10.1002/anie.201909264
Angewandte Chemie International Edition
COMMUNICATION
N/P ratio. Hela cells transfected with pSPN/CRISPR
nanocomplexes were mixed with Matrigel and subcutaneously
injected into two sides of the back of living mice (Figure 4a). The
left side was exposed to 680 nm laser for 20 min and then the
fluorescence images of whole mice were longitudinally recorded.
To avoid the skin damage, temperature was monitored during the
NIR irradiation and the maximum temperature only reached
~36.4°C, far below the threshold to induce cell apoptosis (43°C,
Figure S19 in the Supporting Information). The green
fluorescence signal at the irradiated region was 1.2, 1.5- and 1.8-
fold higher than that without irradiation at 24, 48 and 72 h of post-
treatment, respectively (Figures 4b, c); while the NIR signals from
pSPN at two sides were the same at each time point (Figure S20
in the Supporting Information), showing the stability of pSPN
during the treatment. The tissues containing the implanted cells
were taken out and sliced for fluorescence imaging. The stronger
green fluorescence signals were observed for the irradiated group
(Figure 4d and Figure S21 in the Supporting Information), further
confirming that the occurrence of pSPN-mediated photoregulation
of CRSIPR/Cas9 repair of GFPs. Histological analysis was
conducted on the skins with or without irradiation through
hematoxylin and eosin (H&E) staining. The result showed that
there was no obvious histopathological abnormalities or lesions
appeared on the light-treated skin (Figure 4e). These data
confirmed the feasibility and the biosafety of pSPN-mediated
photoregulation of CRISPR/Cas9 gene editing at designated
location in living mice.
In conclusion, we synthesized
a
photolabile polymeric
nanotransducer (pSPN) for delivery and photoregulation of
CRISPR/Cas9 gene editing. Upon NIR photoirradiation, the
backbone of pSPN generated 1O2 to cleave the 1O2-cleavable
linker, resulting in the efficient release of gene vectors. Such a
photoregulation process was validated in CRISPR/Cas9 gene
editing in the model of homology-directed repair, showing 15- and
1.8-fold enhancements of repaired GFP expression in cells and
living mice, respectively. Because this photoregulation is exerted
on the gene carrier (pSPN), no specific modifications are required
for Cas9 and sgRNA, making it a generic approach for gene
editing. To the best of our knowledge, pSPN represents the first
NIR photoregulatable CRISPR/Cas9 nanocarrier. In view of its
tunable optical properties and purely organic components, pSPN
holds promise for the development of CRISPR/Cas9 mediated
gene therapy.
Acknowledgements
This work was supported by Nanyang Technological University
(Start-up grant: M4081627.120) and Singapore Ministry of
Education Academic Research Fund Tier1 (2017-T1-002-134,
RG147/17) and Tier 2 (MOE2016-T2-1-098&MOE2018-T2-2-
042).
Conflict of Interest
The authors declare no conflict of interest.
Keywords: semiconducting polymer • CRISPR/Cas9 • gene
editing • photoregulation
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