“If all you have is a hammer, everything looks like a nail” is how the saying goes. But does that have to be true? A hammer can actually be a quite versatile instrument and, with some insight and ingenuity, be put to tasks that would previously have been thought outside of its purview. A hammer doesn’t have to be a hammer, if you tilt your head in the right way. That is the sort of philosophical statement that lends itself well to the scientific community.

While it is nice to be able to make completely new and revolutionary discoveries and tools, that very rarely is the case in science. That which is new is developed off the backs and minds of the old, finding things that were missed or applications for an implement that went untouched. There is much originality that can be gleaned from the old.

As should be expected by now, we’re here to talk about CRISPR. Barely a month or a week goes by without some contemporary sighting of a entirely different form or the formation of a tool for use in who knows how many scientific fields. Today’s deals with developmental biology and, to a fair extent, the medical community and disease research as a whole.

Making A Map

There have long been attempts in biology to properly map the development of an organism from a single cell to the trillions they eventually become. Understanding how each cell fits into its place and where it came from can explain not only developmental disorders, but also perhaps why conditions such as cancer seem more prevalent in certain cell types. The ability to make new organs and tissues also relies on being able to differentiate the right kinds of cells from preceding ones, so a map of their relationships to each other is of paramount importance. This sort of mapping is one we’ve talked about before and work continues onward to improve upon prior knowledge.

The most effective methods thus far of tracking individual cellular development have involved cellular labeling with inheritable genetic markers. But these only work for a limited number of cell divisions and can be broken down, altered, or removed entirely by cellular repair systems. Pre-synthesized markers that act as a sort of barcode have seen greater success in a larger amount of cells at once, but they are always only snapshots at one point in time during the development cycle due to their static nature.

With even more recent advances in items like CRISPR, in vivo generation of these barcodes has become a possible reality, allowing for a real-time analysis of development. Though this has been demonstrated to work in cultured cells and in simple invertebrates, there has yet to be an attempt in a comparatively complex organism. Researchers at Harvard University wanted to take that step and confirm its usefulness in broader applications.

A Chimeric Pattern

But it is certainly no easy task. Since gestation of mice, the model organism they chose for this test, takes place within the mother’s womb, the ability to use CRISPR on the zygote is made difficult. The long gestation time of the species, with accompanying significant amounts of cellular division, means any sort of change needs to last through that entire time period to provide a proper picture of embryonal development. The option they came upon uses multiple, independent mouse lines with different barcoding and lineage recording genetic systems in place in each.

The first step was to integrate into a mouse line a unique application of guide RNAs produced last year. These are known as homing guide RNAs (hgRNAs), which are constructed to direct the CRISPR complex to the gene sequence coding for themselves and cut it from the genome. Targeting their own genetic loci, in turn, allows for a larger amount of diverse mutation errors to be introduced than normal single guide RNAs (sgRNAs) can manage. These mutations then work as the expressed genetic barcodes for future generations.

To get these into the founder populations, the hgRNA sequences were transposed into mouse embryonic stem cells in a manner to cause them to be inserted multiple times into a single genome. These transgenic stem cells were then injected into blastocysts that were then themselves placed into the womb of a surrogate mouse mother.

A Unique Tattoo

The children that developed from those blastocysts are chimeric in nature thanks to some of their cells being the transgenic ones. Of the 23 mice that were born, eight of the males exhibited up to 60% of their cells being of the chimeric transgenic ones, which could be seen by the changed coat color. Five of those eight were found to have 20 total hgRNA sequences inserted in their germline cells to pass on to their own progeny. Thus, creating a founder population that they named the MARC1 (Mouse for Actively Recording Cells!) line.

Of these mice, 60 total hgRNA insertions were found across them, with 54 of them being able to be sequenced to find their locations. 26 of those were intergenic, being inside of a gene sequence, and 28 were located in introns nearby to known genes. Luckily, none of them were found to be inside an active exon of a gene, so no gene disruptions were expected to occur.

The founder population was next crossed with a line that had a Cas9 sequence inserted into them. The crossed children had their CRISPR sequences activated due to the presence of hgRNAs, resulting in mutations that created a developmental barcode during the entire period of gestation. Since hgRNAs continue to build up new mutations as gestation continues, it provides a timeline of that development and shows which cells were formed at what time and from what other cells they divided from. No earlier mutations are deleted, so the timeline is kept intact.

An Opening Into Life’s Development

Finally, the way these mutations pile on top of each other are in a recognizable and repeated way, so that similar mutation profiles indicate cells that are more closely related and have a closer cell “ancestor”. Even so, the amount of potential diversity of lineages is such that there are a maximum of 10 to the power of 74 possible combinations with the 41 hgRNAs that ended up in the final lines. This is a high enough number that each individual cell will have a unique barcode and there will be little chance of an accidental overlap to mess up the modeled tree of their connections.

The scientists noted that, more than just embryonic development, this technique can also be applied to cellular signals over time or be used in only specific organs or tissues to follow their particular formation in the body and how cells are replaced in them over time. The insights this can give into which cells are more likely to go cancerous cannot be overstated. They hope that this tool can serve for all those needs and more, as a deep dive mechanism to trace how cells change and what that means for life in general.

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Photo CCs: E15.5 mouse embryonic testes from Wikimedia Commons

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