Description:
Efficient and Scalable tagging of endogenous genes
Homology-independent intron targeting (HITI) for scalable tagging of endogenous genes
Market Need
Fusing endogenous proteins with fluorescence or epitope tags is a widely used and essential approach for studying proteins within their natural regulatory context. The advent of CRISPR tools for modifying the genome has made this easier and even more accessible, yet scalability is still very limited. The need for a gene-specific Homology Directed Repair (HDR) template requires costly synthesis or labor-intensive molecular cloning, and since precise targeting must be achieved in frame with the coding sequence, it necessitates careful design of reagents and screening of clonal cell lines to avoid disruptive editing at the non-tagged allele. An alternative approach for generating endogenous fusions is by random integration of synthetic exons delivered by transposons or retroviral particles7. This approach, known as “protein trapping” or “CD-tagging”8, is inexpensive and scalable, yet the random nature of tag integration precludes its use for the generation of curated libraries of fusion cell lines.
Technology Overview
The Shalem lab has developed Homology-Independent Targeted Integration (HITI) with a synthetic exon donor containing a fluorescence tag to preform targeted protein trapping at intronic locations to insert a donor flanked by splice acceptor and donor sites. In this case the donor is generic, the same one can be used for any gene, the generation of additional fusion cell lines therefore only requires the cloning of additional intron-targeting sgRNAs. dramatically reducing the costs of generating reporter cells lines. Intronic HITI also tolerates: (1) mutations in the non-tagged allele, as those are intronic and typically non-disruptive, and (2) indels that flank the inserted donor as a result of HITI-based editing.
Advantages
• Fast and cost effective
• Scalable
Application
• Fast tagging of endogenous genes
• Diagnostics
• Research
Stage of Development: in vitro proof of concept