Large donor CRISPR for whole-CDS replacement of cell adhesion molecule LRRTM2

Abstract

The cell adhesion molecule LRRTM2 is crucial for synapse development and function. However, our understanding of its endogenous trafficking has been limited due to difficulties in manipulating its coding sequence (CDS) using standard genome editing techniques. We instead replaced the whole LRRTM2 CDS by adapting the recent CRISPR method Targeted Knock-in using Two Guides (TKIT), enabling complete control of LRRTM2. In primary rat hippocampal cultures, N-terminally tagged, endogenous LRRTM2 was found in 80% of synapses, and synaptic LRRTM2 content correlated with PSD-95 and AMPAR levels. LRRTM2 was also enriched with AMPARs outside synapses, demonstrating the sensitivity of this method to detect relevant new biology. Finally, we leveraged total genomic control to increase the synaptic levels of LRRTM2 via simultaneous mutation of its C-terminal domain, which did not correspondingly increase AMPAR enrichment. The coding region of thousands of genes span lengths suitable for whole-CDS replacement, suggesting this simple approach will enable straightforward structure-function analysis in diverse cellular systems.