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Developmental biology - Barcoding Genes|
Recording every cell's history in real-time
The new method developed at the Wyss Institute and Harvard Medical School (HMS) uses gene barcodes to actively record cell division in fetal mice. Barcodes enable every cell line in a mouse to be traced back to its single-cell origin.
The research is published in Science magazine.
Gene barcodes are created by encoding a modified RNA molecule called a homing guide RNA (hgRNA). Developed in previous research, hgRNA molecules are engineered using the enzyme Cas9 of the famous CRISPR-Cas9. hgRNA can guide Cas9 to a targeted hgRNA sequence in the gene and cut that sequence out of the gene. When the cell repairs the cut, it introduces a gene mutation via the hgRNA sequence. Over time, mutations accumulate creating a unique barcode on that gene.
The researchers implemented the hgRNA-Cas9 system in mice by creating a "founder mouse" with 60 different hgRNA sequences scattered throughout its entire genome. They then crossed the founder mouse with mice that expressed the Cas9 protein, producing zygotes (fertilized eggs) whose hgRNA sequences started mutating shortly after fertilization.
Each hgRNA can produce hundreds of mutant alleles. Most genes have two alleles, a dominant allele and a recessive allele. Collectively, they can generate a unique barcode that contains the full developmental lineage of each of the ~10 billion cells in an adult mouse.
"In every single cell that divides to become the zygote, there's a chance that its hgRNAs will mutate," explains first author Reza Kalhor PhD, a postdoctoral research fellow at the Wyss Institute and HMS. "In each generation, all cells acquire their own unique mutations in addition to the ones they inherit from their mother cell, so we can trace how closely related different cells are by comparing which mutations they have."
The ability to continuously record cells' development also allows researchers to resolve a longstanding question regarding the embryonic brain: Does it distinguish its front from its back end first, or its left from its right side first?
By comparing hgRNA mutation barcodes in cells taken from different parts of two mouse brains, researchers found neurons from the left side of each brain are more closely related to neurons from the right side of the brain than to neurons from the left side of neighboring areas - suggesting that front-back brain patterning emerges first before left-right patterning, in the mouse central nervous system.
"This method allows us to take a model organism and reconstruct a full lineage tree all the way back to its single-cell stage." says Church. Researchers are now focusing on improving their readout techniques so they can analyze barcodes of individual cells and reconstruct the lineage trees recorded.
"If applied to disease models, barcoding via homing CRISPR could provide entirely new insights into how diseases such as cancers emerge."
In vivo barcoding using nuclease-induced mutations is a powerful approach for recording biological information, including developmental lineages; however, its application in mammalian systems has been limited. We present in vivo barcoding in the mouse with multiple homing guide RNAs that each generate hundreds of mutant alleles and combine for an exponential diversity of barcodes. Activation upon conception and continued mutagenesis through gestation result in developmentally barcoded mice wherein information is recorded in lineage-specific mutations. We use these recordings for reliable post hoc reconstruction of the earliest lineages and investigating axis development in the brain. Our results provide an enabling and versatile platform for in vivo barcoding and lineage tracing in a mammalian model system.
Authors: Reza Kalhor, Kian Kalhor, Leo Mejia, Kathleen Leeper, Amanda Graveline, Prashant Mali, George M. Church.
This research was supported by the National Institutes of Health and the Intelligence Advanced Research Projects Activity.
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Illustration created for the National Institute of Standards and Technology or NIST),
for their new CRISPR platform called MAGESTIC.