miRNA circuit modules for precise, tunable control of gene expression
Rongrong Du*
1
, Michael J. Flynn*
1
, Monique Honsa
2
, Ralf Jungmann
2
, Michael B. Elowitz
1†
*These authors contributed equally to this work
†
correspondence:
melowitz@caltech.edu
1
Howard Hughes Medical Institute and Division of Biology and Biological Engineering,
California Institute of Technology, Pasadena, CA 91125, USA
2
Max Planck Institute of Biochemistry, Martinsried, Germany; Faculty of Physics, Ludwig
Maximilian University, Munich, Germany
Abstract
The ability to express transgenes at specified levels is critical for understanding cellular
behaviors, and for applications in gene and cell therapy. Transfection, viral vectors, and
other gene delivery methods produce varying protein expression levels, with limited
quantitative control, while targeted knock-in and stable selection are inefficient and slow.
Active compensation mechanisms can improve precision, but the need for additional
proteins or lack of tunability have prevented their widespread use. Here, we introduce a
toolkit of compact, synthetic miRNA-based circuit modules that provide precise, tunable
control of transgenes across diverse cell types. These circuits, termed DIMMERs
(Dosage-Invariant miRNA-Mediated Expression Regulators) use multivalent miRNA
regulatory interactions within an incoherent feed-forward loop architecture to achieve nearly
uniform protein expression over more than two orders of magnitude variation in underlying
gene dosages or transcription rates. They also allow coarse and fine control of expression,
and are portable, functioning across diverse cell types. In addition, a heuristic miRNA design
algorithm enables the creation of orthogonal circuit variants that independently control
multiple genes in the same cell. These circuits allowed dramatically improved CRISPR
imaging, and super-resolution imaging of EGFR receptors with transient transfections. The
toolbox provided here should allow precise, tunable, dosage-invariant expression for
research, gene therapy, and other biotechnology applications.
One sentence description:
Compact synthetic miRNA-based regulatory circuits enable
tunable, orthogonal, and generalizable dosage-invariant gene expression control for
research and biotechnology.
1
.
CC-BY-NC-ND 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 12, 2024.
;
https://doi.org/10.1101/2024.03.12.583048
doi:
bioRxiv preprint
Introduction
Biotechnology and biomedical research rely heavily on ectopic expression of transgenes in
living cells. Popular expression systems produce a broad range of expression levels in
individual cells. This is true for non-integrating approaches such as DNA transfection and
AAV vectors, as well as integrating systems such as lentivirus
1
or piggyBac transposons
2
.
This heterogeneity reflects unavoidable variation in the number of gene copies taken up,
integrated, and expressed by each cell, as well as gene expression noise
3,4
. Variability or
noise may be tolerable or even useful
5
in some situations, but more often presents an
obstacle to accurate analysis and precise control of cell behaviors. Selecting for stable
clones can reduce expression variation but is time-consuming, and can also be susceptible
to stochastic silencing
6
. An ideal gene regulation system would compensate for this
variation, allowing more precise control of expression level, reduced toxicity in gene and cell
therapies, and lower backgrounds with reporters, among other applications (
Figure 1A
).
The incoherent feed-forward loop (IFFL) circuit motif provides an ideal mechanism to provide
these capabilities
7
. In an IFFL, a target gene and its negative regulator are co-regulated by
the same input (
Figure 1B
). Gene dosage can be considered such an input, and
proportionately affects expression of both the regulator and its target. In some parameter
regimes, these two effects cancel out, and target expression approaches a fixed level at high
dosage (
Figure 1C, Supplementary Modeling
). An ideal IFFL system should further allow
tuning of this expression set point, the creation of multiple orthogonal regulation systems for
simultaneous control of multiple genes, and the ability to operate in multiple cell types
(
Figure 1C
).
Earlier work demonstrated IFFL circuits could generate dosage-invariant expression over a
50- or 100-fold range in bacteria and mammalian cells, respectively
8,9
. However, these
systems required expression of additional proteins, complicating their routine use. miRNA
could be an ideal regulator for an IFFL dosage compensation system, as it can be expressed
from within an intron, or from compact transcripts. In pioneering work, Bleris et al.
demonstrated that a miRNA-based IFFL could achieve dosage compensation
10
. Strovas et
al. improved on this by incorporating a natural miRNA and multiple repeats of its target
sequence within the gene
11
. This reduced expression variation in single-copy integrations,
and achieved dosage compensation over a ~20-fold range, at the cost of potential crosstalk
with endogenous genes. Most recently, Yang et al introduced an “equalizer” architecture that
combined transcriptional negative feedback through the TetR protein with miRNA
12
. This
extended the range of effective dosage compensation, but required expression of the
bacterial TetR protein, adding complexity and potential immunogenicity. Despite much work,
it has remained unclear what sequence features are sufficient to enable orthogonal, tunable,
dosage compensating miRNA IFFLs, and as a result a broadly useful toolkit of such circuits
does not yet exist.
Here, we engineered a set of miRNA-based dosage compensation systems termed
DIMMERs (Dosage Invariant miRNA-Mediated Expression Regulators) that fulfill this need.
These circuits use specific configurations of the miRNA expression cassette and its target
sequences, and take advantage of the ability to achieve multivalent miRNA regulation
through the natural TNRC6 scaffold system. They allow systematic tuning of expression
levels by modulating the number of miRNA cassettes, numbers of target binding sites, and
2
.
CC-BY-NC-ND 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 12, 2024.
;
https://doi.org/10.1101/2024.03.12.583048
doi:
bioRxiv preprint
miRNA-target site complementarity. Further, they can be used to orthogonally regulate
multiple genes in the same cell, and operate similarly across different cell types. We
constructed a toolkit of ten mutually orthogonal ready-to-use expression systems that can be
incorporated into diverse systems. Finally, we demonstrated their utility for CRISPR imaging
and super-resolution protein imaging modalities. DIMMERs should allow routine research
and biotechnology applications to operate with greater precision, control, and predictability.
3
.
CC-BY-NC-ND 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 12, 2024.
;
https://doi.org/10.1101/2024.03.12.583048
doi:
bioRxiv preprint
Figure 1. miRNA incoherent feedforward circuits enable dosage-invariant gene expression.
(A)
An ideal gene expression system generates uniform protein expression levels despite variable
gene dosage delivered. The blue gradient shown in the nucleus indicates the copy numbers of gene
delivered, in which darker blue represents higher dosage, and lighter blue represents lower dosage.
Green indicates the desired uniform output protein expression levels.
(B)
The architecture of the incoherent feedforward loop (IFFL). The input is gene dosage in arbitrary
units, which activates the expression of both the mRNA and the microRNA. microRNA inhibits mRNA
translation. Output is the resulting protein expression.
(C)
IFFLs enable tunable
(
①
)
, orthogonal
(
②
)
control of the target and also operate in multiple cell
contexts
(
③
)
.
(
①
)
shows the modeling of the dosage compensation system which permits tuning of
the setpoint levels.
(
②
)
depicts schematic flow cytometry data of two genes of interest regulated by
orthogonal, tunable, miRNA-controlled dosage compensation circuits. Cells are poly-transfected with
two independently-regulated constructs. Each color represents one set of the designs used in the
4
.
CC-BY-NC-ND 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 12, 2024.
;
https://doi.org/10.1101/2024.03.12.583048
doi:
bioRxiv preprint
poly-transfection. Each ellipse indicates where most cells are located. Dashed lines indicate the
centroids of expression.
③
schematically depicts relatively similar expression behavior across diverse
cell types. The central diagram introduces the circuit architecture of the microRNA(miRNA)-based
IFFL, in which the two arrows indicate the divergent promoter, the small rectangle on the left indicates
the miRNA, and the long rectangle on the right indicates the regulated gene, the short rectangle on
the right indicates the miRNA binding sites.
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5
.
CC-BY-NC-ND 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 12, 2024.
;
https://doi.org/10.1101/2024.03.12.583048
doi:
bioRxiv preprint
incorporated one or more target miRNA sites of varying complementarity in the 3’UTR of the
target gene. This compact two-transcript construct allowed systematic analysis of different
miRNAs and target site configurations.
To implement miRNA regulation orthogonal to natural miRNAs, we first used a previously
described miRNA sequence targeting Renilla luciferase (miR-L), together with a single copy
of its fully complementary 21bp target site. We transiently transfected U2OS cells with the
resulting construct, analyzed cells by flow cytometry after 48h, and plotted target EGFP
expression versus gene dosage, as indicated by mRuby3 (
Figure 2C
). Compared to an
unregulated control with no miRNA target site, the IFFL strongly reduced target EGFP
expression, as expected (
Figure 2C
). However, the circuit failed to achieve the dosage
compensation behavior anticipated from mathematical modeling (
Figure 2A
).
We next asked whether the lack of dosage compensation could relate to the strongly
repressing regime produced by full miRNA-target complementarity. We designed a set of
IFFL variants which progressively reduced complementarity of the single target site from 21
to 17 bp (
Table S1
). Designs with reduced 3’ complementarity showed weak or no
repression of target expression, particularly below 19bp (
Figure 2D
), while those that did
efficiently repress retained strong repression comparable to that of the full length 21bp
construct. Nevertheless, within this set, no construct achieved full dosage invariance. Thus,
modulation of complementarity alone was not sufficient to provide dosage compensation.
The loss of regulation at reduced complementarity contrasted with the well known regulatory
capacity of miRNAs with much shorter complementary regions of only ~8bp
13
. One
mechanism to enable specific regulation with short sequences involves multivalent
recognition of multiple target binding sites on the same mRNA
16
. TNRC6 is a scaffold protein
that enables multivalent recognition by simultaneously binding to multiple miRNA-loaded
Argonaute (Ago) complexes (
Figure 2G
)
17,18,19
. Consistent with a role for multivalent
regulation, tandem repeats of two to four copies of the 17bp target site progressively
increased regulation, and strongly reduced dosage sensitivity at higher expression levels
(
Figure 2E
). For example, with 4 tandem binding sites, target expression increased by only
4-fold over a 200-fold range of dosages (
Figure 2F
). A “tail” of elevated expression at the
highest dosages may reflect saturation of miRNA-associated machinery, as observed in
other studies
12
. Together, these results show that a miRNA-based IFFL based on 17bp of
miRNA-target complementarity and 4 tandem binding sites can achieve nearly complete
compensation over more than two orders of magnitude of dosage variation.
To find out whether this compensation behavior was dependent on TNRC6, we took
advantage of a previously identified fragment of the natural TNRC6B protein, the T6B
peptide that competitively inhibits TNRC6 activity (
Figure 2G
)
20
. When co-transfected with
the 4×17bp IFFL, the T6B inhibitory peptide abolished regulation, producing
dosage-dependent expression nearly identical to that produced by an unregulated construct
(
Figure 2H
). By contrast, the T6B inhibitory peptide had little effect on the single fully
complementary 21bp construct, suggesting that it regulates in a TNRC6-independent
manner (
Figure 2I
). Finally, negative control mutant variants of T6B lacking the
Ago2-binding domain failed to abolish regulation, as expected (
Figure S3A
). Together, these
results suggest that the 4×17bp and 1×21bp designs respectively operate through
TNRC6-dependent and TNRC6-independent mechanisms. Because of their ability to limit
6
.
CC-BY-NC-ND 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 12, 2024.
;
https://doi.org/10.1101/2024.03.12.583048
doi:
bioRxiv preprint
expression, we term these circuits DIMMERs. More generally, these results indicate that
multivalent regulation through multiple, individually weak, miRNA binding sites can achieve
strong regulation and dosage compensation within the context of the IFFL circuit.
7
.
CC-BY-NC-ND 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 12, 2024.
;
https://doi.org/10.1101/2024.03.12.583048
doi:
bioRxiv preprint
Figure 2. miRNA-based IFFLs achieve linear regulation and dosage independence through
TNRC6-mediated repression.
(A)
To identify the parameter regimes allowing dosage compensation, we built a simplified model of
miRNA inhibition. Upper panel, gray dashed arrows in the diagram represent the natural dilution and
degradation of the mRNA or miRNA. Black, double-directed arrows represent the association and
dissociation of the mRNA-miRNA complex. Gray arrows represent the decay of the mRNA. The rates
of all the reactions are labeled on the side of the arrows. Lower panel, modeling results of the
miRNA-mRNA interaction. The unregulated curve suggests the expression when there’s no miRNA.
(B)
To implement the miRNA-based dosage compensation system, we designed the divergent
promoter circuit. We varied the target-miRNA complementarity and target site numbers to explore the
parameter space that gives rise to the dosage independence.
(C)
We performed flow cytometry to verify the behavior of the circuit shown in
(A)
with a single, fully
complementary miR-L target site versus an unregulated control, which has neither the miR-L nor the
miR-L target site. We used mRuby3 fluorescent protein as the dosage indicator, and EGFP
fluorescent protein as the target. Cells are gated and binned by mRuby3 intensities. Each dot
corresponds to the geometric mean fluorescence intensity of the mRuby3 bin breaks and median
fluorescence intensity of EGFP in the bin. Shaded regions denote the range from
8
.
CC-BY-NC-ND 4.0 International license
made available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprint
this version posted March 12, 2024.
;
https://doi.org/10.1101/2024.03.12.583048
doi:
bioRxiv preprint