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

• DOI: 10.7554/elife.65202
• Publication Date: 2021-03-10T17:00:38Z
• Authors: Huaqiang Fang; Alexei M Bygrave; Richard H Roth; Richard C Johnson; Richard L Huganir
• Keywords: Gene knockin; Endogenous retrovirus

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 a novel CRISPR/Cas9 based approach for efficient, and precise, genomic knock-in. Through targeting non-coding regions TKIT is resistant INDEL mutations. demonstrate labeling synaptic various tags, efficiencies up 42% mouse primary cultured Utilizing utero electroporation or viral injections mice can AMPAR subunits Super Ecliptic pHluorin, enabling visualization AMPARs vivo using two-photon microscopy. further use assess the mobility fluorescence recovery after photobleaching. Finally, we show that be used tag rat neurons, demonstrating precise genome editing another model organism highlighting broad potential method visualize proteins.