Photocleavable Guide crRNAs for a Light-Controllable CRISPR/Cas9 System

Area of Science:

• Molecular Biology
• Genetic Technology
• Biochemistry

Background:

• CRISPR/Cas9 systems offer precise gene editing but controlling their activity and specificity remains a challenge.
• Developing controllable and precise RNA-targeted CRISPR/Cas9 systems is crucial for advancing molecular biology and genetic technologies.

Purpose of the Study:

• To design and evaluate photocleavable guide CRISPR RNAs (crRNA) for spatiotemporally controlled CRISPR/Cas9 genome editing.
• To investigate the impact of photolabile linkers (PL) on crRNA stability, Cas9 nuclease activity, and DNA cleavage specificity.

Main Methods:

• Synthesis of photocleavable crRNAs with 2'-modified analogs incorporating one or two 1-(2-nitrophenyl)-1,2-ethanediol photolabile linkers (PL).
• Assessment of crRNA photocleavage efficiency and kinetics under UVA irradiation.
• Evaluation of the effect of photocleavage on Cas9 nuclease activity in vitro.
• Analysis of DNA cleavage specificity by Cas9 with modified crRNAs.

Main Results:

• Photocleavable crRNAs were successfully synthesized and demonstrated degradation under mild UVA irradiation (rate constants 0.24-0.77 min⁻¹).
• Photocleavage significantly reduced Cas9 nuclease activity in vitro, with two PLs yielding faster crRNA destruction than one.
• The incorporation of PLs enhanced the specificity of Cas9-mediated DNA cleavage for fully complementary targets.

Conclusions:

• Photocleavable crRNAs enable spatiotemporal control over CRISPR/Cas9 genome editing.
• This approach effectively mitigates off-target effects by allowing the system to be switched off on demand.
• The developed photocleavable crRNA technology represents a significant advancement in precise and controllable genome editing applications.