Monkey Gene

8/18/2019 Monkey Gene

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Generation of Gene-ModiedCynomolgus Monkey viaCas9/RNA-Mediated GeneTargeting in One-CellEmryos


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*!NTRO"#CT!ON

Monkeys have served as one of the most valuable models for

modeling human diseases and developing therapeutic strategies dueto their close similarities to humans in terms of genetic and

physiological features (Chan, 2013)


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Although several transgenic monkeyshave been successfully generated usingretroviral or lentiviral vectors precisegenomic targeting in monkeys is the mostdesired for generating human diseasemodels and has not been achieved so far(Chan, 2013; hen, 2013!"


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C!"#$! (clustered regularly interspaced short palindromic

repeats)% C!"#$!&associated (Cas) ' system confers targeted gene

editing by small !s that guide the Cas' nuclease to the target site

through base pairing (*inek et al, 2012)

+he C!"#$!%Cas' system has been demonstrated as an easy&

handle, highly specic, efcient, and multiple-able approach forengineering eukaryotic genomes (Mali et al,2013a)


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this system has been successfully used to targetgenomic loci in the mammalian cell lines and severalspecies, including mice and rats" #ut $hether it%sfeasible in primates is still unclear"

.e have e-tended the application of the C!"#$!%Cas' system tomultiple- genetic engineering in one cell&stage embryos of monkeys

and successfully obtained founder animals harboring t.o gene

modications


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*$ROCE"#RE


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• Animal

&ealthy femalecynomolgus monkeys('acaca fascicularis!

to )years

3,*2 to /,'0kg

$ere housed at the+unming #iomednternational (+#!


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• Emryo Colle%tion

11 female

cynomolgus monkeys

('acaca fascicularis

regular menstrual cycles

oocyte donors for superovulation

rh# (recombinant human follitropin alfa,

4&, Merck #erono) for 5 days

rhC (recombinant human chorionic gonadotropin

alfa, 6"7!84, Merck #erono) on day '

ocytes .ere collected by laparoscopic follicular

aspiration 3293/ hr after rhC administration

oocytes .ere used to perform intracytoplasmic sperm in:ection

("C#") and the fertili;ation .as confirmed by the presence of

t.o pronuclei


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• Cas9/sgRNA!n&e%tion ofOne-CellEmryos

+he ;ygotes .ere in:ected .ith a mi-ture of Cas' m! (20

ng%ml) and five sg!s (/ ng%ml each)

Microin:ections .ere performed in the cytoplasm of ;ygotes

the ;ygotes .ere cultured

protein&free hamster embryo culture medium&10 (8CM&10)containing 10< fetal calf serum (yclone 4aboratories,

#3005502) at 3= C in /< C2

+he cleaved embryos .ith high >uality at t.o&cell to

blastocyst stage .ere transferred into the oviduct of the

matched recipient monkeys

+.enty&nine monkeys .ere used as surrogate recipient, and

typically, three embryos .ere transferred into each female

+he earliest pregnancy diagnosis .as performed by

ultrasonography about 20930 days after the embryo transfer


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• "NA CON'TR#CT'Cas'&&4#&flag&linker (ddgene o ??=/5), .assynthesi;ed and inserted into p#+13=? vector

+he p@C/=&sg! e-pression vector used for in

vitro transcription of sg!s

p43&@A&sg!&$B&$uro vector, containing the

@A&$B&$uro fragment amplified from

p4B1(ddgene o 5?/3), sg! scaffold

amplified from p@C/=&sg!, and p43&asic

plasmid backbone ($romega 81=/1)

ligos for the generation of sg! e-pression plasmids

.ere annealed and cloned into the sa" sites of p@C/=&sg! or p43&@A&sg!&$B&$uro

v

v

.as deposited in ddgene


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*Cell Culture andEle%tro(oration

*C-./ cells (ACC, C.1*1! $ere cultured in'4'5high glucose (&yClone! $ith 106 7#, penicillinand streptomycin ;

*2 9 10* cells $ere electroporated $ith Cas: epression

plasmids and pA.?


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*!n )itro Trans%ri(tion

* he p13/@.Cas:.>.>.Bag.linker vector $aslinearied by Age1 enyme and transcribed in vitro"

*Cas:.>.>.Bag.linker m>A $as puriDed"

*sg>A oligos $ere annealed into p=C/.sg>A

epression vector $ith / promoter"

* hen epression vectors $ere linearied by ra andtranscribed in vitro" he sg>As $ere puriDed"


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*T*EN+ Cleavage Assay

and 'e,uen%ing

*"ierent sam(les .%ells (la%enta umili%al %ord andear (un%0 tissues1 %olle%ted and digested t0en t0egenomi% "NA 2as e3tra%ted and am(lied 4

*T*EN+ %leavage assay targeted fragments 2eream(lied from e3tra%ted "NA and (uried t0endenatured and reannealed in NE5uer 6 .NE51 using at0ermo%y%ler4

*To dete%t T*EN %leavage (rodu%ts of Nr0b1.lo%ali8ed on%0romosome 1 in %ultured emryos

* :; ng of $CR fragment from 2ild-ty(e %ontrol emryos <+:; ng of $CR fragments from emryos in&e%ted 2it0 Cas9mRNA and sgRNAs4

* $CR (rodu%ts 2it0 mutations dete%ted y T*EN+ %leavageassay 2ere su-%loned into T ve%tor4 =or ea%0 sam(le%olonies 2ere (i%ked u( randomly and se,uen%ed y M+>-

?* (rimer4

http://var/www/apps/conversion/tmp/scratch_6/20140527101154.pdfhttp://var/www/apps/conversion/tmp/scratch_6/20140527101154.pdf


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Target Assay

*All potential oE.target sites $ith homology to the 23 bpseFuence (sg>AG?A'! $ere retrieved by a base.by.base scanof the $hole rhesus genome, allo$ing for ungapped alignments$ith H @ mismatches in the sg>A target seFuence"

* potential oE.target sites $ith H 3 mismatches in the seedseFuence (1 to / base! $ere selected to ?C ampliDcation usingumbilical cord genomic >A as templates"

* he ?C products $ere Drst subIect to /4>1 cleavage assay" he potential oE.target sites yielding typical cleavage bands$ere considered as candidates, then the ?C products of the

candidates $ere cloned and seFuenced to conDrm the oE.targeteEects"


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!8#@4+# 7 7"#C@##"

Cas'%! 8ffectively Mediates ene 7isruptions in Monkey Cell 4ine

• +hree genes, namely Nr0b1 ( Nuclear Receptor Subfamily 0 Group B Member

1), Ppar-g ( Peroxisome Prolifera- tor-Activated Receptor Gamma), and Rag1( Recombiatio Acti- vatig Gee 1), .ere selected as the target genes

• +he efficiency of all sg!s .as first tested by cotransfection .ith Cas' into

the C#&= cell line derived from frican green monkey kidney

• +.o sg!s separated by 11= bp for Nr0b1, 2 sg!s separated by ?' bp for Ppar-g, and 1 sg! targeting Rag1 (igure 1), .ere designed as described

• +he cleavage bands .ere visible in all target genes urther characteri;ation of

the cleavage by se>uencing sho.ed, different inde- .ere detected at all five

target sites .ith various mutation si;es (D33A EF1 bp) at the efficiency of

222< for Nr0b1&sg!1, 20< for Nr0b1&sg!2, 10< for Ppar &g&

sg!1, 2/< for Ppar &g&sg!2, and 235< for Rag1&sg! (igure 1C)


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*Cas9/RNA !ndu%esE@%ient Genomi%

Targeting in MonkeyEmryos

* A total of 1 embryos $ith normal development to morulaor blastocyst stages $ere collected and eamined for thepresence of site.speciDc genome modiDcation analysis by

?C, /4>1 cleavage assay* argeted modiDcation $ith a range of sies (30 G* bp! in

monkey embryos occurred at all three target genes $itheJciency of @51 for >r0b1, /51 for ?par.g, and :51 forag1" ntriguingly, * of 1 embryos (embryos 2, , ), 10,11, and 13! harbored simultaneously mutations in both

?par.g and ag1; $hereas 2 of 1 embryos (embryos 3and @! harbored simultaneously mutations in both >r0b1and ag1y, and seFuencing as described above


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* Cas9/RNA Enales One-'te( Multi(le

GeneModi%ations in Monkeys

* he noninvasively available tissues of thet$o infant monkeys, including placenta,

umbilical cord, and ear punch tissues,$ere collected"

*Cleavage products $ere detected in bothinfants in ag1 and around the secondsg>A target site of ?par.g, indicating thepresence of multiple genomicmodiDcations in the founder monkeys"


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igure 3 sg!GCas'&Mediated Modifications of Ppar-g and Rag1 in ounder Cynomolgus Monkeys

$hotographs of 1?&day&old founder infants and

$C! products of the target region of Ppar-g and Rag1 in founders +argeted region of Ppar-g and Rag1 loci .ere

$C! amplified from the umbilical cord genomic 7 of and founders M, 7 markerH Con, control umbilial

cord from .ild&type cynomolgus monkey, .hich .as born ' days after birth of and

C 7etection of sg!GCas'&mediated on&target cleavage of Ppar-g and Rag1 by +=81 cleavage assay $C! products

from () .ere sub:ected to +=81 cleavage assay

7 #e>uences of modified Ppar-g and Rag1 loci detected in founders t least 15 + clones of the $C! products .ere

analy;ed by 7 se>uencing +he $M se>uences are underlined and highlighted in greenH the targeting se>uences in

redH the mutations in blue, lo.er caseH deletions (D), and insertions (F) % indicates positive colonies out of total

se>uenced #ee also igure #2 and #?


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igure ? sg!GCas'&Mediated Modifications of Nr0b1, Ppar-g, and Rag1 in 8ar and $lacenta

of ounders

()$C! products of the targeted region of Nr0b1, Ppar-g, and Rag1 in founders +arget

regions of Nr0b1, Ppar-g, and Rag1 loci .ere $C! amplified from the ear and placenta

genomic 7 of and founders M, 7 markerH Con, .ild&type control

()7etection of sg!1GCas'&mediated on&target cleavage of Nr0b1, Ppar-g, and Rag1

by +=81 cleavage assay

(C)7 se>uences of Nr0b1, Ppar-g, and Rag1 loci +he $C! products .ere analy;ed by

7 se>uencing +he $M se>uence are underlined and highlighted in greenH the

targeting se>uences in redH the mutations in blue, lo.er caseH deletions (D), and insertions

(F) % indicates positive colonies out of total se>uenced #ee also igure #3 and #?


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*MO'A!C!'M

* he C?5Ca:.mediated cleavage had

occured multiple times at diEerent stageof monkey embriogenesis, and resulted inmosaicism of the modiDcation


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*O==-TargetAnalysis

*a total of )@ potential oE.target sites(-!, including : for site 1 of Nr0b1, 20

for site 2 of Nr0b1, 1@ for site 1 of Ppar-γ ,20 for site 2 of Ppar-γ , and 21 for Rag1,respectively"

*Cas: m>A and sg>As demonstrate thatthe multiple genetic mutations can be

estabilshed at once $ithout detectableoE.target eEects


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* '#MMAR

ere, .e rst applied the C!"#$!%Cas' system, a versatile tool

for editing the genes of different organisms, to target monkey

genomes y coin:ection of Cas' m! and sg!s into one&cell&

stage embryos, .e successfully achieve precise gene targeting in

cynomolgus monkeys

Ie also sho. that this system enables simultaneous disruption oft.o target genes ( Ppar &γ and Rag1) in one step, and no offtarget

mutagenesis .as detected by comprehensive analysis +hus,

coin:ection of one&cell&stage embryos .ith Cas' m! and

sg!s is an efcient and reliable approach for gene&modied

cynomolgus monkey generation


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Monkey Gene

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