A versatile CRISPR-based system for lineage tracing in living plants

Abstract

Individual cells give rise to diverse cell lineages during the development of multicellular organisms. Understanding the contribution of these lineages to mature organisms is a central question of developmental biology. Several techniques to document cell lineages have been used, from marking single cells with mutations that express a visible marker to generating molecular bar codes by CRISPR-induced mutations and subsequent single-cell analysis. Here, we exploit the mutagenic activity of CRISPR to allow lineage tracing within living plants. Cas9-induced mutations are directed to correct a frameshift mutation that restores expression of a nuclear fluorescent protein, labelling the initial cell and all progenitor cells with a strong signal without modifying other phenotypes of the plants. Spatial and temporal control of Cas9 activity can be achieved using tissue-specific and/or inducible promoters. We provide proof of principle for the function of lineage tracing in two model plants. The conserved features of the components and the versatile cloning system, allowing for easy exchange of promoters, are expected to make the system widely applicable.

Where do they come from, where do they go: cell lineage tracing with CRISPR

“…Additionally, it is crucial for lineage tracing that only a few cells express the reporter gene so that lineages of single cells can be observed. In the highlighted publication, Mattia Donà and colleagues used nuclear fluorescent proteins that are expressed depending on the mutagenic activity of CRISPR as cell lineage tracing reporters for living tissue (Donà et al., 2023). The reporter system labels the nuclei of individual cells based on frameshifts induced by CRISPR/Cas9, with mClover fused to Histone 2B (H2B) as a readout.…”

“…(b) H2B‐mClover signal (green) in Arabidopsis root cell lineage induced by Cas9 expressed under the GL2 promoter (upper panel), and in a shoot apical meristem cell lineage induced by Cas9 expressed under the CLV3 promoter (lower panel). (c) H2B‐mClover signal (green) in Marchantia gemmae, induced by Cas9 expressed under the YUC2 promoter; figure modified from (Donà et al., 2023).…”

A history-dependent integrase recorder of plant gene expression with single cell resolution

“…Systems based on CRISPR-Cas9 mediated mutations and subsequent DNA sequencing enabled recording of the Wnt signaling pathway in mammalian cells 15 although this approach lacked spatial resolution. DNA-based recording devices utilizing CRISPR have also been used for lineage tracing, for example in reconstructing lineages in Arabidopsis tissues 16 , zebrafish organs 17 , and in development of a lineage tracing mouse line 18 . Researchers have also utilized the DNA recombination abilities of integrases for similar DNA-based recording approaches.…”