Abstract
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.
Highlights
To learn about protein function in complex systems biologists need tools to visualize endogenous proteins
Homology Independent Target Integration (HITI) based KI strategies have been used to tag endogenous proteins in postmitotic cells such as neurons (Gao et al, 2019; Suzuki et al, 2016; Willems et al, 2020); these approaches are sensitive to INDEL mutations as the coding sequence is directly edited
Strategies to efficiently, and reliably, edit genomic DNA in post mitotic cells, such as neurons, are lacking behind methods based on homology-dependent recombination (HDR) that are routinely applied to dividing cells (Wang et al, 2013; Yang et al, 2013)
Summary
To learn about protein function in complex systems biologists need tools to visualize endogenous proteins. Such approaches have achieved higher KI efficiency in neurons compared to HDR-based methods These approaches directly edit the coding sequence of the target gene, possibly causing (insertion or deletion) INDEL mutations, which commonly occur at CRISPR/Cas mediated editing sites (Suzuki et al, 2016). Even when the KI proceeds as planned, the deletion or addition of some amino acids flanking the tag is unavoidable, and the exact site of KI is still dependent on a (protospacer-adjacent motif) PAM and reliable guide to be present at the right place To address these problems, we designed a new strategy – Targeted Knock-In with Two (TKIT) guides – to enable precise genome editing by utilizing two guide RNAs located in non-coding regions upstream and downstream of the coding sequence to be edited. TKIT provides an efficient, easy to use, and readily scalable approach for the tagging of endogenous proteins to facilitate their study in intact biological systems
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