High Throughout Genome Editing in Human Embryonic Stem Cell-Derived Neurons
Abstract/Contents
- Abstract
- Studying the localization and abundance of neuronal proteins provides valuable insights into their physiological significance and interactions. Labeling of synaptic proteins has been traditionally achieved by immunohistochemistry or viral overexpression of fusion proteins, but these approaches have several limitations. The molecular architecture of synapses comprises hundreds of pre- and post-synaptic molecules, yet only a small fraction of these molecules can be reliably visualized due to the limited availability of specific, high-affinity antibodies. Moreover, immunohistochemistry and overexpression can introduce artifacts and inaccurately recapitulate the distribution of proteins in live cells. Mapping the synaptic proteome with precision and without being encumbered by the availability of quality antibodies, necessitates the use of genetically encoded fluorescent reporters that can be used to tag any molecule of interest. To meet this need, we designed and optimized a CRISPR-HDR strategy to visualize the endogenous expression patterns of neuronal proteins while leaving a minimal genetic footprint. Given the low HDR efficiency in post-mitotic cells like neurons, we leveraged our ability to edit human embryonic stem cells (hESCs) efficiently and induce them into mature, glutamatergic human neurons (iN). Using our approach, we successfully knocked in a HaloTag at the RIMS1 locus in hESCs, isolated pure RIMS1 knock-in cells using a relatively scarless selection strategy, and subsequently differentiated them into iN. We then validated the functionality and correct localization of the HaloTag by microscopy, establishing RIMS1:HaloTag as a reliable presynaptic marker in iN. To demonstrate the generalizability of our approach and the feasibility of a knock-in screen for synaptic molecules, we proceeded to tag other neuronal proteins like FZD3 and DLG4.
Description
| Type of resource | text |
|---|---|
| Date created | June 4, 2022 |
| Publication date | July 10, 2023; June 3, 2022 |
Creators/Contributors
| Author | Dhawan, Sid Suri |
https://orcid.org/0000-0002-2826-955X
(unverified)
|
|---|---|---|
| Thesis advisor | Südhof, Thomas |
Subjects
| Subject | synapse |
|---|---|
| Subject | CRISPR (Genetics) |
| Subject | Neurons |
| Subject | super resolution imaging |
| Genre | Text |
| Genre | Thesis |
Bibliographic information
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- User agrees that, where applicable, content will not be used to identify or to otherwise infringe the privacy or confidentiality rights of individuals. Content distributed via the Stanford Digital Repository may be subject to additional license and use restrictions applied by the depositor.
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 4.0 International license (CC BY-NC).
Preferred citation
- Preferred citation
- Dhawan, S. (2023). High Throughout Genome Editing in Human Embryonic Stem Cell-Derived Neurons . Stanford Digital Repository. Available at https://purl.stanford.edu/wv971kh1334. https://doi.org/10.25740/wv971kh1334.
Collection
Undergraduate Theses, School of Engineering
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- Contact
- sidsd27@stanford.edu
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