The Center for Synthetic Regulatory Genomics (SyRGe)
Institute for Systems Genetics
NYU Langone Health
2026 GENOME WRITING WORKSHOP
What does my Big DNA do?:
readout strategies
Jack Atwater
I have built and integrated my big DNA. Now what?
I have built and integrated my big DNA. Now what?
What sort of information do I want?
DNA
RNA
Protein
Example useful information
Chromatin features
Visualization
RNA quantification
Protein interactions
Visualization
Transcription dynamics
Protein quantification and/or localization
Protein interactions
What sort of information do I want?
DNA
RNA
Protein
Example useful information
Chromatin features
Visualization
RNA quantification
Protein interactions
Visualization
Transcription dynamics
Protein quantification and/or localization
Protein interactions
Phenotype
What sort of information do I want?
DNA
RNA
Protein
Example useful information
Chromatin features
Visualization
RNA quantification
Protein interactions
Visualization
Transcription dynamics
Protein quantification and/or localization
Protein interactions
Phenotype
Too context-dependent
for today’s discussion
What sort of information do I want?
DNA
RNA
Protein
Example useful information
Chromatin features
Visualization
RNA quantification
Protein interactions
Visualization
Transcription dynamics
Protein quantification and/or localization
Protein interactions
Minimal requirement:
- Unique sequence features that
distinguish your genes and products from those in your cells
(eg., SNVs)
Nice-to-have:
(eg., fluorescent reporters)
- Disadvantage: can alter the biology of your gene
Your big DNA needs unique sequences!
Mouse gene X
chrX
My payload
Nucleus
Landing pad
Your big DNA needs unique sequences!
chrX
Nucleus
Mouse gene X
My payload
Your big DNA needs unique sequences!
allele 1
allele 2
chr6
Nucleus
chrX
My payload
endogenous alleles
Mouse gene X
Mouse gene X
Your big DNA needs unique sequences!
allele 1
allele 2
chr6
Nucleus
chrX
My payload
endogenous alleles
Problem: no distinguishing sequences!
Mouse gene X
Mouse gene X
Your big DNA needs unique sequences!
allele 1
allele 2
chr6
Nucleus
chrX
My payload
endogenous alleles
Solution 1: Use completely different sequences (eg., different species)
Human gene X
Mouse gene X
Your big DNA needs unique sequences!
allele 1
allele 2
chr6
Nucleus
chrX
My payload
endogenous alleles
Mouse gene X
Mouse gene X
Solution 2: Design minimal sequence differences for readout of interest
Synonymous
variants
Can distinguish mRNA
Your big DNA needs unique sequences!
allele 1
allele 2
chr6
Nucleus
chrX
My payload
endogenous alleles
Mouse gene X
Mouse gene X
Solution 3: Add synthetic reporters
Fluorescent reporter
DNA readout (completely different sequences)
My payload
Bound proteins
Eg., ChIP-Seq
Histone modifications
Eg., ChIP-Seq
Accessibility
Eg., ATAC-Seq
Chromatin features:
DNA readout (completely different sequences)
My payload
Visualization
Ex. FISH
DNA readout (synthetic reporters)
My payload
Visualization
Ex. TetO array
TetR-GFP
RNA readout (completely different sequences)
mRNA quantification
My payload
products
Endogenous
gene products
Simplest: RT-qPCR
Useful normalization!
RNA readout (completely different sequences)
mRNA quantification
RNA-seq (bulk or single cell)
My payload
products
Endogenous
gene products
RNA readout (completely different sequences)
mRNA quantification
RNA-seq (bulk or single cell)
Splice variant analysis
Long-read sequencing
My payload
products
Endogenous
gene products
RNA readout (completely different sequences)
mRNA quantification
RNA-seq (bulk or single cell)
Splice variant analysis
Long-read sequencing
RNA visualization
Ex. FISH
My payload
products
Endogenous
gene products
RNA readout (completely different sequences)
mRNA quantification
RNA-seq (bulk or single cell)
Splice variant analysis
Long-read sequencing
FISH
RNA visualization
My payload
products
Endogenous
gene products
Bound protein analysis
Ex. RIP-Seq
RNA readout (minimal sequence difference)
mRNA quantification
My payload
products
Endogenous
gene products
Simplest: RT-qPCR
Useful normalization!
Other readouts difficult
RNA readout (synthetic reporters)
pooled integration
BC1
BC2
BC3
BC4
BC5
TCAGTGCCAACG
Barcode (BC) =
unique sequence tag
Ex.
Pooled readout of transcript levels using engineered barcodes
RNA readout (synthetic reporters)
pooled integration
amplify and sequence barcode regions from
mRNA and DNA
BC1
BC2
BC3
BC4
BC5
TCAGTGCCAACG
Barcode (BC) =
unique sequence tag
BC1
BC2
BC3
BC4
BC5
450
37
713
121
334
quantify mRNA barcode counts normalized against DNA counts
Ex.
...
Normalized counts:
Pooled readout of transcript levels using engineered barcodes
RNA readout (synthetic reporters)
Transcription dynamics (eg., bursting)
MS2 hairpins
+ (potentially) all the other readouts
MCP-GFP
Protein readout (completely different sequences)
My payload
products
Endogenous
gene products
Protein quantification and/or localization
Assays: western blot, ELISA, FACS, IF
Species-specific epitope
Protein readout (completely different sequences)
My payload
products
Endogenous
gene products
Protein quantification and/or localization
Assays: western blot, ELISA, FACS, IF
Protein-Protein interactions
Protein-RNA or DNA interactions
RIP-Seq
ChIP-Seq
RNA
DNA
Co-IP mass-spec
Protein readout (synthetic reporters)
Epitope tag
(Eg., HA, Myc, His)
My payload
products
Endogenous
gene products
Protein quantification and/or localization
Assays: western blot, ELISA, FACS, IF
Protein readout (synthetic reporters)
Epitope tag
(Eg., HA, Myc, His)
My payload
products
Endogenous
gene products
Protein quantification and/or localization
Assays: western blot, ELISA, FACS, IF
Fluorescent protein
reporter
OR
Protein readout (synthetic reporters)
Epitope tag
(Eg., HA, Myc, His)
My payload
products
Endogenous
gene products
Protein quantification and/or localization
Assays: western blot, ELISA, FACS, IF
Fluorescent protein
reporter
OR
Protein-Protein interactions
Co-IP mass-spec
Protein-RNA or DNA interactions
RIP-Seq
ChIP-Seq
RNA
DNA
Readouts key points
DNA
RNA
Protein
Key points:
- Add unique sequence features that
enable readout!
- Unique sequence requirements are dependent on desired readout
- Synthetic reporters make readout very easy
BUT, they can alter the biology of your gene
Q&A