CRISPRing our cellular roots

Research Trend   |   Dec 21, 2018

 CRISPRing our Cellular Roots

The remarkable process by which a single totipotent zygote develops into the approximately 37 trillion cells in a human body is orchestrated by a highly controlled sequence of signaling events resulting in a multitude of differentiated cell types. The ability to retrospectively decipher this lineage of each adult cell would have major implications for understanding tissue development, homeostasis, and disease. Such lineage tracing was first realized over 100 years ago [1], and while recent advancements such as the Cre-LoxP inducible system have accelerated our knowledge of stem cell biology, limitations exist [2] and ultimately, current methods are not precise enough to follow a cell through multiple divisions.

Recently, a paper by Kalhor et al harnessed the power of CRISPR “barcoding” to track and reconstruct cellular lineages in mice essentially enabling the tracking of a mammal’s development from a single egg into an embryo [3]. By engineering a Cas9-gRNA that targets the DNA locus of the gRNA itself (homing guide RNA or hgRNA), this homing CRISPR system enables retargeting of the locus leading to a unique sequence that is related to its parent sequence [4-7]. Through the use of single-cell analysis, this self-perpetuating “targeted scar” over subsequent cell divisions potentially allows the delineation of the precise history of all cells in the organism. To accomplish this, a mouse harboring 60 hgRNA loci, was crossed with a Cas9 mouse resulting in offspring that contained “lineage traced” cells whereby closely related cells contain the same barcodes. Analysis of different tissues for barcode frequency revealed that the head and tail samples were most similar, consistent with both tissues being derived from the inner cell mass. Further, analysis of barcoded E12.5 embryos revealed that heart and limb bud cells, which are derived from the epiblast contained related barcodes. Lastly, barcoding was used to show for the first time that, in the brain, the anterior-posterior (A-P) axis is established before commitment to the Lateral (L-R) axis during the development of the central nervous system.

In summary, this work revealed that in vivo barcoding can successfully record the history of cells during development and that it is indeed theoretically possible to label each and every one of the ~ 2 x 1010 cells in a mouse. With further refinement of the system [8-10], such approaches have huge potential to massively expand our knowledge of human development and disease.

VectorBuilder

VectorBuilder offers knockout, CRISPRa, and CRISPRi as well as barcoding libraries in a variety of sizes/scales suitable for a wide variety of functional screens. VectorBuilder’s libraries are available in a variety of formats depending on your needs including E.coli stocks, purified DNA, and even packaged virus for direct use and transduction. With additional downstream services available including library amplification, deconvolution services, and NGS validation, VectorBuilder can meet any library need you might have in a cost-effective manner.

In addition, our award-winning online platform allows you to design and order custom vectors specific to your research needs. Choose from CRISPR/Cas9 vectors, AAVs, lentiviruses, adenovirus, shRNA expression vectors, and more!

References

  1. E.G. Conklin. The organization and cell lineage of the ascidian egg. J. Acad. Nat. Sci. Phila. 1905.
  2. Kai Kretzschmar and Fiona M. Watt. Lineage Tracing. Cell. Volume 148, 1–2, 20. 2012.
  3. Kalhor et al. Developmental barcoding of whole mouse via homing CRISPR. Science 361, 893. 2018.
  4. Kalhor et al. Rapidly evolving homing CRISPR barcodes. Nature methods, Vol.14 No.2 195. 2017.
  5. S. D. Perli et al. Continuous genetic recording with self-targeting CRISPR-Cas in human cells. Science 353. 2016.
  6. S. T. Schmidt et al. Quantitative Analysis of Synthetic Cell Lineage Tracing Using Nuclease Barcoding. ACS Synth. Biol. 6, 936–942. 2017.
  7. B. Raj et al. Simultaneous single-cell profiling of lineages and cell types in the vertebrate brain. Nat. Biotechnol. 36, 442-450. 2018.
  8. Platt, R.J. et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440–455.2014.
  9. Certo, M.T. et al. Tracking genome engineering outcome at individual DNA breakpoints. Nat. Methods 8, 671–676. 2011.
  10. Komor, A.C et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424. 2016.
您可以通过 我的载体发送设计咨询 订购各种分子克隆服务,您可以在首页菜单栏上找到我们提供的服务和产品。
想了解更多的最新技术和发现?请留下反馈联系我们
点击查看最新促销活动信息