High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion.

Haining Zhong,Tianyi Mao,Cesar C Ceballos,Lei Ma,Maozhen Qin,Crystian I Massengill,Michael A Muniak,Stefanie Kaech Petrie

doi:10.7554/elife.64911

Abstract

Precise and efficient insertion of large DNA fragments into somatic cells using gene editing technologies to label or modify endogenous proteins remains challenging. Non-specific insertions/deletions (INDELs) resulting from the non-homologous end joining pathway make the process error-prone. Further, the insert is not readily removable. Here, we describe a method called CRISPR-mediated insertion of exon (CRISPIE) that can precisely and reversibly label endogenous proteins using CRISPR/Cas9-based editing. CRISPIE inserts a designer donor module, which consists of an exon encoding the protein sequence flanked by intron sequences, into an intronic location in the target gene. INDELs at the insertion junction will be spliced out, leaving mRNAs nearly error-free. We used CRISPIE to fluorescently label endogenous proteins in mammalian neurons in vivo with previously unachieved efficiency. We demonstrate that this method is broadly applicable, and that the insert can be readily removed later. CRISPIE permits protein sequence insertion with high fidelity, efficiency, and flexibility.

Highlights

The CRISPR/Cas9 technology has revolutionized genomic editing (Cong et al, 2013; Doudna, 2020; Heidenreich and Zhang, 2016; Jinek et al, 2012; Mali et al, 2013)
To achieve high-fidelity fluorescent protein (FP) labeling, we developed the CRISPR-mediated insertion of exon (CRISPIE) method, which inserts a designer exon module that consists of an exogenous exon encoding the FP flanked by intronic sequences, including the splicing acceptor and donor sites, into the intronic region of the target gene via the non-homologous end joining (NHEJ) pathway (Figure 1A, Figure 1—figure supplement 1)
NHEJ-mediated donor DNA insertion can result in unwanted integration with inverted orientation, and INDELs can occur at loci where the donor fails to insert

Summary

Introduction

The CRISPR/Cas technology has revolutionized genomic editing (Cong et al, 2013; Doudna, 2020; Heidenreich and Zhang, 2016; Jinek et al, 2012; Mali et al, 2013). One of the major unresolved challenges is to use CRISPR-based technologies to precisely and efficiently knock in large DNA fragments in somatic cells of living animals (i.e., without generating transgenic animals) to label or modify endogenous proteins. Such technology holds promise for both studying biological mechanisms and gene therapy.

Methods

Results

Conclusion