Digital barcodes are a pervasive and highly utile means of tracking just about anything that you can imagine. Through the application of barcode technology we can create a grand overview of the journey each book, beer, burger, or boat took to travel from A to Z. Scientists would love a similar system to track cells and their progeny in multicellular animals, although most commonly used labeling strategies (dyes, radioactive tracers, genetic markers, and genetic recombination etc.) suffer from limitations that restrict their utility. However, this may all now change, and it’s our old friend CRISPR/Cas9 to thank yet again!
Several reports from Science and the arXiv and bioRxiv preprint servers have described the first steps towards the creation of evolving genetic “barcodes” to track cell lineages in multicellular systems based on CRISPR/Cas9 gene-editing technology!
GESTALT: The Shape of Things to Come
The first report from Jay Shendure (Howard Hughes Medical Institute, Seattle, USA) describes a strategy the authors named GESTALT, for “genome editing of synthetic target arrays for lineage tracing”. Initially assessed in a human embryonic kidney cell line, this strategy took advantage of combinatorial and cumulative CRISPR/Cas9-based genome editing to introduce mutations in a DNA barcode.
In this case, the barcode consisted of a contiguous artificial array of CRISPR/Cas9 target sites that can be easily read through single cell sequencing. The first target site represented a perfect match for a single guide RNA (sgRNA), with the following sites modified to be progressively more off-target in order to allow for barcode evolution following each cell division. This strategy can generate thousands of unique barcodes encoding information about the relationships between cells and can, therefore, provide us with sufficient data to reconstruct cell lineage relationships.
The authors applied this strategy to zebrafish (Danio rerio) embryos to demonstrate that most cells in adult organs derive from a small number of embryonic progenitors and that progenitors do not contribute equally to germ layers and organ systems. Handily, the authors have made their tree reconstructions for cell culture, zebrafish embryo, and zebrafish adult experiments available for us all to discover at gestalt.gs.washington.edu. For more details, see the full study at Science, May 2016.
Worming Around CRISPR-based Lineage Trees
Using a similar strategy, researchers from the laboratory of Stephen R. Quake (Stanford) applied CRISPR/Cas9 to barcode cells in the nematode worm C. elegans, an organism with a completely known lineage tree. This time, the group targeted ten sites within the enhanced green fluorescent protein (EGFP) gene by injecting Cas9 and 10 specific sgRNAs into the gonad of worms from an EGFP-expressing strain. This, therefore, would allow DNA barcoding to start in the following worm progeny with EGFP knockdown used as a marker for successful CRISPR/Cas9 genome editing. Along with their proof-of-concept, the group also applied a simulation and employed an analytical model to understand the overall information coding capacity of the system. See all the highly technical, but highly interesting, details at arXiv.
Scartrace: Say Hello to My Little Friend
Now, our next study doesn’t come from Tony Montana (Cuba), but rather the (probably) more sedate pairing of Jan Philipp Junker and Alexander van Oudenaarden (Hubrecht Institute/University Medical Center Utrecht, Netherlands). Their paper introduces “scartrace”: a strategy for whole-organism lineage tracing based on the ability of our “little friend” Cas9 to induce short insertions or deletions (indels), or as they put it, genetic “scars”.
Their tactic involved targeting multiple histone-GFP transgene loci in a zebrafish line by injecting GFP-sgRNA and Cas9 into 1-cell stage embryos and later sequencing GFP from resultant progeny to elaborate cell lineage maps. They also demonstrated the utility of this system by applying cell tracing to study the regeneration of the caudal fin in the adult; a structure consisting of about a dozen different cell types derived from cell-type specific precursors. See all the scar-y details at bioRxiv.
Homing CRISPR-Cas9 for Evolving Barcodes
The study presented by Prashant Mali and George M. Church (Harvard) also attempted to generate DNA barcodes using CRISPR/Cas9, but they applied a different tactic. They applied a homing guide RNA (hgRNA) scaffold that directs the CRISPR/Cas9 to target the hgRNA DNA locus itself. This made CRISPR/Cas9 target a different site after each cell duplication due to a change in the hgRNA specificity, a nice alternative! They also demonstrated the use of in situ sequencing technologies to read specific RNAs derived from these barcodes, allowing cell lineage tracing without the loss of information on position and cell type. See all the info on this study at bioRxiv.
mSCRIBE: Creating Biological Memory
Researchers from the laboratory of Timothy K. Lu (MIT) also targeted the guide RNA locus in their study, which they describe the development of a tool named the “Mammalian Synthetic Cellular Recorder Integrating Biological Events (mSCRIBE)”. The authors previously reported on the development of genomically encoded analog memory in Escherichia coli based on dynamic genome editing with retrons, and their new study applies CRISPR/Cas9-based genome editing to record biologically relevant information in vitro and in vivo.
To show the enormous potential of mSCRIBE, the authors adapted an acute inflammation model in which lipopolysaccharide (LPS) causes immune cells to release tumor necrosis factor alpha (TNFa) that then activates the NF-kB pathway. The introduction of an NF-kB-inducible Cas9 expression cassette into human embryonic kidney cells carrying the self-targeting guide RNA (stgRNA) locus allowed the researchers to analyze LPS-induced acute inflammation over time. Using both in vitro and in vivo assays, the authors found that increased LPS dosage boosted Cas9 expression and led to a surge in the levels of stgRNA mutation. Therefore, the stgRNA mutations act as a type of memory to record biological signals in vivo! This paper is sure to get lodged in your memory, so get all the info you can at bioRxiv.
CRISPR/Cas9-based Barcoding: The Future
These exciting strategies represent a bold step in the right direction, but as always, the technology needs improvement to make it more effective and efficient. Keep scanning Epigenie.com to see all the new findings on CRISPR/Cas9-based genetic barcoding!