A Singular System with Precise Dosing and Spatiotemporal Control of CRISPR-Cas9

Article abstract of DOI:10.1002/anie.201900788

Several genome engineering applications of CRISPR-Cas9, an RNA-guided DNA endonuclease, require precision control of Cas9 activity over dosage, timing, and targeted site in an organism. While some control of Cas9 activity over dose and time have been achieved using small molecules, and spatial control using light, no singular system with control over all the three attributes exists. Furthermore, the reported small-molecule systems lack wide dynamic range, have background activity in the absence of the small-molecule controller, and are not biologically inert, while the optogenetic systems require prolonged exposure to high-intensity light. We previously reported a small-molecule-controlled Cas9 system with some dosage and temporal control. By photocaging this Cas9 activator to render it biologically inert and photoactivatable, and employing next-generation protein engineering approaches, we have built a system with a wide dynamic range, low background, and fast photoactivation using a low-intensity light while rendering the small-molecule activator biologically inert. We anticipate these precision controls will propel the development of practical applications of Cas9.

Full text of DOI:10.1002/anie.201900788

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Accepted Article
Title: A singular system with precise dosing and spatiotemporal control
of CRISPR-Cas9
Authors: Amit Choudhary, Debasish Manna, Basudeb Maji,
Soumyashree Ashok Gangopadhyay, Kurt J Cox, Qingxuan
Zhou, Benjamin Law, and Ralph Mazitschek
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201900788
Angew. Chem. 10.1002/ange.201900788
Link to VoR: http://dx.doi.org/10.1002/anie.201900788
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10.1002/anie.201900788
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COMMUNICATION
A singular system with precise dosing and spatiotemporal
control of CRISPR-Cas9
Debasish Manna[a],[b],[c], Basudeb Maji[a],[b],[c], Soumyashree A. Gangopadhyay[a],[b],[c], Kurt J. Cox[a],
Qingxuan Zhou[a], Benjamin K. Law[a], Ralph Mazitschek[a],[b],[d] and Amit Choudhary*[a],[b],[c]
This work is dedicated to Professor Ronald T. Raines on the occasion of his 60th birthday.
Abstract: Several genome engineering applications of CRISPR-Cas9, strand-break-triggered genotoxicity[6] at elevated Cas9 activity.
an RNA-guided DNA endonuclease, require precision control of Cas9
activity over dosage, timing, and targeted site in an organism. While
some control of Cas9 activity over dose and time have been achieved
using small molecules, and spatial control using light, no singular
system with control over all the three attributes exists. Furthermore,
the reported small-molecule systems lack wide-dynamic range, have
background activity in absence of the small-molecule controller, and
are not biologically inert, while the optogenetic systems require
prolonged exposure of high-intensity light. We previously reported a
small-molecule-controlled Cas9 system with some dosage and
temporal control. By photocaging this Cas9 activator to render it
biologically inert and photoactivatable, and employing next-
generation protein engineering approaches, we have built a system
with wide-dynamic range, low background, and fast photoactivation
using a low-intensity light while rendering the small molecule activator
biologically inert. We anticipate these precision controls will propel the
development of practical applications of Cas9.
Furthermore, as the off-target activity of Cas9 generally occurs at
a slower rate than the on-target activity,[7] restricting the nuclease
activity to a narrow temporal window is highly desired. Finally,
several delivery systems accumulate Cas9 in specific off-target
tissues,[8] so spatial restriction of Cas9 activity could avert off-
target genotoxicity. Beyond genome editing, the fusion of
catalytically-impaired Cas9 to effector domains has enabled
targeted transcriptional activation or repression, base editing,
[3b, 9]
epigenetic modification, and chromatin imaging.
Equipping
these transformative tools with precision control over the
aforementioned dimensions will also be highly desirable and
propel many biological studies.
Controlling the dosage and timing of Cas9 activity has been
accomplished using small-molecule-modulated systems,[3b] and
spatial control with light-activated protein domains[10] or
photoactivatable residues,[11] however, a singular system with
built-in control over all three attributes does not exist.
Unfortunately, a simple combination of the existing systems does
not provide an adequate solution since the precision of such
CRISPR-Cas9 is an RNA-guided DNA endonuclease[1] that
has facilitated genome engineering applications in biotechnology,
medicine, and agriculture. [2] These applications require precision
control of Cas9 activity over multiple dimensions, including dose,
temporal, and spatial.[3] Controlling the dose is particularly
important for Cas9, as the targeted DNA sequence is present at
a much lower concentration than the enzyme, causing significant
off-target effects,[3b, 4] chromosomal translocations,[4a, 5] and DNA-
system is insufficient for several biotechnological and therapeutic
2d, 3b, 12]
applications.[2b,
For example, small-molecule-activated
systems have low dynamic range of Cas9 activity and retain
significant background activity even in the absence of the small-
molecule modulator, preventing precise temporal control.[3b] As an
additional drawback, the currently employed small-molecule
activators (e.g., trimethoprim, hydroxytamoxifen) are not
biologically inert and in some cases can adversely impact the
organism’s microbiome. The precise control of Cas9 systems
using light requires: (i) the rapid activation of Cas9 on timescale
of minutes upon illumination with minimal background, (ii) low light
intensities and short exposure times, (iii) reversible dosing with
multi-wavelength control of Cas9 activity, and (iv) cost-
effectiveness. Unfortunately, the reported photoactivatable Cas9
systems require long exposure times (often on the timescale of
hours) and/or high light intensities (e.g., laser-based light source).
Additionally, these systems are restricted to a narrow spectral
range, are irreversible in certain cases, and can be expensive if
photocaged RNA residues[11b] are employed. Therefore, while the
reported systems display some of the aforementioned required
characteristics, a singular system encompassing all of the desired
attributes has not been reported.
[a]
Dr. D. Manna, Dr. B. Maji, Dr. S. A. Gangopadhyay, Dr. K. J. Cox, Dr.
Q. Zhou, B. K. Law, Dr. R. Mazitschek , Dr. A. Choudhary Chemical
Biology and Therapeutics Science, Broad Institute of MIT and
Harvard, Cambridge, MA 02142, USA
E-mail: achoudhary@bwh.harvard.edu
[b]
[c]
[d]
Dr. D. Manna, Dr. B. Maji, Dr. S. A. Gangopadhyay, Dr. R.
Mazitschek , Dr. A. Choudhary
Department of Medicine, Harvard Medical School, Boston, MA
02115, USA
Dr. D. Manna, Dr. B. Maji, Dr. S. A. Gangopadhyay, Dr. A.
Choudhary
Divisions of Renal Medicine and Engineering, Brigham and Women’s
Hospital, Boston, MA 02115, USA
Dr. R. Mazitschek
Harvard T. H. Chan School of Public Health,
Boston, MA 02115, USA
Center for Systems Biology, Massachusetts General Hospital
Boston MA 02114, USA
We previously reported a small-molecule-controlled Cas9
system[4b] that exhibited dosage and temporal control by fusing
Cas9 to the destabilized domains (DDs) of E. coli’s dihydrofolate
reductase (DHFR).[13] These domains are unstable and rapidly
target the fusion protein for proteasomal degradation, unless it is
stabilized by the small molecule trimethoprim (TMP), thereby
linking the activity of the fusion protein directly to the presence of
Supporting information for this article is given via a link at the end of
the document
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10.1002/anie.201900788
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TMP in a rapid, reversible, and dose-dependent manner (Fig. 1A).
By controlling the concentration and time of administration or
removal of TMP, both the dosage and temporal control of Cas9
activity was achieved. Though this system meets some of the
requirements of the aforementioned singular system, it still lacks
several others. First, the system exhibits significant background
activity (~20%) in the absence of TMP, preventing precise
temporal control of Cas9 activity. Second, the small molecule
activator, TMP, is a potent anti-bacterial drug[14] that perturbs the
organism’s microbiota,[15] limiting in vivo applications. Finally, this
system lacks spatial control of Cas9 activity.
We hypothesized that next-generation protein engineering
approaches could reduce the background activity in the absence
of TMP. Furthermore, we hypothesized that photocaging TMP
would both abrogate systemic anti-bacterial activity and also allow
spatial control using light. Photocaged small molecules can be
readily delivered to cells at much higher concentrations than those
attainable for light-activated protein domains or photocaged
residues, and small-molecule photocages that can be efficiently
uncaged at various wavelengths are readily available. As such,
we postulated that using a photocaged TMP would enable rapid
Cas9 activation at low-light intensities and at multiple
wavelengths. Herein, we report a Cas9 system that can be
activated by a low-intensity light (non-laser based) of multiple
wavelengths with exposure times of only minutes. This system
has a low background in the absence of TMP, is dosable and
reversible, and is non-toxic to both mammalian and bacterial cells.
Finally, our system can be deployed for optochemically controlling
transcription using catalytically inactive Cas9 fused to
transcription activator.
a
In our previously reported system (Fig. 1A), the Cas9 protein
contained DHFR domains on both the N- and C-termini
(DHFR•Cas9•DHFR), though still retained ~20% background
activity in the absence of TMP.[4b] In parallel, Savage and co-
workers[16] identified loops on Cas9 that tolerated the insertion of
protein domains without
a loss of activity. We therefore
hypothesized that grafting an additional DHFR domain on the
Cas9 loop itself (termed “L-DHFR”) should further reduce the
background activity in the absence of TMP (Fig. 1B). We tested
this 3DHFR construct (Fig. 1B) in an eGFP-disruption assay[4b] in
which Cas9-mediated eGFP-knockout causes loss of
fluorescence. We observed background reduction by 5–7 fold and
dose-dependent activation of Cas9, though the dynamic range
was also reduced, with Cas9 exhibiting only 62% eGFP-disruption
at a full-dose of TMP (Fig. 1C). Given the pronounced effect of
the L-DHFR on background reduction, we hypothesized that
systematically removing DHFR domains at the other sites (N-term,
C-term, or both) of 3DHFR should yield a construct with a large
enough dynamic range and minimal background activity. Indeed,
the construct containing both an N-DHFR and L-DHFR domain
had the minimal background and retained a dynamic range similar
to that of wildtype Cas9 (Fig. 1C). Next, we confirmed the
reversible nature of TMP-mediated activation in a “washout”
experiment (Fig. 1D, E). Briefly, the cells were nucleofected with
the various destabilized domain constructs and treated with TMP.
The media was then exchanged for fresh media without TMP at
different time intervals over 48 h (Fig. 1D). The cells exposed to
TMP for longer durations showed a higher eGFP-disruption and
Figure 1. (A) Schematic representation of destabilized DHFR domains fused to
Cas9, leading to proteosomal degradation. TMP stabilizes the DHFR•Cas9
fusion protein to generate active Cas9. (B) Schematic representation of different
DHFR-fused Cas9 constructs. (C) Dose-dependent activation of different
DHFR•Cas9 systems in the eGFP-disruption assay in U2OS cells (D)
Schematic representation of reversible activation of Cas9. Cells were
nucleofected with different DHFR•Cas9 constructs and treated with TMP (37
nM). The media was then removed and fresh media without TMP was added at
different time intervals over 48 h, and the cells were imaged after 48 h. (E)
Comparison of reversible and dose dependent activation of three different
DHFR•Cas9 constructs by TMP (37 nM).
Cas9 activity, and such reversibility was observed for a wide
range of TMP concentrations from 4 nM to 1 µM (Fig. S1).
After developing this improved inducible Cas9 system with
minimal background activity, we proceeded to introduce optical
control and abrogate the anti-bacterial activity of TMP that can
adversely impact the microbiome.[15] We hypothesized that
appending photocleavable moieties to TMP would render it
inactive and that exposure to light would remove the photocage
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Figure 2. (A) Crystal structure of the TMP-DHFR complex showing that the NH2 groups of TMP are deeply buried inside the binding pocket. (B) Chemical structure
of the photocaged TMP derivatives 1 and 2 and unmasking of photocaging group under UV and visible light, respectively.
nm for only a few minutes caused a dose-dependent eGFP-
to release the free activator. Before embarking on any
photocaging experiments, we confirmed that TMP was
photostable at various wavelengths and light intensities. Indeed,
no functional modification or detectable degradation of TMP was
observed via liquid chromatography-mass spectrometry across
the range of wavelengths and intensities that were relevant for our
photocages (Fig. S2).
knockdown that would be expected for
a
successful
photoactivation of a caged molecule (Fig. 3B, 3F, S7B, S8B). Next,
we varied the light exposure time using a fixed concentration of
the photocaged TMP, and we found that the extent of eGFP-
knockdown was proportional to the light exposure time for both
compound 1 and 2 (Fig. 3C, 3G, S7C, S8C). Both compounds
showed a two-dimensional activation control that could be
achieved by either varying the concentration of the photocaged
TMP or the duration of light exposure (Fig. S7, 8). We confirmed
comparable activation of Cas9 when cells were first treated with
compound 1 or 2 that was then removed through media swap
before light exposure (Fig. S9, 10). In addition to short exposure
times of 12 minutes, our system employs low intensity LED lights
with the low power (8 mW/cm2 for 385 nm and 14.7 mW/cm2 for
470 nm LEDs). This low light intensity and miniature light source
will allow facile optical control of Cas9 in vivo using various
devices, including optical fibers[21] or implantable wireless
photonic devices.[22] We were also able to conduct the
experiments in a 96-well plate by placing a photomask patterned
to spell “CAS” and observed eGFP-disruption in only the light
exposed wells of the microplate, resulting in a spelled-out “CAS”
pattern (Fig. S11).
To design photocaged TMP, we examined the co-crystal
structure of TMP bound to DHFR (Fig. 2A) showing that both
aromatic amino groups of TMP are buried inside the binding
pocket of the DHFR. A bulky photocaging group installed on these
amino groups should therefore adversely impact the ability of
TMP to bind to and destabilize DHFR. We explored two types of
photocaging moieties (Fig. 2B) reported for their use in the precise
photoregulation of various biological processes.[17] For example,
photocaging group 2-(2-nitrophenyl)-propoxycarbonyl (NPPOC)
has been used for spatiotemporal control of cells but long
exposures to wavelengths employed for NPPOC uncaging can
induce genomic lesions,[18] and the tissue penetration of such
wavelengths is also limited. As such, we decided to employ the
photocaging group thiocoumarine, which can be removed by
irradiation with visible light that is safer.[19] We synthesized the
photocaged TMP compounds 1 and 2 (Fig. 2B), which are
uncaged at wavelengths of 385 nm and 470 nm, respectively. We
confirmed that photocaged TMP did not prevent bacterial growth,
supporting our hypothesis that the photocaged molecule would
not adversely impact the microbiome as done by TMP (Fig. S3).
Exposure of the photocaged TMP to light removed the
photocaging moiety and released active TMP (Fig. S4). We have
used the LED device previously reported[20] by some of us for our
experiments.
We next investigated the utility of our system to control
catalytically-dead Cas9 (dCas9) for precision optochemical
control of transcriptional activation. We used our previously
reported[4b] transcriptional activation system (DHFR•PP7•VP64)
consisting of transcriptional activation domain VP64 and PP7,
which binds to a specific sequence displayed on the gRNA. Upon
treatment with varying doses of TMP, we observed robust and
dose-dependent upregulation of IL1RN as well as minimal basal
activity in the absence of TMP (Fig. S12). We then tested the
optical control of IL1RN transcription using the DHFR•PP7•VP64
system with various doses of compound 1. Upon light illumination
for 12 minutes, we observed upregulation of IL1RN transcription
proportional to the dose of the compound (Fig. S13A). Further, to
investigate the effect of light exposure time on transcriptional
upregulation, we treated cells transfected with dCas9,
DHFR•PP7•VP64, and gRNA with 500 nM of 1 and exposed them
to 385 nm of light for varying periods (Fig. S13B). We observed
that
Next, we used compound 1 or 2 to control DHFR-based
Cas9 systems using light in the same eGFP-disruption assay that
was used to quantify Cas9 activity. The cells were nucleofected
with different destabilized domain constructs along with the
eGFP-targeting gRNA plasmid and then incubated with
compound 1 (Fig. 3A, S7A) or 2 (Fig. 3E, S8A). There was no
noticeable loss of eGFP signal in the absence of light exposure,
even after 48 h, though exposure to light of either 385 nm or 470
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Figure 3: (A) Two-dimensional photo-chemical activation of NL-DHFR•Cas9 activity in the eGFP-disruption assay in U2OS cells. Cells nucleofected with NL-
DHFR•Cas9 and eGFP-targeting gRNA plasmids were incubated with at various concentrations of compound 1 and exposed to increasing amount of UVA light
followed by 48 h of incubation. (B) Dose-dependent photoactivation of the NL-DHFR•Cas9 system with compound 1 in the eGFP-disruption assay in U2OS cells
upon exposure to 12 min of UVA light (C) UVA light exposure time-dependent photoactivation of the NL-DHFR•Cas9 system in the eGFP-disruption assay in U2OS
cells in the presence of 125 nM of compound 1. (D) Representative images of the eGFP-disruption assay by NL-DHFR•Cas9 under various conditions: no gRNA,
dark, 500 nM of compound 1 and 12 min of UVA exposure, or 8 min UVA exposure and 125 nM of compound 1. (E) Two-dimensional photochemical activation of
NL-DHFR•Cas9 by compound 2 and visible light. (F) Dose-dependent photoactivation of the NL-DHFR•Cas9 system with compound 2 in the eGFP-disruption assay
upon exposure to 12 min of visible light. (G) Visible-light-dependent photoactivation of the NL-DHFR•Cas9 system in the eGFP-disruption assay in U2OS cells in
the presence of 8 nM of compound 2. (H) Representative images of the eGFP-disruption assay by NL-DHFR•Cas9 under various conditions: no gRNA, dark, 8 nM
of compound 2 and 12 min of visible light exposure, or 6 min of visible light exposure and 8 nM of compound 2.
the upregulation of IL1RN was proportional to the duration of light
exposure. Notably, we observed minimal basal activation of
Acknowledgements
IL1RN transcription in the absence of either the compound 1 or
light exposure (Fig. S13C). Similarly, we were able to control the
transcription of IL1RN by varying the concentration of compound
2 or varying the exposure to visible light (Fig. S14). These studies
suggest that our optochemical system can be used for precision
control of both catalytically-active Cas9 and technologies based
on catalytically-impaired Cas9. Finally, we note that the
transcriptional activation system based on those reported by
Dahlman et al.[23] failed to provide an optochemical system with
low background and high dynamic range (Fig. S15-17).
This work was supported by the Burroughs Wellcome Fund
(Career Award at the Scientific Interface), DARPA (N66001-17-2-
4055), NIH (R21AI126239), and Army Research Office Award
W911NF1610586.
Conflict of interest
Broad Institute has filed a patent application including work from
this paper
In summary, we developed a singular system with dose,
temporal, and spatial control over Cas9 activity. This system
possesses a low background activity in the absence of TMP and
has an activity comparable to wild type Cas9 at a full dose of TMP.
Furthermore, this system is dosable, reversible, non-toxic to both
mammalian and bacterial cells, and afford precision optical
control of Cas9 activity by allowing photoactivation in minutes
using low-intensity light. Our system can be deployed for
optochemical control of transcription using catalytically inactive
Cas9 fused to a transcriptional activator. Finally, our approach is
extendable to other reported small-molecule regulated Cas9
systems. These studies will propel the development of therapeutic
and biotechnological applications of CRISPR-Cas9.
Keywords: CRISPR-Cas9 • genome editing • transcription •
photocaging • trimethoprim
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10.1002/anie.201900788
Angewandte Chemie International Edition
COMMUNICATION
COMMUNICATION
Debasish Manna, Basudeb Maji,
Soumyashree A. Gangopadhyay, Kurt J.
Cox, Qingxuan Zhou, Benjamin K. Law,
Ralph Mazitschek and Amit Choudhary*
A singular system for precision control
of Cas9 activity over dosage, timing
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Page No. – Page No.
Title
This article is protected by copyright. All rights reserved.