   No. 57 (2018 October)2018  10 

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生命化学研究会の 10 年+フロンティア生命化学研究会の 10 年
大阪府立大学 大学院理学系研究科
円谷 健

4 



4 細胞外微粒子を追跡する遺伝子制御分子〜非コード RNA を操って生命現象を操る〜

山吉 麻子

長崎大学 大学院医歯薬学総合研究科（薬学系）

9 金ナノ粒子を用いた分子検出〜一分子計測を駆使した検出法開発〜
愛媛大学 大学院理工学研究科

座古保

15   
大阪大学 大学院薬学研究科創成薬学専攻
九州大学 大学院工学府応用化学部門
千葉大学 工学部共生応用化学科第二分野

24 

堀場 昌彦

博士後期課程３年

博士後期課程２年

佐々木 光一

日本学術振興会 PD

上田 大次郎

アルバータ大学留学体験記
九州大学 大学院工学府

長尾 匡憲

28   
第 45 回 国際核酸化学シンポジウム（ISNAC2018）、日本核酸医薬学会 生物セッシ
ョン 第３回サテライトシンポジウム、KUMP International Symposium

受賞、異動

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ǕŭǠǐǀǧǋÅJĩAeȣ÷ȡþ?jo ð8ºȤǕäǀ¢ŞƹǋǅǞǇƲǞǋƱÈĒĝǕµǩǎǐǄǎǋ&ǐǖ'KĒĝıǕ¢ŞÿǅǂǞǇƲÃǕƱ112ǕúR=oĒĝȞȃȢƴÛǕǔǦŜ»Ƶ
Ǖ\Ģǖ×ǪƻǐǄǎǋjƂĞjoǖž"#úƱǊǅǐUƱÈěǖ\Ģ×ǪƻǐǀǌǄǎǋíÈjooųǖC¹A#úǕäǀĕÿǅǂǞǇƲ
6. D İ » ô
1)Mattick J.S., EMBO Rep., 2, 986–991 (2001).

2)

Krol, J. et al., Nat. Rev. Genet., 11, 597-610 (2010).

3)

Ma, L., Breast Cancer Res., 12, 210 (2010).

4)

Osaki, M., et al.,5)

Teplova, M., et al.,Nature Structural Biology, 6, 535 - 539 (1999).

6)

Stenvang, J., et al.,Silence, 3, 1 (2012).

7)

Obika, S., et al., Tetrahedron Lett., 38, 8735-8738 (1997).

8)

Ma, J.-B., et al., Nature, 434, 666-670 (2005).

9)

Ariyoshi, J., et al., Bioconjugate Chem., 26, 2454-2460 (2015).

Biomarkers, 13, 658-670 (2008).

10) Ariyoshi, J., et al., Chem. Lett., 46, 143-145 (2017).
11) Ariyoshi, J. et al., Nucleic Acids Ther., 27, 303-308 (2017).
12) Valadi et al., Nat. Cell Biol., 6, 654-659 (2007).
13) Kosaka et al., J. Biol. Chem., 285, 17442-17452 (2010).
14) Toda et al., Biochem. Biophys. Res. Commun., 456, 768-773 (2015).
15) Hoshino et al., Nature, 527, 329-335 (2015).

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Ɛ đ40 nmƺůǭǰĦtŁƁǍȐÒűDNAƵ

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-10-ĂXCvĖĞȈǤȌƐ Ɛ No. 57 (2018 October)
a)

b)

ǍƭǊƮǊƺDNAôƹƧƴæǃƯƶƤǋƔôuĎƹǭǰĦt0źƺĚƻk@ƧƯƕDNAǍï@Ƨƴƚ
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0

0

100

200

+100 nM Complementary DNA

c)

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0.1
0.05
0



0

100

200

Intensity (a.u.)
3σ line

+100 nM mismatched DNA

Target DNA concentration (pM)ƸƚƔǲǧǘǙȅǒȊǬƺ$ƹðxœ3"Ǎ@ģƧƯ$(3ƓȅǑȊ)ƶƺ

0.05

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0.1

Intensity (a.u.)

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

0.1
0.05

20 µm

0

0

100

200

Intensity (a.u.)

 GC $58@<\_`:fg2?]

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Aβ Ɛ ůǭǰĦt0źƺ2tŋðƹǆǈƔDNAƺś
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20 μm

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0
0

FrequencyǔǶȅǵȊT(ThT)ǅǛȊǜȈǧǬƸƷƺļĩƟƹ
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0FrequencyƹZƆƟƙƲƯƕ°ǍăƚǉƤƶƵƎ©Ú
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0.75
0.5
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0

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0.05

Aggregate ratio

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0.04

50 100 150 200 250
500 nM00.001

Background
0.1

10

1000

Aβ fibril concentration (nM)
0
0

50 100 150 200 250

Intensity (a.u.) HC :fg2?]

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80 nm AuNP
/40 nm AuNP

0

1

10

100

ȖȏƐ ů ǭ ǰĦ t 0 ź ƺ  2 t ŋ ð ƹ ǆǉÚ 1 © Ɛ ůǭǰĦt0źƺ2tŋðƹǆǈƔ0ź«ǍƝƤ
10 μm

Ʃ2tƺƎ©Ú1ƟPĸƵƙǉƤƶƟ2ƞƲƯƕƵƻ

60
50

%

ƷƺơǇƚƺƎ©CƟPĸƵƙǋƛƞșƭǊƹƻǁƪ

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30ůǭǰĦt0źƺÚ1©ǍÉǇƞƹƩǉ¡ŅƟƙǉƕ

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20

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10

ƭƺƯǃƹƔ40 nmƺůǭǰĦtòìƹƔ0źƺȁǪȇ

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1080 nm AuNP IC 'R[

50

100

(%)

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Frequency (%)Ɛ ƵƻƔƷƺơǇƚƺǝǑǡƺ0źǁƵÚ1PĸƵƙǋ
15

15

10

10

10

5

5

5

0

0 4 8 12 16 20 24 28 32 36 40

Scattering intensity (a.u.)

1

15

0

0
0 4 8 12 16 20 24 28 32 36 40

0 4 8 12 16 20 24 28 32 36 40

Scattering intensity (a.u.)

Scattering intensity (a.u.)

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-13-ĂXCvĖĞȈǤȌƐ Ɛ No. 57 (2018 October)ěĄpvƝǆƾÔ

ŠpƵƺǼǠǬǘÊƺĖĞĮƍƟpƠơ|ƧƴƚǁƩƕĻêƎ¢½¶ȍþÔpvpvŸĽvħ½¶ȎƔƋýĄâÁ½¶ƺƥ³ƹíŕƚƯƧǁƩƕÍƹƔdġƺßǍƜƴƦƲƯƔJ¼I½¶ȍøÒpvȎƹĘĆƧƣǁƩƕ

KĵÁü

1.

O. (2016). 2tŋð¯ŀǍăƚƯǲǑǔ2ÕCv.

2.

O. (2016). ůǭǰĦtǍăƚƯÚ1ȋŏÃ¯ŀ.  -- 34, 190-7.

3.

Ghosh, S. K. & Pal, T. (2007). Interparticle coupling effect on the surface plasmon resonance of gold

497, 173-4.

nanoparticles: from theory to applications. Chem. Rev. 107, 4797-862.
4.

Sato, K., Hosokawa, K. & Maeda, M. (2003). Rapid aggregation of gold nanoparticles induced by
non-cross-linking DNA hybridization. J. Am. Chem. Soc. 125, 8102-8103.

5.

yĄ

& :ĄĀq. (2015). DNA

ŬǇƫǎ±²ůǭǰĦtǍăƚǉ2ÕCv.  64,

15-23.
6.

Jans, H., Liu, X., Austin, L., Maes, G. & Huo, Q. (2009). Dynamic Light Scattering as a Powerful
Tool for Gold Nanoparticle Bioconjugation and Biomolecular Binding Studies. Anal. Chem. 81,
9425-32.

7.

Bu, T., Zako, T., Fujita, M. & Maeda, M. (2013). Detection of DNA induced gold nanoparticle
aggregation with dark field imaging. Chem. Commun. 49, 7531-3.

8.

Bu, T., Zako, T. & Maeda, M. (2016). Dark Field Microscopic Sensitive Detection of Amyloid
Fibrils Using Gold Nanoparticles Modified with Antibody. Anal. Sci. 32, 307-11.

9.

Zako, T. & Maeda, M. (2014). Application of biomaterials for the detection of amyloid aggregates.
Biomater. Sci. 2, 951-5.

10.

Sakono, M., Zako, T. & Maeda, M. (2012). Naked-eye detection of amyloid aggregates using gold
nanoparticles modified with amyloid beta antibody. Anal. Sci. 28, 73-6.

11.

Ohshiro, T., Zako, T., Watanabe-Tamaki, R., Tanaka, T. & Maeda, M. (2010). A facile method
towards cyclic assembly of gold nanoparticles using DNA template alone. Chem. Commun. 46,
6132-4.

12.

Watanabe-Tamaki, R., Ishikawa, A., Tanaka, T., Zako, T. & Maeda, M. (2012). DNA-Templating
Mass Production of Gold Trimer Rings for Optical Metamaterials. J. Phys. Chem. C. 116, 15028–33

13.

Wang, G., Bu, T., Zako, T., Watanabe-Tamaki, R., Tanaka, T. & Maeda, M. (2017). Dark fieldmicroscopic analysis of discrete Au nanostructures: Understanding the correlation of scattering withstoichiometry. Chem. Phys. Lett. 684, 310-5.

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Wei, H., Li, B., Li, J., Wang, E. & Dong, S. (2007). Simple and sensitive aptamer-based colorimetricsensing of protein using unmodified gold nanoparticle probes. Chem. Commun., 3735-7.

-14-

¬8-O¹ÃƃūƆğ ğ No. 57 (2018 October)CEeğ ƇŊœŅ ŋıņįƈ
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Yin, H.; Song, C.-Q.; Suresh, S.; Kwan, S.-Y.; Wu, Q.; Walsh, S.; Ding, J.; Bogorad, R. L.; Zhu, L. J.; Wolfe,

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with theCas9tracrRDNA
5 G G G C G A G G A G C T G T U C A C C Greplacementof DNA nucleotides in crRNA is tolerated by Cas9, the percent
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promoter-GFP were transfected with a crRNA targeting GFP and the tracrRNA. Cas9-mediated frame-shift NHEJ yields GFP-negative cells. W
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ARTICLE....

ARTICLE

Published on 06 February 2018. Downloa

Published on 06 February 2018. Downloaded by Osaka University on 7/30/

guidehaving
diﬀerent
G-quadruplex(Tm = 60 1C at 100 mM KCl) as seenin RNAs
previous
reports
from our
10
ends
and
performed
an
in
vitro
lab. To functionally validate the modifications, we chose ancleavage re
0
that
while
sgRNA that targets the eGFP gene(Fig.
1).both 3 G-quadruplex motif-contaand G3U3)
the not
targetDNA
as eﬃc
NATURECHEMICAL
BIOLOGY
DOI:
10.1038/NCH
Although chemical
modificationsof cleaved
bases
do
alter
0
¬8-O¹ÃƃūƆğ
ğ
No.
57
(2018
October)sgRNAs, theaddition oftoa DNA,
5 G-quadrupl
sgRNA:Cas9 association or subsequentlocalization
its cleavage
eﬃciency
2Aaba structure abolishedmodifying the sgRNAmight potentiallyhindereitherb (Fig.
Fig.VEGFA2
Cas9őĻĽŝŹūƆŢŭŰć#ňŃïõŘs%ľĬŔĿñŘ0RNAsimilarresultswith
3
G-quadruplex
motifs50
A 700 bt
DNA replacementof theseorderreporterto testDNA
TracrRNA steps. In
293T-GFP
cells the eﬃcacy of G-quadruplexÄĽŋĳĸġķįľĠŸŰŢŵžłIĭŃŝŹūƆŢŭŰć
loci, Tert andTransfect:c-mycGFP-crRNA(Fig. S1, ESI†), protein
while
GFP crRNA
DNAprotein
mismatch complex
containing sgRNAs andCas9
activity on a given 40
EFs
Cas9+ VEGFA
TracrRNA
crRNA minimal
RNA mismatch
containing sgRNAs
showedcleav
perform#īN>ĴŔ VEGFA ĄLŘ³Ŀĳĸ
crRNAŘëDNA target, we purified the Cas9 protein, in vitro synthesized 300
5end
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A C C C CC
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guide
RNAs
having
diﬀerent
G-quadruplex
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and3
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10
DNA
G
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C
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Tolerated
crRNA
with
resolved
NATURE CHEMICAL BIOLOGY DOI:10.1038/NCHEMBIO.2559
the formation of the active Cas9–sgRNAcom
DNA replacement
10
andperformed5 Gan
in vitro cleavage reaction. We observed
sgRNA.
ĦĽóĳĽĦŋĴ
ŃŌľ
Tail (= 2)ġķŕłőĻĽĠRNASeed
3 TracrRNAends
interacting
regionG G C C C C T C C A C C C C G C C T C OT1of the complex to bindand cleave a targe
Top off-target
reactionC A C C C C G C C T C OT2
5 G C C C C C A C Cmotif-containingthat whileboth
3 0 G-quadruplex
sgRNAs00 sites(G2U1
pıŕĸ crRNA(Native
crRNA)
Ŀ
10
FBŘ
DNA
ċ
investigate
whether
5
modification
to sg
d
c
b
DNA replacement at 5’ of GFP crRNA
50
OT3sgRNAs5 C G C C C T C C C C A C C C C G C C T C
and
G3U3)
cleavedthetargetDNA
aseﬃcientlyasunmodified
NATURECHEMICAL
NATURECHEMICAL
BIOLOGY
DOI:10.1038/NCHEMBIO.2559
łÒyĳĸ crRNA(10 DNA)ń VEGFA ĄLłVĳ
TracrRNA
293T-GFP reporter
cells
crRNA
2.
crRNA 0VEGFAC C G Native
theaddition5 G G G C G A G G A G C U G U U C AsgRNAs,
40 of a 5 G-quadruplex motif completely
Transfect: GFP-crRNAEFs( (Fig. 2A). We also observed
Ľ4ÇŃŢŵžÏĕ,ªŘ»ĴľĠŝŹūƆŢŭŰG A G G A G C U G U U C Aabolished
C+CTracrRNAG 2 DNA its cleavage eﬃciency
5 G G G C Cas9bcba
VEGFA on-target
off-targ
Cas9
d 0 )eVEGFA
50
DNA
G G G C G A
G G10
A GDNAC U G RNAU U C Asimilar
C C G 4 results
5EF1a30G-quadruplex motifs
GFP2
crRNA
formatwith3
targetingendogenous
GFP
ć#łVĴŔŢŵžÏĕń
łĩĦĽJ_łs%
50
On-t
50
replacement
AM
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40
0
G A G C Ureporter
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A C C G 6 DNA Tolerated crRNA
5 G G G C G A G 293T-GFP
Gwith
G C A
CG G
G C A G while
C U U G C theC G G 5Native
5 G
crRNA
we
per
loci,
Tert
and
c-myc
(Fig.
S1,
ESI†),
G-quadruplex
40
Transfect:
GFP-crRNA
ıŕŔįĿīŒĪłŁœŋĳĸġ
ŃÍĪŒĠCRISPR/Cas9ťŧŮžīuĨŔŝŹūƆŢŭŰć#łV
DNA mismatch
40
DNA
replacement
20G G C A C G G G C A G C U U G C C G G 10 DNA
G C U G Cas9U U C A C C G 8 DNA
TracrRNA interacting region
5 G
5 G G G C G A G G A EFs
30+
TracrRNA
VEGFA
crRNA
RNA
mismatchwiththO
containing sgRNAs showed minimal30cleavage.
OT2
30 OT1ĴŔŢŵžÏĕĿĦħ9ĚŘ;ąĴŔĸŁqī»ıŕŋĳĸġCRISPR/Cas9
10 DNAU U G G C G G RNA mut-1
5 G G G C A C G G G C A G CťŧŮžŘ.±l­ĴŔ
5 G G G C G A G G A G C U G U U C A C C G
20
crRNA
A C C C CCU C C AWeC C Creasoned
C G C C U C Native
EF1a
5 G
GFP
catalyt
that
this
could
be
eitherdue
tothe
failureind
GFP crRNA
50
10
C U U G G C G G 10 DNA mut-1 20
5 G G G C A C G G G C A G20
PAM
A GG AC CT CGCUCUC CT AC CC CA G
5 G G G C G A G G 5
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the formationof the Gactive
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and elongation
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VEGFA
crRNA.
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hinder
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a GcrRNA
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determine
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ĈŘXĳĠÉ×
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replacement
thesequenceof GFP crRNAs.
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10 DNA(c) IllustrationRNA
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with
20
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10
DNA
are
shown
in
black
and
red,
respectively.
(d)
HEK293T
cells
described
above
were
incubatedwiththetracrRNAanda
GFP-targeting
TA TGCG AA CGCCGand(d) Illustration of mismatch mutationsof GFP2 sequences. Arr
5 G G G C A C G G G C A G C U U G G C G G activity.
10 DNA mut-1
12 DNAT G18UDNAU C A C C G*
10illustrated.
TIDE analysis was performed
to calculate the percent of indels
at mut-2
GFPnlocus
day 3. n = 9independent
biologically samples,
independent
samples.
Error
analyze
indels.
= 3–5atbiologicallyas indicated.
*P
<0
RNA
TA TGCGAACGCCGT G20
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U C A C C G 14 DNA
00mean o s.d. Purple color indicates mock-transfection-treatedsamples.
Black
dots
indicate
nativecrRNAtransfected
samples.
Red
dots
indicattargetanalysisofnativeand
10
DNA
crRNAsfromthreeendogenous
gc
Fig.
1
RepresentationofvariousCas9–sgRNA
improv
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each typeof crRNA.
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Tail
Seed
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factor 1-AūƆŢŭŰć#ŃŢŵžÏĕŐ-O
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motifsreplacingthetwo to ten RNA nucleotides with DNA
ind
hypothesized
that
the guide sequence
tolerate
DNA indicatestolerated byWe
Cas9,
the percent of
GFP-negative
cells will bemay
retained.
Thepartial
blue arrowhead
10 PAM,
25,26
ement at theguideregion
of
a
GFP
crRNA
inducesgene
editinginhumancells.
(a)Diagramofthe
CRISPRsystem.
Figure3|PartialDNA
replacement
at
the
guide
regionreduces
off-target
effectsinhumancells.
(a)Illu
We
performed
GUIDE-seqtosystematicallycompareunmod2).Commun., 2018, 54, 2377--2380
replacement
basedon thestructureof TheCas9
and guide RNA
. To
Chem.
ĦĻĸ9ĚŘMřľĦŋĴġÝÔŒńĠ
NA replacement
at the guidesequenceof GFP
crRNAs.
20-nucleotide(nt) guideregion(Supplementary
is shown. 2378
RNA | Fig.
b) HEK293Ttest
cells
stablyexpressing
bothelongation
factor-1
short
(EFs)
promoter-SpCas9
andelongation
factor
1-A(EF1A)
genome-wide
off-target
activityofnative
and
10
DNA
crR
sequence of unmodified
VEGFA crRNA.
Arrows(native
denote mismatches
betweenwhether
target andoff-target(b,c) HEK293
To investigate
partialDNAsites.
replacementcantourcells
hypothesis,
wefirst weresynthesized
crRNAsmore
tively. (d) HEK293T
described
above
incubated with the tracrRNA and a GFP-targeting crRNA
crRNA
tracrRNAed with a crRNAtargeting
GFP
andtracrRNA.
Cas9-mediated
NHEJ
yields
When
Three
guide
sequenceswerechosen:
mouse
Pcsk9
34),
hu
with theidentified
tracrRNA guideand aframe-shift
VEGFA-targeting
crRNA
with
10 DNAnt
replacement
at the 5`
end of(ref.
the
guide
se
byGFP-negative
sgRNA,
we cells.
synthesizedsgRNAstargeting
GFP,
EMX
crRNA)
onĿthebasis
oftheaŘÐĮĽċĿpreviously
sequence targetsgRNA
alculate the percent of27 indels at GFP locus at day 3. n = 9 biologically independent samples. Error bars show
des in crRNAtolerated
Cas9,
thepercent
of
GFP-negative
cells will
betoretained.
blue humanarrowhead
the
EMX1,
and
293indicatessite
4 (ref.
10).
Mouse
Hepa1-6
liver
cularendothelial
growthfactorA(VEGFA),withandw
ingisĳĸGFP. A bynumberof crRNAs
withthe same
guidesequence,
but Theindels
Surveyor
assay
were performed
determineatthe VEGFA
locus
(b)
and
3 top
off-target
(OT)
sites
sgRNA
łVĳĠÉ×łRĔł
sfection-treated samples. Black dots indicate native crRNA transfected samples. Red dots indicate DNA–RNA
until 624
lustration ofvarying
DNA replacement
the substitutions
guidesequenceof
GFP
crRNAs.
The
20-nucleotide
(nt)
guide
regionisstably
shown.
RNA
human
HEK293cells
expressing
weretransfedegrees of atDNAatindependent
the5` end,were
synthesized. Aftertransfecting
theSpCas9
sgRNAsin
HEK29
biologicallysamples.
*P <or
0.01
byreplacement
one-way
ANOVA
with
Tukey
post hoc
test.
(d,e)
Partial
DN
N>ĴŔšŚųƄ:ĉċĀŘĝNex
withGuide-seq
oligos,tracrRNA,
andsgRNAs(1)
nativeor(2
and red, respectively.
HEK293T
cells describedabove
werenative
incubatedwithtargetthe
tracrRNAand
GFP-targeting(Fig. 1c).(d)Whenevaluatedinactivity.
HEK293T
cells,
crRNA
cells,
weasequences.
found
that
thecrRNAnativeandcrRNAsgRNAswi(d)Illustrationof mismatch
mutationsof GFP2
Arrowsdenotepoint
mutations.
(e)
TID
modifiDNA
crRNA.
Analysisoftheoff-target
peaks
revealed
that
allperformed tothe percent
of indels
at
GFP
locus
at
day
3.
n
=
9
biologicallyindependent
samples.
Error
bars
show
ĴŔįĿľĠmŁŀŃ9ĚŘ;ąĴ
ingcalculate
GFP generated
27%
o 2%
GFP-negativecells(Supplementary
tion
of
eight
DNA
and
ten
DNA
nucleotides
in
the
5`
en
of indels. n = 3–5 biologically independent samples, as indicated. *P < 0.05 by unpaired, two-tailed Studentt
10 DNA
crRNAshaddetectable
off-target
sites
using
the
thr
sgRNA
3’
Fig.
1a,b).
We
found
that
replacing
four,nativesix,3.1eight
ten5’RNAsimilarlevelsofno
indelsinDNA–RNA
cellsThe
forchart
all
three
guide
sequenc
ates mock-transfection-treatedsamples.
Black
dotstwo,indicate
nativecrRNAtransfectedRed
dots
indicate
injectetargetand
10orDNA
crRNAsfromthreeendogenous
indicates
the
numbe
Fig.
Representation
ofsamples.
variousCas9–sgRNAcomplexes.
sgRNAsarece may tolerateŔĿłĠŴŠƃŚƆũÕmŃ
partial DNA replacing
twoanalysistoī
ten ofRNA
nucleotides
withDNA
inducedindels genes.
00
Cas9–sgRNA
(
)
old
of
six
or
fewer
total
mismatches
between
the
guide
sequ
nucleotides
withDNA
nucleotides
starting
from
the
5`
end
of
the
These
data
show
that
partialDNA
replacementof
bothrately iappendedwitheither 3 orG-quadruplex
motifs.
forCRISPR
each typeof 2).
crRNA.
Six total
mismatches
are5 allowedin the guideand PAM. (g) Number of GUIDE-seq
esamples.
editing
humancells.
system.
PAM,
25,26(a) Diagram of theCas9andinguideRNA. To (SupplementaryFig.
2ØľĥŔĿÓĨŋĳĸġ
and
PAM
(Fig.
3f,g
and
Supplementary
DatameanSet o1),s.d.
althoughguide
sequences alsoand
generated
similarlevelsof off-target
GFP-negative
cells
sgRNA
retains
on-target
genomeediting.
site.
OT1–OT6
are
top
sites
in
the
native
crRNA
data
set.
Error
bars
represent
or-1
short
(EFs)
promoter-SpCas9
elongation
factor
1-A(EF1A)
unmodified crRNAs (native
To investigate whether partial DNA replacement
canGUIDE-seq
be tolerateddata for the EMX1 target site is subopt
depth of2 the(ranging
from22%31%) Ń
(Supplementary
1a,b), indicating
To investigate whether
DNA–RNA chimeric crRNAğ ŋĵÝÔŒńĠsgRNA5’ÅĩőŇ
3’Åłć#Ń¯ŁŔÀěŃšŚųƄ:ĉċĀŘĝĳŋ
mediated
frame-shift
NHEJ
yieldsto
GFP-negative
cells.
When Fig.
ified guidesequence
targetby
sgRNA,
wesynthesizedsgRNAstargeting
GFP,
EMX1
andconclusions
vas-in therapeutictomakedefinitivefor indels
this sequence.
Consistentthat
partialreplacementofuptoten-nucleotide
DNA
at
the
5`
end
can
be
applieddelivery, we
incubatreplacing
two
to
ten
RNA
nucleotides
withDNA
inducedguide
sequencemaytolerate
partialDNA
egativewill
be retained.
The bluearrowhead
indicatesthee samecells
guidesequence,but
cular
endothelial
growthfactorA (VEGFA),
withandwithout
DNA
10 theĳĸ(=3)ġîÉłļĦĽń'oĦĸĳŋĴīĠ3’ÅłšŚųƄ:ĉċĀŘĝĳĸE3łĩĦĽŴ
25,26
VEGFA
off-target
analysis,
these
data
collected
via
an alter
16DNA
design(16-nuc
We
performed
GUIDE-seqtosystematicallycompare
the
the
guide
sequencewastolerated.
However,
replacement
with
10
DNA
(replacingtenRNA
nucleotides
with
DNA
nu
(SupplementaryFig.
2).
structure
of
Cas9
and
guide
RNA
.
To
2378
|
Chem.
Commun.,2018,
54,2377--2380
24
GFP5`crRNAs.
Thesynthesized
20-nucleotide (nt)guide regionis shown.
RNAtheend, were
replacement. Aftertransfecting
thesgRNAsin
HEK293T
reporterapproachindicate
that
10
DNA
crRNA
substantiallyreduces
12-nucleotide
DNAgeneratedsignificantly
fewer
GFP-negative
the
5’
end)
crRNA
and
a
tracrRNA
with
Cas9
proteinavoiding
the
Cas9
bindi
genome-wide
off-target
activityofnativeand
10
DNA
crRNA.
To
investigate
whetherpartialDNA
replacementcanbetoleratedtsynthesized
unmodified
crRNAs(nativeŠƃŚƆũÕmīĳĠľŏŤśŨŃYıĦ
G2U1 :ĉċľŢŵžÏĕ,ªŃ5īºïıŕŋĳĸġ
e cells,
were incubatedwith thetracrRNA
andwe
a GFP-targeting
crRNA
nativecrRNAtargetcells,
foundthat thenativesgRNAsandsgRNAswithtargetediting.
cells
thanguidethenativecrRNA,
andreplacement
withor moreDNA
10targeting
DNA
sgRNA
with
Cas9
protein to form
ribonuThree
guidesequenceswerechosen:mouse
Pcsk9substitu(ref.
34),EMX1
humancomparable
to nativecrby sgRNA,
we 14
synthesizedsgRNAsGFP,
and vaseviously identified
sequence
targetsativeat day3. n(Supplementary= įŃÍłļĦĽÝÔŒńĠšŚųƄ:ĉċĀŃČıŐĀī
9 biologically independent
samples.
Error
bars
show
sgRNA
ŃØłfĘŘĨĽĦŔ
cells
tion
of
eight
DNA
and
ten
DNA
nucleotides
in
the
5`
end
generatednucleotides
reduced
GFP-negativecells
tobackgroundlevels(~2%)(RNP).
We
found
that
they
each
inducedlevelsofinand human
293 growthsite 4 (ref.
10).
Hepa1-6
livercells DNA
sequently tested whethe
endothelial
factorA Mouse(VEGFA),
with andwithout
RNAs with the same guide sequence,EMX1,
but cular
catenativecrRNAortransfected
samples.
Red
dots
DNA–RNA
four,
six, eightten RNA Fig.
similarlevelsofindicate
indelsinfor allthreeexpressing
guidesequences
(Fig.
2b).
24
Structure-guided
substitution
3`RNP
ofDNA
crRNA
1a,b).
The
efficiency
ofcells
genomeediting
after
toSpCas9
those
ofwerenativecrRNAor sgRNA
after
electroor
human
HEK293cells
stablytransfectedwithat5`the
and
3`
sub
replacement
ubstitutions(SupplementaryatĿÓUĳĽĦŋĴġĠ5’ÅłšŚųƄ:ĉċĀŘĝĳĸE3ńĠŢŵžÏĕīÝĳĭđSıŕĽ
the 5` end, weresynthesized. Aftertransfecting
thesgRNAsinDNA
HEK293T
reporterting from the5` nucleotide
end of the replacement
These data
show
that
partialDNA
replacement
of
both
crRNA
and
To
explorewhetherthe3`
tracrRNA-interacting
regionof crR
DNA
at
the
guide
sequence
was
confirmed
Jurkat
T
cells
(Fig.
2c).
with Guide-seq
oligos,that
tracrRNA,and(1)
native
crRNA
or
(2)
10
both
8DNA
and
16DNA
in HEK293TĳŋħĿĦħÍłŁĻĽĦŋĴġįĺŒłļĦĽńĠ¢mŁ
cells, native crRNA targetcells, we foundthe native
sgRNAsandsgRNAswith
substituCas9-sgRNA
ã3ŃdpĠŋĸńĠ³Ą
28
levels of GFP-negativesgRNA
retainscrRNA.
on-target
genomecantolerateDNA
we synthesized
a withseriesotherof chby tracking
ofcells
indelsby decomposition
Torevealed
test whether
replacement
worksC
ofoff-target
theDNAof editing.
the
peaks
that5`DNA
allendthree
in 5` and16-nucleotide
2% GFP-negativecells(Supplementarytion(TIDE)
of Analysis
eightanalysisDNAandten PCR
DNA
nucleotides
inreplacement,
thegenerated
21
ntary Fig. amplicons
1a,b),
indicatingTo
investigate
whether
DNA–RNA
chimeric
crRNAsor
sgRNA
ric
crRNAstargeting
GFP
(Fig.
4a).
U2OS-GFP-PEST
cells
wesynthesized
Acidaminococcus
sp.
Cpf1(AsCpf1)
na
from
the
GFP
genomic
locus
(Fig.
1d).
LŃïõī$łŁĻĽĦŔįĿī0<ľńŁĦĪĿÓUĳĽĦŋĴġŋĸĠŝŹūƆŢŭŰć#łVĴ10
DNA
crRNAshadnodetectable
off-target
sites
using
the
threshing
(Fig.
4b).
Of
note,
t
replacing two, four, six, eight or ten RNA similar levels of indels in cells for all three guide sequences (Fig. 2b).
ing two DNA
to tenRNA
nucleotides
withDNA
inducedindelsucleotide
at
the
5`
end
can
be
appliedintherapeutic
delivery,weincubated
VEGFA
bly
expressing
Cas9
were
transfected
withcrRNAsandtracrRtargeting
DNMT1
(ref.
31)
To
study
the
effectsofDNA
nucleotide
replacement
on
an
or
crRNA
with
an
eight-nucl
or datafewer
totalthatmismatches
betweenthe guide
sequenceof DNA basecleotides startingfrom the 5` end of old
the of six
These
show
partialDNA
replacementof bothcrRNA comprisedandŔŢŵžÏĕńĠšŚųƄ:ĉċĀŘ3’ÅłĴŔsgRNA
łĩĦĽ§łùįĻĽĩŒĵĠĝ®
plementaryFig.
2). with
However, replacement
10 DNA(replacing
ten RNA
nucleotides
withDNA
nucleotides
in 3` end
GFP-negative
cells
bythe
Cas9-mediatedframeshiftN
endogenous
gene, wesynthesized
crRNAstargetingthe human
replacement
atcausedthe(as theguide
sequence
of Cp
and
PAM
(Fig.
3f,g
and
Supplementary
Data
Set
1),
althoughDNAbases
are moret
rated similarlevels of GFP-negative
cells
sgRNA
retains
on-target
genomeediting.
29tolerated
cantlyfewer
GFP-negative
the5’ end)
crRNA
and
awith
tracrRNA
with
Cas9
proteinor by
VEGFAŃmŏåŒŕĽĦŁĦĿÿŉĽĩœŋĴġ
ŃőħłĠŴŠƃŚƆũÕmĿŢŵžÏĕ,ªŃÍĪŒĠ
investigate
whetherpartialDNA
replacement
can
be
were
measured
FACS
at
day
3
to
reportgenomeeditinggene
EMX1(emptyspiracles
homeobox
1),
variouslevels
of
at
3`
end).
HEK293T
cells
weretransfected
with
a
plasm
depth of thedata for the
EMX1 target
site is crRNAs
suboptimal
oligo synthesis, the cos%) (Supplementary Fig. 1a,b), indicating
ToGUIDE-seq
investigate whether
DNA–RNA
chimericor sgRNA
ment with
14 or more
DNA
DNA
sgRNA
with andCas9vasto
form ribonucleoproteinRNA,synthesizedsgRNAstargeting
GFP,
EMX1As AsCpf1
shownConsistentin
Figure4b,allless22
RNA
nucleoDNAreplacement
at10
the
After
transfecting
aproteincrRNAtargetingproteinfollowed
by~60%
crRNAsafter
24 thath.
Thetodefinitive
conclusions
forciency.
this sequence.
withreplacing
than
forC
f up toweten-nucleotide
DNA atthe 5`
5` end.
endmakecanbe applied
in-16therapeutic
delivery,weincubated
VEGFA
to backgroundlevelsfactor(~2%)(RNP). We
foundthat theyeach
inducedlevelsof regionindelssimilar
in
the
3`
with
DNA
(22DNA)abolishedgenomeedi
ing
EMX1
and thetracrRNA
into
HEK293T
cells
stablyexpressing
withpartialDNA
replacement
fully
maintainsits
activitendothelial
growthA(VEGFA),withandwithout
DNA
the VEGFA
off-target
analysis,thesenucleotides
data collected
viaDNAan alternate
these data pr
as tolerated.
However, replacement with
10 DNA(replacing
ten RNA
with
nucleotidesTogether,
in
24
ncy
of genome
editing
tosgRNAs
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inthe5` endgenerated¬8-O¹ÃƃūƆğ ğ No. 57 (2018 October)šŚųƄ:ĉċĀī sgRNA ŃĝĿĳĽŕĸ"LľĥŔįĿī»ıŕŋĳĸġŃôĿ4ĠK¥®ŃĀŘ\Ōłl­ĴŔįĿľ sgRNA ŃØ5łp*ĳĽĩœĠ­mŃĞıŐţŧŰėĪŒŏĖ
Journal of the American Chemical Society^łŃĥŔ¹ÃĿèĨŋĴġ

Receptor-mediated delivery of CRISPR-Cas9 endonuclease for cell-type-specific gene editing
Rouet, R.; Thuma, B. A.; Roy, M. D.; Lintner, N. G.; Rubitski, D. M.; Finley, J. E.; Wisniewska, H. M.;
Mendonsa, R.; Hirsh, A.; De Oñate, L.; Barrón, J. C.; McLellan, T. J.; Bellenger, J.; Feng, X.; Varghese, A.;
Chrunyk, B. A.; Borzilleri, K.; Hesp, K. D.; Zhou, K.; Ma, N.; Tu, M.; Dullea, R.; McClure, K. F.; Wilson, R.
Journal of the American Chemical Society

C.; Liras, S.; Mascitti, V.; Doudna, J. A. J. Am. Chem. Soc. 2018, 140, 6596–6603.
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Selective targeting of engineered T cells using orthogonal IL-2 cytokine-receptor complexes
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Fig. 4 orthoT cell ƕ orthoIL-2 ƘƥƨǝǢ
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orthoT cellƕŶǚưǀīÛƮǤǓǛǨĪK3A10 (MSA-3A10) ƬîżgűƬĬƗƑƏŷĖ»ƕƉƓŶMSA3A10ƚorthoT cellƕîƉƏ[KƘƙƠŶWTIL-2 + WT T cellǗƮLÇƙġõ<»ƬāƉƏ (Fig.4)ŷ
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ƬāƋìùƗ^òƚŝ÷ƈƩƗƀƑƏƁŶ

UƙZNƚŶIL-2ƙŘčƗ7îƬUœƉƒƒACTƕĔƠKƫƍƨƕżŽúÈƬŏƉƓĳƍƏŷCAR-TĒĝöÖ
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Antigen recognition-triggered drug delivery mediated by nanocapsule-functionalized cytotoxic T-cells
R. B. Jones et al., Biomaterials 117, 44-53 (2017)ų Ĩ6¢.ƙ­ĈŞ3ƬúƉŶÈùĔĘCTL

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Target2îƔƂƨáƔa^°±ƔŻƨŷħĜƦƚŎ
DƘŶğłǖǪǀƙǊǍĎcƬĒĝťhT

Fig.5 ų CTL ƘƥƨCĻĿƬǈǣƶǪƕƉƏĨ6¢.ñĒĝǬCTLǭĮŨƙĢǃǨǏƸłƕ+°ĖKƋƨƆƕƔŶCTLƘĨ6->ĎcƬōƈƍƓƂƏ (Nat.
Med 16, 1035-1041 (2010), Sci. Transl. Med 7, 291ra94 (2015))ŷƎƙŤŶCTLƙ-=ƤÈùĒĝƙÐ
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ǨǏƸł (CD45, LFA-1, Thy1) ƘůƈƩƨƆƕƣāƉƓżƨ (Biomaterials 33, 5776-5787 (2012))ŷƬŅƟžµľ¥ƔƚŶCĻĿƘŽǏǪǒƳǣǨƙ¢.ƬǈǣƶǪƕƉƓŶCTLĮŨƘ

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NC-CTLƬ+YŰƋƨƕŶNC-CTLƚAlexa647-OVAï¸ƙĩ'~}ƬÜrƈƍƨƕ+ƘŶCD107aƬĒĝĢƘĮ.ƈƍƏŷƆƙĖ»Ƙ;žŶǗǔǄǉŧǏǤǀCD4+TĒĝƕƙ+YŰƔƚLÇƙëŀƁĳƦƩƗƀƑƏƆƕŶǏǪǒƳǣǨdĒĝ%hĕńƙŠh6Ƭ;žƨƕŶĨ6¢.Ɓ3ƈƩƏƆƕƣKƫƍŶ
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(IL-15Sa; IL-15ƕIL-15RαƙıK)ƁƆƙ2·ƬÝƏƋƕāRƉƓżƨ (Nat. Med 16, 1035-1041 (2010),%HIV-Gag+

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