Large Donor CRISPR for Whole-Coding Sequence Replacement of Cell Adhesion Molecule LRRTM2.

Area of Science:

• Neuroscience
• Molecular Biology
• Genetics

Background:

• The cell adhesion molecule leucine-rich repeat transmembrane neuronal protein 2 (LRRTM2) is vital for synapse development and function.
• Understanding endogenous LRRTM2 trafficking is challenging due to difficulties in standard genome editing of its coding sequence (CDS).

Purpose of the Study:

• To develop a novel method for complete control of endogenous LRRTM2 using CRISPR technology.
• To investigate the synaptic localization and function of LRRTM2 in primary rat hippocampal neurons.

Main Methods:

• Adaptation of a two-guide CRISPR knock-in method for whole-CDS replacement of LRRTM2.
• Utilizing N-terminally tagged endogenous LRRTM2 in primary rat hippocampal cultures.
• Employing total genomic control for targeted manipulation of LRRTM2's C-terminal domain.

Main Results:

• Endogenous LRRTM2 was detected in 80% of synapses, correlating with PSD-95 and AMPAR levels.
• LRRTM2 enrichment was observed with AMPARs both inside and outside synapses.
• Increasing synaptic LRRTM2 levels via C-terminal mutation did not proportionally increase AMPAR enrichment.

Conclusions:

• The developed CRISPR approach enables precise manipulation and study of LRRTM2.
• This method facilitates the detection of novel biological insights into synaptic protein interactions.
• The whole-CDS replacement strategy is applicable to a wide range of genes for neuronal structure-function analysis.