Cas13 RNA design for knockdown of chicken cells

I have experiences in RNAi and Cas9 knockout experiments in mammalian cells, but am now facing a little challenge.

I have primary chicken and duck endothelial cells that require knockdowns of genes - knockouts of these genes are impossible due to their importance for these cells.

I have seen 1-2 papers looking at CMV/Ef1a promoters and they show that they work reasonably well in chicken fibroblasts, but I really haven't seen any promoters specific for avian work in the lentivirus system.

Any input would be highly appreciated!

Julian


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Blonde Centaur9 months ago

I guess I will keep uploading here with updates in the unlikely scenario another researcher will find themselves in the same position

1. Decided to use Wessels et al. program as they are able to screen much easier than other programs such as chop chop
2. This means I've resigned to using the plasmids from Sanjana lab as they have CMV promoters that a random paper has shown worked in avian cells (for expression of lenti genes), and Ef1a promoter (for expression of crRNA and human optimised cas13)

3. This isn't too bad because Wessels et. al improved upon lenticrisprV2, which is the current cas13 platform and the one I use anyways

4. crRNA design had to be done in duplicates (1 for chicken and 1 for duck) and then cross referenced to see which sites were perfectly complementary followed by some raggedy ass NCBI blast to ensure nothing comes too close to complementary and possibly knocking down of other essential genes - this is twofold; 1. to reduce the number of constructs, 2. to reduce the variability and efficiency of knockdown based on sequence design
5. Cross checked these crRNAs with Omar's C2C2 design website to ensure I know what in the world I am doing

Fingers crossed and will come back to this thread in 3 months time (god forbid COVID-related delivery times) with either a successful or unsuccessful attempt

Blonde Fairy9 months ago

Greetings and thanks for your email. I'm replying with an interest in creating a #RealisticVirtualEarth for evolutionary biology and genetics and species and STEM - https://twitter.com/WorldUnivAndSch/status/1489383134109917185 (and in digitizing single molecules! per a Harvard Medical School Prof of Genetics' George Church Tweet here) and in developing CC-4 MIT OCW-centric wiki World Univ & Sch planned in all ~200 countries and in all their main languages for genetic research especially.

Brainstorming-wise, and doing what you're exploring, Julian, I'm interested in adding genes of live chickens (and possibly ducks), as well as of human beings, dogs, mice, (and regarding species' genomes for flight) honey bees, and drosophila flies - and again chickens (6 species), to a realistic virtual earth for genetics (and think Google Street View with time slider, Maps, Earth, TensorFlowAI and realistic species' avatar bots, and also at the cellular and molecular levels too - so big data-genetics-genomics projects). See the proposed academic paper here - http://scottmacleod.com - regarding these 6 species.

So I searched on

'adding genes to Google's Street View' -

and found, amazingly ...

Building a ‘Google Street View’ for cancer
https://blogs.biomedcentral.com/on-medicine/2016/01/25/building-google-street-view-cancer/

and
Publish & connect 360 photos with the Street View app
https://support.google.com/maps/answer/7011737?hl=en

Here's the proposed 'brainstorming' academic paper -

Upcoming Paper (2024?) ~
Comparative Genomics' paper - Modeling (beyond Sequencing) their Genomes together: Human, Dog, Mouse, Chicken, Honey Bee, Drosophila fly (D. melanogaster), in a #RealisticVirtualEarthForGenetics and for Longevity Genetics, using machine learning to predict life spans beyond oldest individuals in these species, in Google Street View with time slider, Maps, Earth, TensorFlowAI, Translate, and with realistic avatar bots at the cellular and molecular levels too; Developing this for aging reversal genetics' engineering and research too.

http://scottmacleod.com/

One aspect of WUaS World Univ & Sch's seeking to develop a realistic virtual earth for genetics in Google Street View with time slider is WUaS's plans to facilitate academic publishing in new ways, potentially publishing in 3D for example, and with text-in-the-sidebar as well, both to paper, as well as to something like #wuAsVR on a new digital platform too (possibly a new computer, and even with new chips).

An aspect of this 'gene adding' to information technologies regarding Google Street View is that we can add photos and videos to Street View with time slider already (and also, in a different vein, regarding my Physical-Digital Harbin Hot Springs' ethnographic field site project - and a new social science method I'm developing called 'ethno-wiki-virtual-world-graphy' - https://scott-macleod.blogspot.com/search/label/ethno-wiki-virtual-world-graphy ... And Visit the Harbin Gate here in Google Street View, and "walk" down the road - http://tinyurl.com/p62rpcg - and add pictures or videos if you have them, or are planning to head to Harbin, and if you do, take some samples from along the road, and upload digitized genetic samples of species' individuals there, if you can ).

And all of this aligns with Google's mission of organizing the world's information too, and see -

"In terms of organizing the World's information, WUaS seeks to develop -

adding all genes, and All libraries, all museums, in all languages - ever - by iteratively adding them to Google Street View with TIME SLIDER, Maps, Earth, TensorFlow AI + in 1 realistic virtual earth for libraries and museums (and everything), especially genetics ...

in all languages - https://wiki.worlduniversityandschool.org/wiki/Languages (planning all 7,139 known living languages)

With a #FilmTo3D App, e.g. - https://twitter.com/sgkmacleod/status/1261073678566998017?s=20 - so that people can add more genes (or libraries, and museum objects), to their Google Street View STEM field sites"

https://scott-macleod.blogspot.com/2021/08/asian-arowana-google-alignment-with_01213412819.html

Also found ...
(https://www.addgene.org/52961/
lentiCRISPR v2
(Plasmid #52961) - which is far too expensive ...

AND
Sequencing of 53,831 diverse genomes from the NHLBI TOPMed Program
https://www.nature.com/articles/s41586-021-03205-y )

So, one of the questions I'm asking here further, Julian and all, is how to go from physical chickens (and humans, mice, dogs, drosophila, and honey bees) to cellular and molecular samples (from a hypothetical little farm) I could then add to Google Street View with time slider, and begin to sequence, model, and work with genetically and with CRISPR CaS13, and other related scissor-like gene editing technologies ... and especially for longevity genetics' questions, beyond the outer age limit for all of these 6 species. Thoughts, questions, ideas, suggestions?

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