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

The efficient knock-in of large DNA fragments to label endogenous proteins remains especially challenging in non-dividing cells such as neurons. We developed Targeted Knock-In with Two (TKIT) guides as a novel CRISPR/Cas9 based approach for efficient, and precise, genomic knock-in. Through targeting non-coding regions TKIT is resistant to INDEL mutations. We demonstrate TKIT labeling of endogenous synaptic proteins with various tags, with efficiencies up to 42% in mouse primary cultured neurons. Utilizing in utero electroporation or viral injections in mice TKIT can label AMPAR subunits with Super Ecliptic pHluorin, enabling visualization of endogenous AMPARs in vivo using two-photon microscopy. We further use TKIT to assess the mobility of endogenous AMPARs using fluorescence recovery after photobleaching. Finally, we show that TKIT can be used to tag AMPARs in rat neurons, demonstrating precise genome editing in another model organism and highlighting the broad potential of TKIT as a method to visualize endogenous proteins.

Automated summary

The novel CRISPR/Cas9 based approach TKIT allows efficient and precise genomic knock-in of large DNA fragments, particularly challenging in non-dividing cells like neurons. By targeting non-coding regions, TKIT is resistant to INDEL mutations and has shown high efficiency, up to 42%, in labeling endogenous synaptic proteins. Utilizing in utero electroporation or viral injections in mouse models, TKIT can be used to visualize endogenous proteins and assess their mobility, highlighting its broad potential for precise genome editing in various model organisms.