a
Lorenzo Olivi1, Cleo Bagchus1,2, Victor Pool1,2
1Laboratory of Microbiology, Wageningen University & Research, Wageningen, The Netherlands.
Abstract:
CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the last years, rational engineering led to the creation of PAM-relaxed variants SpRYCas9 and impLbCas12a to broaden the targetable DNA space. By employing their catalytically inactive variants (dCas9/dCas12a), we quantified how the protein-specific characteristics impact the target search process. To allow quantification, we fused these nucleases to the photoactivatable fluorescent protein PAmCherry2.1 and performed single-particle tracking in cells of Escherichia coli. From our tracking analysis, we derived kinetic parameters for each nuclease with a non-targeting RNA guide, strongly suggesting that interrogation of DNA by LbdCas12a variants proceeds faster than that of SpydCas9. In the presence of a targeting RNA guide, both simulations and imaging of cells confirmed that LbdCas12a variants are faster and more efficient in finding a specific target site. Our work demonstrates the trade-off of relaxing PAM requirements in SpydCas9 and LbdCas12a using a powerful framework, which can be applied to other nucleases to quantify their DNA target search.
Insights
CRISPR-Cas genome editing nucleases, SpyCas9 and LbCas12a, were studied for their DNA target search efficiency. LbCas12a variants demonstrate faster and more efficient DNA targeting compared to SpyCas9 variants, especially when PAM requirements are relaxed.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas systems are essential genome editing tools utilizing Cas nucleases like SpyCas9 and LbCas12a.
- These nucleases differ in PAM recognition, guide structure, and DNA cleavage mechanisms.
- Engineered variants, SpRYCas9 and impLbCas12a, expand the DNA targeting range by relaxing PAM constraints.
Purpose of the Study:
- To quantify the impact of protein-specific characteristics on DNA target search kinetics for CRISPR-Cas nucleases.
- To compare the DNA search efficiency between wild-type and engineered SpyCas9 and LbCas12a variants.
- To establish a framework for analyzing DNA target search dynamics in other nucleases.
Main Methods:
- Utilized catalytically inactive dCas9 and dCas12a variants fused to PAmCherry2.1 for single-particle tracking in Escherichia coli.
- Performed single-particle tracking experiments to derive kinetic parameters for DNA target interrogation.
- Employed simulations and live-cell imaging to validate findings on target search efficiency.
Main Results:
- Kinetic analysis revealed that LbCas12a variants interrogate DNA faster than SpyCas9 variants when using non-targeting RNA guides.
- In the presence of a specific target DNA site, LbCas12a variants exhibited superior speed and efficiency in target acquisition compared to SpyCas9 variants.
- The study quantified the trade-offs associated with relaxing PAM requirements in both SpyCas9 and LbCas12a systems.
Conclusions:
- LbCas12a nucleases demonstrate inherent advantages in DNA target search speed and efficiency over SpyCas9.
- Relaxing PAM specificity in engineered variants impacts DNA search dynamics, presenting a trade-off between target accessibility and search kinetics.
- The developed single-particle tracking framework provides a versatile method for quantifying DNA search processes in various nucleases.
