Genome editing owes a huge debt to genome sequencing With efforts like @humancellatlas, we’re sequencing cells like mad. What is the "Cas9" of single cell? With @omarabudayyeh @insitubiology: RADARS, for programmable cell-specific sensing/expression 🧵👇 Tweet added by Jonathan Gootenberg @jgooten

Jonathan Gootenberg

2 years

Genome editing owes a huge debt to genome sequencing With efforts like @humancellatlas , we’re sequencing cells like mad. What is the "Cas9" of single cell? With @omarabudayyeh @insitubiology : RADARS, for programmable cell-specific sensing/expression 🧵👇

Programmable eukaryotic protein expression with RNA sensors

The diversity of cell types and states can be scalably measured and defined by expressed RNA transcripts. However, approaches to programmably sense and respond to the presence of specific RNAs within...

www.biorxiv.org

8

100

494

Jonathan Gootenberg

@jgooten

2 years

Targeting a cell type has typically required use of specific regulatory elements or promoters. RADARS makes cell type or state-specific expression of protein payloads (GFP, caspase, Cre, etc) as easy as designing a guide RNA to a transcript of interest 2/13

1

0

3

Jonathan Gootenberg

@jgooten

2 years

How does RADARS work? We were inspired by RNA editing tools, including our work on the REPAIR editing system, which use a guide RNA to direct RNA editing (A->I) on a transcript in the cell. Quite useful, especially for removal of stop codons in genetic disease (UAG -> UIG) 3/13

1

0

7

Jonathan Gootenberg

@jgooten

2 years

RADARS flips this around: instead of guide editing the transcript, what if the transcript edited the guide? Put a stop codon in the guide, blocking expression of a “payload” gene downstream. When the transcript is present, binding->guide editing->viola, payload is expressed 4/13

2

0

11

Jonathan Gootenberg

@jgooten

2 years

RADARS works well with both luciferase and fluorescent outputs, with our first experiments yielding >50-fold activation. Optimization of designs, such as hairpins to drop readthrough noise, brought fold change >150-fold! 5/13

1

0

4

Jonathan Gootenberg

@jgooten

2 years

Extra bonus, RADARS is quantitative over a 25-fold range (R^2 = .96). We had to redo this assay a couple times, since it seemed unbelievable at first. 6/13

1

0

5

Jonathan Gootenberg

@jgooten

2 years

Testing RADARS on endogenous genes, we found that both in downregulation (siRNA) and upregulation (heat shock) models, RADARS tracked well with gold standard (qPCR) changes in expression (non-destructively, of course) 7/13

1

0

4

Jonathan Gootenberg

@jgooten

2 years

RADARS can be used for more than just sensing – by swapping out the payload for a caspase, we can do transcript-dependent cell killing. Ever wanted to “knock out” a cell in your population as a perturbation? Now you can! (Also, tons of therapeutic applications here) 8/13

1

0

11

Jonathan Gootenberg

@jgooten

2 years

Of course, a single transcript isn’t often enough to characterize a cell state or type. What about some logic? We show both OR and AND logic. (We also find that 2-input logic is enough to distinguish 35/37 tissue types according to GTEx) 9/13

1

8

Jonathan Gootenberg

@jgooten

2 years

RADARS is versatile – you don’t even have to supply an exogenous ADAR protein (though doing so can increase fold activation). Natural ADAR levels are high enough to edit RADARS guides and lead to expression...even in vivo (see below) 10/13

1

0

3

Jonathan Gootenberg

@jgooten

2 years

RADARS can be used for mRNA too–where transgene expression can be controlled from cell type specific promoters, there’s no analogous way to “turn on” mRNA in cell types of interest. We design mRNA RADARS (mRADARS) and deliver them to mice for transcript-specific expression. 11/13

1

0

3

Jonathan Gootenberg

@jgooten

2 years

We hope that RADARS will be useful to the community for basic research, diagnostics, and therapeutics. @omarabudayyeh are actively growing our team to apply across multiple applications – reach out if you’d like to be a part of it! 12/13

1

12

Jonathan Gootenberg

@jgooten

2 years

Developing RADARS was a really fun ride with an amazing team with @insitubiology @omarabudayyeh at @broadinstitute @mcgovernmit . Thanks to authors @idmjky , Jeremy Koob, @dawnchenx , @RohanKrajeski , and Yifan Zhang with @lukas_villiger and @91_naynay ! 13/13

1

2

6

Jonathan Gootenberg

@jgooten

2 years

This work wouldn't have been possible without support from Impetus Grants @MartinBJensen , NIH @NIBIBgov @genome_gov , K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics, G. Harold & Leila Y. Mathers Charitable Foundation, @CF_Foundation , and all our other supporters!

0

9

Giuseppe Testa

@gtesta72

2 years

@jgooten @humancellatlas @omarabudayyeh @insitubiology @threadreaderapp unroll

1

0

Yair Einhorn

@yaireinhorn

2 years

@jgooten @humancellatlas @omarabudayyeh @insitubiology An amazing find that can definitely help treat many genetic diseases caused by #Stopcodons such as Inherited retinal diseases (IRDs) - #Choroideremia and #RetinitisPigmentosa which each of them has a huge amount of #PTC pathogenic variants. Super exciting!

0

3

Bobby Bowman

@bowman_rl

2 years

@jgooten @humancellatlas @omarabudayyeh @insitubiology Any indication of whether guide expression influences mRNA stability of its endogenous activator? Great technology!

1

0

Debojyoti Chakraborty

@Debojyoti_C

2 years

@jgooten @humancellatlas @omarabudayyeh @insitubiology Really cool work @jgooten ! Was wondering within what time frame do you see the protein perturbation happening (compared to say degrons)? As a therapy it would be remarkable have a burst of protein loss that is both effective and persistent. Wonderful work.

0

1