generation of gene-modified cynomolgus monkey via cas9

8/16/2019 Generation of Gene-Modified Cynomolgus Monkey via Cas9

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GENERATION OF GENE-MODIFIE

CYNOMOLGUS MONKEY VIACAS9/RNA-MEDIATED GENE

TARGETING IN ONE-CELL EMBRY


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INTRODUCTION


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• Monkeys ~ h!"n #ene$%& "n' (hys%o)o#%&") *e"$+es !o'e) *o'%se"ses "n' 'e,e)o(%n# $he+"(e$%& s$+"$e#%es

• The #ene$%& !o'%.&"$%on %s %n,")")e *o+ #ene+"$%on o* !onkey !A)$ho#h se,e+") $+"ns#en%& !onkeys h",e een s&&ess*))y #en

s%n# +e$+o,%+") o+ )en$%,%+") ,e&$o+s0 (+e&%se #eno!%& $"+#e$%n# %n !%s $he !os$ 'es%+e' *o+ #ene+"$%n# h!"n '%se"se !o'e)s "n' h"seen "&h%e,e' so *"+

• The +e&en$)y 'es&+%e' &)s$e+e' +e#)"+)y %n$e+s("&e' sho+$ (")%n+e(e"$s 1CRIS2R3/CRIS2R-"sso&%"$e' 1C"s3 9 sys$e! &on*e+s $"+#e$e'%$%n# y s!")) RNAs $h"$ #%'e $he C"s9 n&)e"se $o $he $"+#e$ s$h+o#h "se ("%+%n#

• The CRIS2R/C"s9 sys$e! h"s een 'e!ons$+"$e' "s "n e"sy-h"n'h%#h)y s(e&%.&0 e4&%en$0 "n' !)$%()e5")e "((+o"&h *o+ en#%nee+ek"+yo$%& #eno!es

• By no60 $h%s sys$e! h"s een s&&ess*))y se' $o $"+#e$ #eno!%&$he !"!!")%"n &e)) )%nes "n' se,e+") s(e&%es0 %n&)'%n# !%&e "n' 6he$he+ %$7s *e"s%)e %n (+%!"$es %s s$%)) n&)e"+


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• By $"k%n# $he "',"n$"#es o* CRIS2R/C"s90 #ene $"+#e$

!%&e "n' +"$s y &o%n8e&$%on o* one-&e))-s$"#e e!+yosC"s9 !RNA "n' s#RNAs s&&ess

• En&o+"#e' y o+ s&&esses %n CRIS2R/C"s9-!e'%"$e$"+#e$%n#0 "s 6e)) "s #ene !"n%()"$%on %n e"+)y-&)e","s$"#e e!+yos o* !onkeys0 he+e0 6e h",e e5$en'e' $"(()%&"$%on o* $he CRIS2R/C"s9 sys$e! $o !)$%()e5 #e

en#%nee+%n# %n one-&e))-s$"#e e!+yos o* !onkeys


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E2ERIMENTAL 2ROCEDURES


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ANIMALS

• :e")$hy *e!")e &yno!o)#s !onkeys 1Macaca fascicu+"n#%n# %n "#e *+o! ; $o < ye"+s "n' h",%n# o'y 6e%#=>? $o ;9@ k#0 6e+e se)e&$e' *o+ se %n $h%s s$'y

• A)) "n%!")s 6e+e hose' "$ $he Kn!%n# B%o!e' In$e+n1KBI3 A)) "n%!") (+o$o&o)s "+e "((+o,e' %n "',"n&e yIns$%$$%on") An%!") C"+e "n' Use Co!!%$$ee o* Kn!B%o!e' In$e+n"$%on")


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EMBRYO COLLECTION

• he")$hy *e!")e &yno!o)#s !onkeys "#e' ;< ye"+s 6%$h

!ens$+") &y&)es 6e+e se)e&$e' "s oo&y$e 'ono+s *o+ superov6h%&h 6e+e (e+*o+!e' y %n$+"!s&)"+ %n8e&$%on 6%$h +hFS:

1+e&o!%n"n$ h!"n *o))%$+o(%n ")*"0 GONAL-F0 Me+&k Se+ono3< '"ys0 $hen +hCG 1+e&o!%n"n$ h!"n &ho+%on%& #on"'o$+o(

OVIDREL0 Me+&k Se+ono3 on '"y 9

• Oo&y$es 6e+e &o))e&$e' y )"("+os&o(%& *o))%&)"+ "s(%+"$%on ="*$e+ +hCG "'!%n%s$+"$%on MII 1.+s$ (o)"+ o'y (+esen$3 oo&y$

se' $o (e+*o+! %n$+"&y$o()"s!%& s(e+! %n8e&$%on 1ICSI3 "n' $

*e+$%)%"$%on 6"s &on.+!e' y $he (+esen&e o* $6o (+on&)e%


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CAS9/SGRNA INECTION OF ONE-CELLEMBRYOS

• The y#o$es 6e+e %n8e&$e' 6%$h " !%5$+e o* C"s9 !RNA 1?@ n#/)3 "n' *%,e s#

1; n#/) e"&h3 M%&+o%n8e&$%ons 6e+e (e+*o+!e' %n $he &y$o()"s! o* y#o$es s%

!%&+o%n8e&$%on sys$e! n'e+ s$"n'"+' &on'%$%ons

• The y#o$es $hen 6e+e &)$+e' %n $he &he!%&"))y 'e.ne'0 (+o$e%n-*+ee h"!s$e

&)$+e !e'%!-@ 1:ECM-@3 &on$"%n%n# @ *e$") &")* se+! 1:y&)one L"o+

S:=@@


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DNA CONSTRUCTS

• Co'on o($%!%e' C"s9 e5(+ess%on &ons$+&$0 C"s9-N-N)%nke+ 1A''#ene No H;-2G*+"#!en$ "!()%.e' *+o! (LKO 1A''#ene No -s#RNA-2GK-2+o (GL=-U>-s2GK-2+o 6"s 'e(os%$e' %n A''#ene 1A''#ene NO ;

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CELL CULTURE AND ELECTRO2ORATIO

• COS-H &e))s 1ATCC0 CRL->;3 6e+e &)$+e' %n DMEM/h%#h

#)&ose 1:yC)one3 6%$h @ FBS0 (en%&%))%n "n' s$+e($o!y

• ? @> &e))s 6e+e e)e&$+o(o+"$e' 6%$h C"s9 e5(+ess%on (

"n' (GL=-U>-s#RNA-2GK-2+o 1P?3

• E!($y (GL=-U>-s#RNA-2GK-2+o ()"s!%' 6"s se' "s &on

• Ce))s 6e+e &o))e&$e' H? h+ (os$e)e&$+o(o+"$%on


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IN VITRO TRANSCRI2TION

• The (ST=H-C"s9-N-NLS-"#-)%nke+ ,e&$o+ 6"s )%ne"+%e' y A#e

"n' %n ,%$+o $+"ns&+%e' s%n# TH U)$+" K%$ C"s9-N-NLS-"#-)%nke+ !

(+%.e' y RNe"sy M%n% K%$

• s#RNA o)%#os 6e+e "nne")e' %n$o (UC;H-s#RNA e5(+ess%on ,e&$o+ 6

TH (+o!o$e+

• Then e5(+ess%on ,e&$o+s 6e+e )%ne"+%e' y D+" I "n' $+"ns&+%e' y

MEGAsho+$s&+%($ K%$ %n ,%$+o

• The s#RNAs 6e+e (+%.e' y MEGA&)e"+ K%$ "n' +e&o,e+e' y ")&oh

(+e&%(%$"$%on


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THEN CLEAVAGE ASSAY ANDSEQUENCING• Diferent samples, including cells, placenta, umbilical cord, and ear punch

were collected and digested in lysis buer (10 μM ris!"#l, 0$% M &a#l, ' μ

1* +D+, and 100 μgml -roteinase ./$ he genomic D&) was etracted an

amplied $

• 2&1 cleavage assay was perormed$ targeted ragments were amplied

etracted D&), and puried then denatured and reannealed in &3ufer '

using a thermocycler$ "ybridi4ed -#5 products were digested with 2&1

and separated by '$7* agarose gel$

• o detect 2& cleavage products o Nr0b1(locali4ed on chromosome 8/ in

embryos, 70 ng o -#5 ragment rom wild!type control embryos was mie

170 ng o -#5 ragments rom embryos in9ected with #as: m5&) and sg5&

• -#5 products with mutations detected by 2&1 cleavage assay were sub

into vector$ ;or each sample, colonies were pic


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OFF-TARGET ASSAY

•

A)) (o$en$%") o-$"+#e$ s%$es 6%$h ho!o)o#y $o $he ?= ( seJen&e 1s#RN6e+e +e$+%e,e' y " "se-y-"se s&"n o* $he 6ho)e +hess #eno!e0 "))

n#"((e' ")%#n!en$s 6%$h ( $o *o+ !%s!"$&hes %n $he s#RNA $"+#e$ s

• In $he o$($ o* $he s&"n0 (o$en$%") o-$"+#e$ s%$es 6%$h )ess $h"n $h+ee

!%s!"$&hes %n $he see' seJen&e 1 $o H "se3 6e+e se)e&$e' $o 2CR

"!()%.&"$%on s%n# !%)%&") &o+' #eno!%& DNA "s $e!()"$es

• The 2CR (+o'&$s 6e+e .+s$ s8e&$ $o THEN &)e","#e "ss"y The (o$e$"+#e$ s%$es y%e)'%n# $y(%&") &)e","#e "n's 6e+e &ons%'e+e' "s &"n'%'"

$he 2CR (+o'&$s o* $he &"n'%'"$es 6e+e &)one' "n' seJen&e' $o &on.

o-$"+#e$ ee&$s


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5+>?+ )&D D@+#>++@A&


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CAS9/RNA EFFECTIVELY MEDIATES GDISRU2TIONS IN MONKEY CELL LINE

• e se)e&$e' &yno!o)#s !onkey 1Macaca fascicularis3 "s $he !o'e) "n%!") e&"

s%e0 ","%)"%)%$y0 s%!%)"+ !ens$+") &y&)e $o h!"n0 "n' e4&%en$ +e(+o'&$%on "%)%

?@@


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#)+:5&) @&D>#+ ;;@#@& B&AM@)5B@&B @& MA&.C M35CA+

• A)$ho#h !%&+o%n8e&$%on o* WFN o+ TALEN !RNA %n$o e!+yo h"s een s&&ess*))y se' *o+ &+e

#ene $"+#e$ "n%!")s0 $ $hey h",e no$ een *e"s%)e %n !onkeys so *"+

• To $es$ 6he$he+ $he CRIS2R/C"s9 sys$e! 6o+ks %n !onkey e!+yos0 $he C"s9 1A''#ene No

s#RNAs 6e+e $+"ns&+%e' y TH RNA (o)y!e+"se %n ,%$+o

• T6en$y n"no#+"!/) C"s9 !RNA "n' ?; n"no#+"!/) o* !%5$+es &on$"%n%n# eJ") "!on$ o*

s#RNAs 6e+e (oo)e' "n' !%&+o%n8e&$e' %n$o ?? one-&e)) *e+$%)%e' e##s o* &yno!o)#s !onkeys

6e+e *+$he+ &)$+e' "$ =HC %n ; CO? A $o$") o* ; e!+yos 6%$h no+!") 'e,e)o(!en$ $o !

)"s$o&ys$ s$"#es 6e+e &o))e&$e' "n' e5"!%ne' *o+ $he (+esen&e o* s%$e-s(e&%.& #eno!e !o'%."n")ys%s y 2CR0 THEN &)e","#e "ss"y0 "n' seJen&%n# "s 'es&+%e' "o,e

• D%e+en$ s#RNAs *n&$%on y '%e+en$ e4&%en&y T"+#e$e' !o'%.&"$%on 6%$h " +"n#e o* s%es 1

%n !onkey e!+yos o&&++e' "$ ")) $h+ee $"+#e$ #enes 6%$h e4&%en&y o* /; *o+ Nr0b10 H/; *o

9/; *o+ Ra1 In$+%#%n#)y0 > o* ; e!+yos 1e!+yos ?0 ;0


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#)+:5&) &)3?+ A&!+- M>?@-? B& MAD@;@#)@A&+ @& MA

• %$h $hese s&&esses0 6e se$ o$ $o #ene+"$e #ene$%& !o'%.e' &yno!o)#s !onkeys A $o$") o* 9< MII oo

&o))e&$e' A*$e+ *e+$%)%"$%on y %n$+"&y$o()"s!%& s(e+! %n8e&$%on 1ICSI30 C"s9 !RNA "n' s#RNA !%5$+es o*

6e+e %n8e&$e' "s 'es&+%e' "o,e A $o$")


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• The (+esen&e o* #ene !o'%.&"$%on 6"s *+$he+ "n")ye' s%n# #en

*+o! e"+ (n&h $%sses "n' ()"&en$" The s"!e 2CR "n's0 &)e","

"n' !o'%.&"$%ons 6e+e 'e$e&$e' %n Ra1 "n' Ppar-γ #enes %n o$h

1F%#+e 30 *+$he+ 'e!ons$+"$%n# $he $"+#e$%n# s&&ess "n' &on.+!

CRIS2R/C"s9 %n'&es #)o") #eno!e !o'%.&"$%on %n !onkey e!+

%!(+ess%,e)y0 no 6%)'-$y(e Ra1 seJen&e 6"s 'e$e&$e' %n $he e"+

*on'e+ B 1F%#+e C30 'e!ons$+"$%n# $h"$ $he $"+#e$ !o'%.&"$%on

%J%$os)y "n' e4&%en$)y %n$e#+"$e' %n$o '%e+en$ $%sses0 !os$ )

%n&)'%n# $he #e+!)%ne

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• e ")so *+$he+ ss$"n$%"$e' $he "))e)%& $"+#e$%n# ee&$s y $"##%n# s%n#)e-n&)e

(o)y!o+(h%s!s 1SN2s3 o* ("+en$ !onkeys A =< k *+"#!en$ h"+o+%n# Ra1-s#R

s%$e 6"s 2CR "!()%.e' *+o! e"+ #eno!%& DNA o* $he ("+en$s "n' seJen&e' T6o

&o!%n"$%ons o* SN2s $"##%n# $he ("+en$s 'e+%,"$%on 6e+e 'e$e&$e' 'o6ns$+e"

$"+#e$ s%$e o* Ra1-s#RNA 1F%#+e S=A0 ables +1 "n' +73 The $"##%n# SN2 &o!

o* $he ("+en$s "n' $he *on'e+ $6%ns 6e+e *+$he+ 'e$e+!%ne' y TA &)on%n# "n'

1F%#+es S=B "n' S=C3 The +es)$s sho6e' $h"$ $6o $"##%n# SN2 &o!%n"$%ons se

"&&o+'"n&e 6%$h Men'e)7s )"6s The Ra1-s#RNA $"+#e$ s%$e %n $he e"+ o* *on'e+

h%#h $"+#e$ e4&%en&y 6"s *+$he+ seJen&e' The +es)$s 1F%#+e S=D3 sho6e' $h

"))e)es %'en$%.e' y $"##%n# SN2s h"+o+e' $"+#e$ !o'%.&"$%ons0 %n'%&"$%n# $6o "

o$h ("+en$s &o)' e !o'%.e' y C"s9/RNA-!e'%"$e' $"+#e$%n# %n !onkeys

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• S+(+%s%n#)y0 on)y one #eno$y(e 6%$h " s%n#)e-n&)eo$%'e %nse+$%on *o+

'%e+en$ $%sses o* o$h *on'e+ "n%!")s 6"s 'e$e&$e' 1F%#+es =D "n

e5&)'e $he (oss%%)%$y $h"$ $h%s s%n#)e-n&)eo$%'e %nse+$%on 6"s " SN2

$h"n " +e") !$"$%on0 $he $"+#e$ s%$es o* $he ("+en$s "n' s++o#"$e !o

6e+e "!()%.e' $o (e+*o+! THEN &)e","#e "ss"y "n' seJen&%n# 1F%#

The +es)$s e5&)'e' $he (+esen&e o* $he s"!e s%n#)e n&)eo$%'e0 &on

$h"$ $he %nse+$%on 6"s %n'ee' &"se' y CRIS2R/C"s9 !o'%.&"$%on $o

γ #ene T"ken $o#e$he+0 6e h",e s&&ess*))y "&h%e,e' C"s9/RNA-!e

s%$e-s(e&%.& !o'%.&"$%ons %n !onkey #eno!e y one-&e)) e!+yo

!%&+o%n8e&$%on

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MA+)@#@+M

• I$ %s 6o+$h no$%*y%n# $h"$ $he seJen&e '"$" o* o$h &)$+e' e!+yos "n' *o

"n%!")s sho6e' !)$%()e #eno$y(es 1F%#+es ?B0 =D0 "n' C30 s##es$%n# $he

CRIS2R/C"s9-!e'%"$e' &)e","#e h"' o&&++e' !)$%()e $%!es "$ '%e+en$ s$"

!onkey e!+yo#enes%s "n' +es)$e' %n !os"%&%s! o* $he !o'%.&"$%on0 "s h"

ose+,e' %n o$he+ s(e&%es 1Sn# e$ ")0 ?@=0 Tesson e$ ")0 ?@3 C++en$)y0

*on'e+ "%es "+e hose' %n 'e'%&"$e' *"&%)%$%es "n' 'e,e)o(%n# no+!"))y D

)%!%$e' "&&ess o* $%sses *+o! $he *on'e+ %n*"n$s0 !o+e $ho+o#h &h"+"&$e+%$he #eno!%& !o'%.&"$%ons "s 6e)) "s (heno$y(e +e!"%ns $o e (e+*o+!e' T

e "6"%$e' n$%) $he *on'e+ !onkeys h",e 'e,e)o(e' %n$o "')$hoo' In "''

!o+e *))-$e+! *on'e+s 6%)) e o+n "n' (+o,%'e !o+e s"!()es *o+ *+$he+ "s

o* CRIS2R/C"s9-!e'%"$e' #eno!e !o'%.&"$%on %n !onkeys

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A;;!)5B )&)?C+@+• The o-$"+#e$ ee&$ %s o* " !"8o+ &on&e+n *o+ $he CRIS2R/C"

sys$e! 1F e$ ")0 ?@=0 :s e$ ")0 ?@=0 2"$$"n"y"k e$ ")0 e ose+,e' CRIS2R/C"s9 %n'&e' he+%$")e o-$"+#e$ !$"

!%&e 1BS0 Wh"n#0 Wh"n#0 Who0 0 L Chen0 L "n

:o'#k%ns0 V Iye+0 :0 "n' C Sk"+nes0 n()%she' '"$"3

6he$he+ o $"+#e$ o&&++e' %n $hese #ene$%& !o'%.e' !onk

s&+eene' $he !onkey #eno!e "n' (+e'%&$e' " $o$") o*


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SGRNAPCAS9-MEDIATED CLEAVAGE OF NR0B1 IN FOUNDERS A AERE DETECTED BY 2CR AND THEN CLEAVAGE ASSAY USING UMCORD GENOMIC DNA AS TEM2LATE M0 DNA MARKER CON0 ILD-TCONTROL


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• The o-$"+#e$ ee&$s 6e+e &o!(+ehens%,e)y "ssesse'

$"+#e$ ee&$ "n")ys%s s%n# #eno!%& DNA *+o! !%)%&

The *+"#!en$s "+on' ")) $he (o$en$%") o-$"+#e$ )o&% 6

2CR "!()%.e'0 $hen s8e&$e' $o THEN &)e","#e "ss"Se,en$een 2CR (+o'&$s y%e)'e' &)e","#e "n's 6e+e

(+e&%se)y e5"!%ne' y TA seJen&%n# S+(+%s%n#)y0 ")

&)e","#e 6e+e &"se' y SN2 o+ +e(e"$ seJen&es0 "

"$hen$%& !$"$%on 6"s 'e$e&$e' 1able +63

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1A3 S&he!"$%& '%"#+"! o* )o&"$%on o* $he s%n#)e-n&)eo$%'e (o)y!o+(h%s! 1SN23 'o6ns$+e"! o* Ra1 s#R*on'e+ $6%ns7 ("+en$s The Ra1 "))e)e 6"s "!()%.e' y (+%!e+s =


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• These +es)$s 'e!ons$+"$e' $h"$ C"s9/RNA 'oes no$ %n

'e$e&$")e o-$"+#e$ !$"$%on %n o+ s$'y Cons%'e+%n

$he o-$"+#e$ ee&$ %s s%$e-'e(en'en$0 "n' !o+e s(e&%

s$+"$e#%es s%n# !$"$e' C"s9 h",e ")+e"'y een es$"

1R"n e$ ")0 ?@=30 $he o-$"+#e$ !$"#enes%s &"n e

!%n%!%e' y o($%!%%n# $he (+o&e'+e0 s##es$%n#

CRIS2R/C"s9 &o)' e " +e)%")e #eno!e $"+#e$ $oo) *o

!onkeys

generation of gene-modified cynomolgus monkey via cas9

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