An optimized CRISPR/Cas9 approach for precise genome editing in neurons

Area of Science:

• Molecular Biology
• Genetics
• Neuroscience

Background:

• Efficiently knocking in large DNA fragments to label endogenous proteins is difficult, especially in non-dividing cells like neurons.
• Existing CRISPR/Cas9 methods face challenges with precision and efficiency for genomic knock-in in complex cellular environments.

Purpose of the Study:

• To develop a novel CRISPR/Cas9-based approach for efficient and precise knock-in of large DNA fragments into non-dividing cells.
• To demonstrate the utility of this method for labeling endogenous synaptic proteins and studying their dynamics in vivo.

Main Methods:

• Development of Targeted Knock-In with Two (TKIT) guides, a CRISPR/Cas9 system targeting non-coding regions to ensure resistance to INDEL mutations.
• Application of TKIT for labeling endogenous synaptic proteins with various tags in mouse primary cultured neurons (up to 42% efficiency).
• In vivo validation using in utero electroporation and viral injections in mice to label AMPAR subunits with Super Ecliptic pHluorin for two-photon microscopy and FRAP analysis.

Main Results:

• TKIT achieved high efficiency (up to 42%) in labeling endogenous synaptic proteins in mouse neurons.
• Successfully visualized endogenous AMPARs in vivo and assessed their mobility using TKIT.
• Demonstrated TKIT's efficacy in rat neurons, highlighting its potential across different model organisms.

Conclusions:

• TKIT is a robust and precise CRISPR/Cas9-based method for efficient knock-in of large DNA fragments, particularly in challenging non-dividing cells.
• TKIT enables visualization and functional studies of endogenous proteins in vivo, advancing neuroscience research.
• The broad applicability of TKIT across species underscores its potential as a versatile tool for genome editing and protein labeling.