Site-specific recombinatorics: in situ cellular barcoding with the Cre Lox system

TL;DR

This paper introduces a novel in situ cellular barcoding method using the Cre Lox system, enabling high-diversity, stable genetic labels within native tissue environments, surpassing previous approaches in diversity and feasibility.

Contribution

The authors design a new Cre Lox-based barcode construct that achieves significantly higher diversity by exploiting sequence constraints during recombination, suitable for in vivo tissue applications.

Findings

Achieves orders of magnitude higher barcode diversity than previous methods.

Can be implemented with existing technology.

Exceeds the number of lymphocytes in mice.

Abstract

Cellular barcoding is a significant, recently developed, biotechnology tool that enables the familial identification of progeny of individual cells in vivo. Most existing approaches rely on ex vivo viral transduction of cells with barcodes, followed by adoptive transfer into an animal, which works well for some systems, but precludes barcoding cells in their native environment, such as those inside solid tissues. With a view to overcoming this limitation, we propose a new design for a genetic barcoding construct based on the Cre Lox system that induces randomly created stable barcodes in cells in situ by exploiting inherent sequence distance constraints during site-specific recombination. Leveraging this previously unused feature, we identify the cassette with maximal code diversity. This proves to be orders of magnitude higher than what is attainable with previously considered Cre Lox barcoding approaches and is well suited for its intended applications as it exceeds the number of lymphocytes or hematopoietic progenitor cells in mice. Moreover, it can be built using established technology.