Large-scale parallel characterization of RNA-guided nuclease activity and specificity

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Rapid advancements in RNA-guided nucleases (RGNs) necessitate systematic performance evaluations.
• Existing comparisons often lag behind the development of new RGN systems.

Purpose of the Study:

• To comprehensively evaluate the efficiency, specificity, and genomic structural impacts of 50 RGN systems.
• To characterize off-target effects and cytotoxicity of high-efficiency RGNs.
• To establish a multidimensional framework for RGN performance assessment.

Main Methods:

• Systematic screening of 50 RNA-guided nucleases (RGNs).
• Characterization of off-target effects and genomic structural impacts for a subset of RGNs.
• Assessment of cytotoxicity and cell proliferation inhibition.

Main Results:

• AsCas12a-Ultra, LbCpf1, and AsCas12a-Plus showed comparable or superior efficiency to SpCas9.
• enOsCas12f1 demonstrated high efficiency and small size, suitable for in vivo delivery.
• AsCpf1-YH and FnCpf1 had the lowest off-target risks, while DpFNuc had the highest.
• enCas12f-HKRA frequently caused chromosomal translocations; Cas12j-SF05 posed a lower risk.
• High-efficiency RGNs were linked to translocation hotspots.
• enRhCas12f1 and SpaCas12f1 exhibited low cytotoxicity; enAsCpf1-HF inhibited cell proliferation.

Conclusions:

• A multidimensional performance evaluation framework for RGNs was established.
• The study provides a data-driven tool to aid in selecting appropriate RGNs for precise genome editing.
• Specific RGNs were identified with favorable profiles for efficiency, safety, and reduced off-target effects.