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

The cell adhesion molecule Leucine-Rich Repeat Transmembrane neuronal protein 2 (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. Instead, we replaced the entire LRRTM2 CDS by adapting a two-guide CRISPR knock-in method, enabling complete control of LRRTM2. In primary rat hippocampal cultures dissociated from embryos of both sexes, 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 neurons. Synaptic cell adhesion molecules are transmembrane proteins vital for neurotransmission, and their genes are frequently linked to disease. Mechanistic studies are challenging, however, because overexpression alters their protein trafficking and perturbs neuronal function, yet traditional gene-editing techniques typically permit manipulation at just single sites. Here, we replaced the entire coding sequence (CDS) of the critical adhesion molecule Leucine-Rich Repeat Transmembrane protein 2 (LRRTM2) with a freely editable custom donor sequence, and made the approach even more versatile through introduction of knock-in-dependent cell markers. Using whole-CDS replacement, we discover new aspects of LRRTM2 subcellular distribution and test its role in regulating synaptic proteins. The approach should be suited to structure-function analysis of many other neuronal proteins in their endogenous genetic locus.