7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 1/31

Leslie Tetteh Research Project and Report 13/04/15 2015

A Qualitative Risk

Assessent o! Possi"le

#colo$ical and

 Therapeutic %a&ards

arisin$ !ro the use o! 

'R()PR/'as*

1 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 2/31

Leslie Tetteh Research Project and Report 13/04/15 2015

Contents

2

A"stract

3%+pothesis

3

(ntroduction 3

,ethods 5

Results -

.iscussion

12

'onclusion

14

2 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 3/31

Leslie Tetteh Research Project and Report 13/04/15 2015

Re!erences

14

Appendi (

1

Appendi ((

22Appendi (((

2

Appendi (

2

Abstract:

A ualitative risk assessent as carried out on 'R()PR'as*6 (t as !ound that

there ere several ha&ards hich could result in an unintended $enetic seuenceor novel $enetic co"ination and that the pro"a"ilities o! these occurrin$ ere

ver+ hi$h6 (t as also !ound that several iproveents associated ith the 'as*

technolo$+ ere capa"le o! controllin$/iti$atin$ these risks7 "ut the+ had not

+et pro$ressed to the point here 'as* as suita"le !or in vivo therapeutic

applications or idescale release to the environent6 (ntentional ha&ards that

a+ arise !ro the use o! 'as* ere also "rie8+ suari&ed6

Aims: 

 The ai o! this paper is to evaluate the e9ect o! 'R()PR/'as* in ters o! the

ha&ards associated ith its use7 and the risks o! those ha&ards occurrin$6 This

ill "e esta"lished "+ a short revie o! the current and pendin$ applications o!

3 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 4/31

Leslie Tetteh Research Project and Report 13/04/15 2015

'R()PR/'as*7 and a ualitative risk assessent o! ho these could potentiall+

ipact huan/anial health7 and ho the use o! this technolo$+ could ipact

the environent6

Hypothesis:

As the 'R()PR/'as* cople is $enerall+ used !or $enoe editin$7 it is

h+pothesi&ed that a ke+ part in assessin$ ha&ards that arise !ro its use ill

involve accurate $enoe seuencin$7 and as such the risk ana$eent o!

'R()PR/'as* use ill rel+ to an etent on the accurac+ o! seuencin$ technolo$+6

(n ters o! risk assessent "ased on hat has alread+ "een seen so !ar7 it is

also h+pothesi&ed that a "i$ part in iti$atin$ the risks and avoidin$ the ha&ards

that arise !ro this !or o! $enoe editin$ ill arise in the !or o!

iproveents o! the technolo$+6

Introduction:

'R()PR'as* is a relativel+ ne "reakthrou$h in "iotechnolo$+ that has led to a

sall la"s revolution via the ease o! $enoic anipulation6 (nitiall+ discovered

"+ (shino et al6 in 1*7 clustered regularly interspaced palindromic repeats 

:'R()PRs;7 ere descri"ed as a set o! 2*"p repeats interspaced "+ 32"p

seuences :spacers; located in the $enoe o! Escherichia Coli. The !ail+ o! 'as

:CRISPR-associated; proteins< !ound in over 40= o! >acteria seuences and

around *0= o! Archaea7 as descri"ed soon a!ter :,ojica et al6 2000;6 Throu$h

stud+ o! their putative !unction as endonucleases it as discovered that the+

ere capa"le o! actin$ as an innate adaptive !or o! >acterial iunit+ :,ojica

et al67 2005< Pourcel et al67 2005;7 and in 200 ,arra?ni et al6 discovered that the32nt @spacers direct 'as nuclease activit+ a$ainst .BA< con?rin$ a h+pothesis

?rst put !orard "+ >olotin in 20056

(n 2012 a e"er o! the t+pe (('as !ail+7 'as* as !ound to !unction as a

sin$le endonuclease !or the editin$ o! .BA in cople ith to pieces o! RBA6

 Tered CRISPR-RNA/ crRBA and transactivatin$ crRBA/tracrRBA C these to can

"e co"ined !or a sin$le $uide RBA or s$RBA :Dinek et al6 2012;6 The

'as*/s$RBA cople as !ound to "e potentiall+ pro$raa"le< "+ chan$in$ a

short 2021 nt seuence at the 5 end o! the s$RBA7 it as possi"le to tar$et the

'as* cople to cleave a piece o! .BA in vitro via Eatson'rick "ase pairin$6 This occurred "eteen the 2021nt ‘spacer  seuence on the s$RBA and a

correspondin$ 2021"p stretch o! .BA7 a!ter reco$nition o! an initial PA,

seuence :protospacer adjacent oti!;6 (ts eistence as a sin$le non!usion

protein7 opened up the idea that it could potentiall+ "e used !or !uture $enoic

en$ineerin$ projects6

(n Danuar+ 2013 it as deonstrated "+ Fhan$ and collea$ues that a codon

optii&ed huani&ed version o! 'as* !ro Streptococcus Pyogenes as capa"le

o! cleavin$ $enoic .BA in vivo6 The+ also deonstrated that "+ aino acid

su"stitution in to nuclease doains o! 'as*7 .10A in Ruv' and %40A in %B%7

a catal+ticall+ dead !urther pro$raa"le version o! 'as* tered d'as* thatretained "indin$ speci?cit+ via s$RBA could also "e created :>ikard et al6 2013;6

4 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 5/31

Leslie Tetteh Research Project and Report 13/04/15 2015

(n the sae onth to ore papers deonstratin$ soon !olloed :Dinek et al6

20137 ,ali et6 al 2013; and the era o! 'as* $enetic en$ineerin$ as "orn6

(n contrast to earlier attepts at creatin$ pro$raa"le endonucleases7 the !act

that 'as* speci?cit+ as controlled "+ a nucleotide seuence as opposed to a

protein hich needed su"stantial reen$ineerin$ as in the case o! FGBs :FincGin$er Bucleases; and TAL#Bs :TAL #9ector Bucleases; ushered in an ease o!

$enetic pro$rain$ previousl+ unseen6 >ein$ "oth coste9ective and

rearka"l+ uick to construct7 the $eneration o! trans$enic anial odels usin$

'as* soon !olloed as earl+ as Ge"ruar+ 20137 and it as soon deonstratin$ its

utilit+ in a variet+ o! or$aniss such as ice7 &e"ra?sh and +east :)hen et al6

2013< 'han$ et al6 2013< .i'arlo et al6 201306

(n !our short onths 'as* had proven its utilit+ as a potential transcriptional

activator via the recruitent and crosslinkin$ o! transcriptional activation

doains to the !unctionall+ dead d'as* version o! the protein7 si!tl+ !olloed "+

a nel+ discovered a"ilit+ !or ultipleed activation :Pinera et al6 20137 'hen$ et

al 2013;6 'oupled ith its previousl+ deonstrated !unctionalit+ as a

transcriptional repressor :>ikard et al6 2013;7 'as* soon shoed that it held

potential !or a ran$e o! "iolo$ical !unctions past its initial classi?cation as a .BA

endonuclease6

 These novel uses include "ut are not liited to< use as a potential .BA "indin$

a$ent7 due to a hi$h level o! "indin$ ener$+ :reuirin$ , urea to dissociate it

!ro attached .BA;7 !or use in connectin$ strands in the construction o! .BA

ori$ai and ultiatel+ in the desi$n o! nanoolecular achiner+ :)tern"er$ et

al6 20147 Rotheund 200-;< use in recruitin$ and editin$ epi$enetic control

!actors :%su et al6 2014;< and use as an RBA nuclease usin$ scaRBA or PA,er

oli$onucleotides :)apson et al 20137 H 'onnell et al 2014;6 This ide ran$e o!

uses stes priaril+ !ro its pro$raa"le a"ilit+ to colocali&e to all three

seuence dependent "iopol+ers< proteins7 .BA and RBA7 and in this a+

essentiall+ direct their actions :,ali et al6 2013;6

As one o! the ajor potential uses !or the 'as* protein involves editin$ o! an

or$aniss $enoic data and/or $enoic epression7 it as decided in the

contet o! a risk assessent to !ocus on the possi"le ecolo$ical and therapeutic

ha&ards that i$ht arise !ro this use6 At the tie o! ritin$7 $iven that thetechnolo$+ is onl+ ver+ recent7 ver+ little data as availa"le on an+ practical

applications o! the technolo$+7 "e+ond the initial $eneration o! odel or$aniss

!or research6 .ue to this !act it as decided to ake to ake this a ualitative

rather than a uantitative risk assessent6

Ehat as hoever knon as that previous $enoe editin$ technolo$ies such

as &inc ?n$er nucleases su9er !ro o9tar$et e9ects due to speci?cit+ :Ia"riel

et al67 2011;7 and ith the advent o! this $enoe editin$ technolo$+7 an+

$enoe en$ineerin$ projects previousl+ postulated ere no via"le :,ali et al6

2013;6 Eith this in ind it as possi"le to proceed on a putative risk assessent

"ased on the data seen so !ar6

5 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 6/31

Leslie Tetteh Research Project and Report 13/04/15 2015

Methods

Selection Criteria

 The project "rie! $iven as the @identi?cation o! possi"le risks that i$ht arise

!ro s+nthetic en$ineerin$6 .ue in part to tie constraints and in part to theauthors !ailiarit+ ith the ediu it as decided to use the popular science

site httpJ//ph+s6or$ to $et an initial understandin$ o! the current technolo$+ in

this area6 Phys.org presents a data"ase o! short suar+ articles o! recent

scienti?c advances7 each "ased priaril+ upon a hi$hlevel journal article in a

?eld knon to the Phys.org researcher6 Ksin$ this site it as decided to !ocus on

the >iotechnolo$+ section as it ould contain articles relevant to + "rie!7 and to

read throu$h all articles !ro the past +ear6

A"out -0 or so articles ere read throu$h "e!ore decidin$ on 'as* as a su"ject

area :see Appendi (;6 This as due in part to its proinence as a $enoe

en$ineerin$ technolo$+ ithin the articles ( had vieed7 and the !act that ost o! 

the short revie articles on 'as* pu"lished on Phys.org ere "ased on peer

revieed research articles in Science and Nature7 "oth ver+ hi$hl+ re$arded and

reputa"le source journals6

(t as decided to read throu$h all relevant articles containin$ the search ter

@'as* on ph+s6or$ :around 5055 articles;7 and an+ other linked e"sites or

short revie articles !ound throu$h $oo$le search and then to read the relevant

peerrevieed research articles linked to each revie< these are all contained in

Appendi ((6 These articles ere accessed !or !ree via either the universit+s

li"rar+ service hich includes a su"scription to Nature7 Research$ate6co7 open

access or via short ter purchase :around 4; !ro Eile+s online Li"rar+6

H! the 5 research articles o"tained a!ter the initial ph+s6or$ search 34 total ere

o"tained !ro hi$hl+ re$arded peerrevieed journals Science and Nature7 24

ere !ro Nature and 10 ere !ro Science7 hich had "een noted durin$ the

initial search6 H! the rest o! the articles - ere o"tained !ro Cell another hi$hl+

re$arded journal7 - ere o"tained !ro ellre$arded journal Proceedings o! the

National Academy o! Sciences7 and 3 !ro the specialist journal Nucleic Acids

Research. 

 The reainin$ articles ostl+ cae !ro specialist journals ith one

contri"ution each !ro "ly# $enes and %evelopment# ACS Synthetic &iology#

 Applied and Environmental 'icro(iology# &io)Riv# $enetics# National Science

Revie* and E+i!e. These articles ere assessed on the erit o! their content7 and

one article Non-coding RNAs as potent tools !or crop improvement  !ro National

Science revie* as ecluded on this "asis7 leavin$ a total o! 5- articles6

Althou$h the "rie! as !or a risk assessent7 the authors prior knoled$e o! the

su"ject as noneistent< all o! the articles contained in!oration on 'as*< had

previousl+ "een peerrevieed "+ reputa"le journals< and a$ain "+ a+ o!

suarisation and inclusion on Ph+s6or$ so it as decided to read and akenotes on all o! the articles6

6 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 7/31

Leslie Tetteh Research Project and Report 13/04/15 2015

Gro this initial process another 12 articles ere !ound7 and !ro these another

- articles ere !ound deeed relevant to the process o! risk assessent :see

Appendi (((;6 (n contrast to the previous articles these articles also contained

in!oration on $uidelines !or research and "iosa!et+ easures hich could "e

taken hen dealin$ ith $eneticall+ odi?ed or$aniss6 Botes suari&in$ thepapers read aounted to around 557000 ords7 hich contained soe

in!oration unrelated to a risk assessent6 ia use o! the 'arta$ena Protocol

:contained in Appendi (((; it as decided to select !or in!oration that relate to

each o! the !olloin$ sections ‘ha,ards# ‘riss# and controls/mitigation# hich

reduced the nu"er o! notes to a ore speci?c 157000 ords relatin$ to a risk

assessent.

A ?nal search !or relevant risk assessents as undertaken and !urther peer

revieed articles detailin$ ualitative risk assessents not necessaril+ related to

'as* or "iolo$+ ere !ound6 Althou$h these ere not re!erenced directl+ in thepaper ( ill include these in Appendi (6

Ris Assessment 

#tensivel+ re!erred to throu$hout this risk assessent is the ‘"inal Report o! the

 Ad-hoc echnical $roup on Ris Assessment and Ris 'anagement under the

Cartagena Protocol on &iosa!ety hich ill "e re!erred to as the @'arta$ena

Protocol !or short :httpJ//"ch6c"d6int/protocol/ tet/;6 This is due to the use o!

'as* as a $enoe editin$ tool7 and the !act that the 'arta$ena Protocol deals

ith the production o! hat it ters @Livin$ odi?ed or$aniss6 The ?rst stepas to set the contet o! the risk assessent and identi!+ the eistin$ strate$ies

and policies that appl+ to the use o! 'as*6

.urin$ the course o! the investi$ation it as !ound that there ere ultiple

eistin$ strate$ies that appl+ to the use o! 'as*6 (n the contet o! therapeutic

use and ecolo$ical ipact it as !ound that the eistin$ ain uses o! 'as * so

!ar are< the $eneration o! trans$enic anial odels !or the stud+ o! $enetic

diseases such as the $eneration and !unctional cure o! a ouse odel !or

yrinosemia :Min et al6 2014;< in vitro/in vivo testin$ a$ainst "iolo$ical ori$in

patho$ens includin$ $eneration o! cellular iunit+ a$ainst %( and the reoval

o! anti"iotic resistance in ,R)A and Entero(acteriaceae in anials :#"ina et al6

2013< %u et al6 2014< >ikard et al6 2013< 'itorik et al6 2014;< the desi$n o!

$enoicall+ edited crops :Araki et al6 2015;< strate$ies !or use a$ainst pest

species and disease vectors such as $ene drives :.i'arlo et al 2015< Iant& and

>ier 2015;< hole $enoe $enetic !unction screens :Ean$ et al6 2014< )hale et

al 2013;< and use in en$ineerin$ industrial icro"ial or$aniss !or optiised

production :Fhan$ et al 2014;6

 These uses ere !ocused on in evaluatin$ potential ha&ards that a+ arise !ro

the use o! 'as*7 and in li$ht o! its potential therapeutic usa$e in huans this

as also evaluated as a source o! potential ha&ards6

7 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 8/31

Leslie Tetteh Research Project and Report 13/04/15 2015

esu!ts

(t as ?rst !ound that it as possi"le to o"tain 'as* under liited license !ro a

ide variet+ o! "iolo$ical retail e"sites such as 'lontech :6clontech6co;

and )+ste >iosciences :6s+ste"io6co;6 A saple user $uide !ro one o!

these e"sites7 as ell as providin$ re!erenced instructions on vector use7$enerall+ arns that 'as* is not licensed !or therapeutic/dia$nostic use or use in

huans7 that it is $ranted under a liited use license7 preventin$ odi?cation

!or resale7 and that an+ use o! the product is su"ject to the B(% $uidelines !or

reco"inant .BA and $enetic research :B(% 2013;6

 The B(% $uidelines $enerall+ reuire that an eperienter ?rst carries out an

initial risk assessent "ased on the risk $roup o! the "iolo$ical a$ent as

classi?ed "+ its potential ha&ard6 This is de?ned as Nthe potential e9ect o! a

"iolo$ical a$ent on a health+ huan adultO6 (t is !urther reuired that this risk

assessent is !urther developed upon accordin$ to the enhanced risk that a+

arise !ro the novel co"inations o! certain seuences in the or$anis or the

s+ner$istic e9ects o! trans$enes6 As such in this risk assessent consideration

ill "e on unintended potential ha&ards that a+ arise !ro novel co"inations

o! $enetic seuences or the s+ner$istic e9ect o! trans$enes6 Althou$h the B(%

$uidelines do not cover therapeutic use in huans7 this ill "e covered in the

risk assessent as one o! the ajor putative uses6

A ta"le ill "e constructed at the end o! the results section suarisin$ the risk

that could occur !ro unintended e9ects6 Gor the consideration o! initial risk e

?rst consider the pro"a"ilit+ o! a ha&ard occurrin$7 and then the likelihood o!

huan/environental eposure to that ha&ard6 Gor the pro"a"ilit+ o! a ha&ardoccurrin$ si cate$ories ill "e used< AC Almost Certain# 0P 0uite Possi(le#

1P 1nliely possi(ility# RP Rare possi(ility# C- Conceiva(le# PI Practically

impossi(le6 Gor huan/environental eposure to a ha&ard the sae si

cate$ories ill "e used6

Gro a co"ination o! these to !actors a ?ve cate$or+ s+ste !or the

pro"a"ilit+ o! a ha&ard occurrin$ ill "e used6 Risk classi?cations ill "e +o*#

'oderate# Su(stantial# 2igh and 3ery 2igh6

Gor the secondar+ consideration o! the conseuences o! a ha&ard a ?ve cate$or+

classi?cation s+ste ill also "e used6 .aa$e classi?cations ill "e 'inor#

'oderate# Su(stantial# Severe# and 3ery Severe

2a,ard 45 Chromosomal ranslocations

 The ?rst instance that ill "e considered is the possi"ilit+ o! unintended $enetic

reco"ination arisin$ !ro a 'as* $RBA cleavin$ its intended tar$et7 and

another .)> "ein$ $enerated elsehere in the $enoe6 'hroosoal

translocations occur via the nonhoolo$ous end joinin$ :B%#D; patha+ in cells7

hich is one o! to .)> repair echaniss located ithin cells7 the other "ein$

hoolo$+ directed repair :%.R; :Ihe&raoui7 %6 et al 2014< Tsai et al 2014;6 Thispatha+ is used in canonical $enoe editin$ to achieve ecient disruption o!

" | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 9/31

Leslie Tetteh Research Project and Report 13/04/15 2015

$enes via the $eneration o! indels6 (! an indel is not $enerated the cut site

reains unchan$ed7 and ith enou$h en&+e7 the site ill $enerall+ "e

repeatedl+ cut until an indel is produced6 %oever i! a .)> occurs elsehere in

the $enoe there is the possi"ilit+ o! a chroosoal translocation occurrin$

here"+ to separate .)>s are repaired to each other6

Ehilst in vitro this is ost likel+ to result in the reduced via"ilit+ o! the cell7 in

vivo this carries an increased risk o! carcino$enesis7 hich can "e har!ul to the

or$anis in uestion :,a et al6 2015;6 This also presents a potential risk to the

environent i! used in I, crops7 via the possi"le creation o! ne traits :Araki et

al6 2015;6 The likelihood o! this occurrin$ is dependent on the possi"ilit+ o! other

dou"lestranded "reaks occurrin$ ithin the $enoe7 hich7 past "asal level7 is

proportional to the nu"er o! o9tar$et .)> cuts that occur ithin the $enoe6

 This has "een shon to "e proportional to the aount o! epression o! the 'as*

cople :Pattana+ak et al6 2013;6 (n vivo hoever7 in order !or increased

ecienc+ in cuttin$ the tar$eted site7 relativel+ hi$h epression levels o! proteinare needed :%su et al 2013;6

2a,ard 65 78-target indels

Related to the $eneration o! chroosoal translocations is the $eneration o! o9

tar$et indels6 As has "een entioned these are also !ored via the B%#D

patha+6 (! an indel is not inserted into a site and it reains unchan$ed7

repeated cleava$e o! the site a+ take place until an indel is !ored6 This can

result in $ene disruptions7 or the disruption o! other !unctional noncodin$

$enoic seuences6 This is at a su"stantiall+ hi$her risk o! occurrin$ than

chroosoal translocations ith soe o9tar$et sites knon to !or indels athi$her rates than the tar$et site itsel!7 and at sites di9erin$ "+ as an+ as 5

nucleotides !ro the tar$et seuence :Tsai et al 2014;6 In vivo this could cause

serious har "+ eans o! $ene disruption6

B%#Ds have also "een shon to cause o9tar$et e9ects in I, crops hich

presents a potential risk to the environent6 (n $eneticall+ odi?ed rice7 o9

tar$et e9ects caused "+ indels ere seen ithin the Hs,P2 $ene :Araki et al6

2015;6 Potentiall+ this could lead to an increase in iuno$enicit+ and toicit+

ithin an or$anis7 hich could then cross"reed ith a ild variet+ o! the plant7

su"stantiall+ increasin$ the aount o! ecolo$ical har that could occur6

A possi"le iti$ation to the pro"le o! o9tar$et e9ects $eneratin$ 2a,ard 4 

and 2a,ard 6 has "een created in the !or o! dual 'as* nickases7 in hich one o! 

the nuclease doains Ruv' o! 'as* is deactivated via the .10A aino acid

su"stitution to !or a nickase6 To 'as* nickases can then "e used on dual

strands to $enerate a dou"le stranded "reak reuirin$ to $RBAs to ake a

.)>7 reducin$ o9 tar$et e9ects si$ni?cantl+ :,ali et al6 2013< Grock et al 2014;6

%oever thou$h the nickase ethod7 initiall+ seeed !ree o! indels7 since then it

has "een shon since still "een shon to $enerate si$ni?cant indels :Tsai et al

2014;6

# | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 10/31

Leslie Tetteh Research Project and Report 13/04/15 2015

 This ethod as re?ned !urther in 2014 via the production o! a dieric Gok(

'as* nuclease in hich dieri&ation o! an RGB :RBA$uided Gok( Buclease; as

needed to achieve a dou"lestranded cut :Tsai et al 2014:2;;6 This enhanced

speci?cit+ at a inor cost o! activit+6 Gor use in ver+ hi$hl+ speci?c therapeutic

applications the ethod could possi"l+ "e re?ned !urther to the point o!

ne$li$i"le o9tar$et cuts "+ adaptation o! the Finc Gin$er nickase techniuehere"+ an B450A utation in one Gok( doain $enerates a catal+ticall+

inactive doain to the point here dieri&ation ust occur to !or a nickase

:i et al 2012;6 Thus in co"ination ith 'as* !our $RBAs ould "e reuired to

produce a .)>6 Althou$h the Finc Gin$er nickase ethod su9ers !ro ecienc+

pro"les7 activit+ is knon to increase ith overepression6

2a,ard 95 78-target (inding

 The catal+ticall+ dead d'as* protein7 used currentl+ !or transcriptional activation

and repression is also knon to su9er !ro su"stantial o9tar$et e9ects7 ith a

'hroatin (unoprecipitation assa+ :'h(Pse;7 ?ndin$ that potential "indin$

took place at an+ ore sites than as !ound !or o9tar$et cleava$e :.oudna

and 'harpentier 2014< Tsai et al 2014;6 Althou$h it is possi"le that a lot o! this

"indin$ as erel+ transient7 it does till raise issues a"out possi"le o9tar$et

e9ects that ould a$ain constitute a therapeutic ha&ard and produce a level o!

risk hen considerin$ in vivo applications :Iil"ert et al6 2014;6 Kpre$ulation and

don re$ulation o! certain $enes7 ould not "e considered to have as "i$ a

potential o! har as translocations or deletions7 due in part to the ease o!

reversi"ilit+

 There is a relativel+ sall indo o! "indin$ opportunit+ !or the ro"usttranscriptional activation/repression o! a codin$ seuence< 50 to 300 "p

relative to the transcriptional start site !or $ene repression< 400 to 50 "p

upstrea !ro the T)) !or $ene activation :Iil"ert et al6 2014;6 This decreases

the possi"ilit+ o! risk $reatl+7 as there is a ver+ sall "indin$ site copared to

the potential B%#Dediated disruption o! the HRG o! a $ene hich can "e

several kilo"ases lon$ and a+ possess seuence hoolo$+ ith other $enes in

the $enoe6 Also the !act that several "indin$ odules $enerall+ usuall+ have to

"e used to achieve ro"ust e9ects7 decreases the risk o! unintended har :'hen$

et al6 2014;

Iiven the potential utilit+ o! this ethod in industrial "iotechnolo$+7 via the

"indin$ o! di9erent potential transcription/repression odules to the catal+ticall+

inactive d'as* protein7 it also constitutes a possi"le industrial ha&ard7 $iven the

possi"ilit+ o! an unknon product "ein$ !ored :onerann et al 2014;6 A$ain

hoever7 ver+ sall indos o! codin$ seuence eist !or e9ective activation

and repression o! $enes7 reducin$ the possi"ilit+ o! occurrence6 I, plant

"reedin$ and vector control $enerall+ ai at creatin$ lon$standin$ inherita"le

traits7 so there is less o! an ecolo$ical consideration !or o9tar$et e9ects o! this

technolo$+6

1$ | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 11/31

Leslie Tetteh Research Project and Report 13/04/15 2015

As previous use o! dieri&ation is not applica"le7 a possi"le iti$ation o! this risk

can "e !ound throu$h tru$RBA6 (t as h+pothesi&ed in 2014 "+ Doun$ and

collea$ues that the ecess anit+ o! crRBA !or the "indin$ re$ion o! .BA as a

preeptive response to potential alterations in viral $enoic seuence7 a+ "e

increasin$ the likelihood o! o9tar$et e9ects occurrin$6 (n testin$ this the+

discovered that use o! a 1nt truncated $RBA :tered tru$RBA; could reduceo9tar$et e9ects "+ ore than 5000!old7 and iprove speci?cit+ ith no overall

loss in tar$et ecienc+6

2a,ard :5 Ine;ciencies o! 2%R path*ay 

%oolo$+ .irected Repair or %.R is one o! 2 .BA repair echaniss contained

ithin the cell7 and !unctions in direct copetition ith B%#D6 Therapeutic

!unctions pertainin$ to the replaceent o! !ault+ $enes or the iplantation o!

ne trans$enes ithin the $enoe tend to ake use o! this !unction7 as ell as

crop $enoic en$ineerin$6 >asal !reuenc+ o! %.R is etreel+ lo7 around 1 in

10-C10* cells :'apecchi 1**;7 hoever this rate can "e iproved via the use o!

techniues such as 8ankin$ hoolo$+ ars :at least 1k" in len$th is desira"le;

:Iant& and >ier 2015;6 #ver+ reco"ination event carries risks in $eneratin$ an

unintended $enetic construct via translocation7 as ell as $eneratin$ an indel

that utates the tar$et site "e+ond reco$nition "+ the $RBA seuence

#speciall+ in the use o! $ene drives this presents a particular pro"le as the

desi$n o! the $ene drive reuires that it "e relia"l+ inserted into a speci?ed

seuence in order to "ias inheritance toards its production :#svelt et al 2014;6

(proper ipleentation o! a $ene drive s+ste a+ lead to undesira"le e9ects

and the increased risk o! ecolo$ical/environental har6 Gor instance a !ault+$ene drive7 a!ter the introduction o! a su"stantial I, osuito population7 could

potentiall+ drive a population eplosion7 ith no iti$ation o! the disease state6

(n order to co"at these e9ects it ould "e desira"le to don re$ulate

coponents o! the B%#j patha+ hilst upre$ulatin$ or e9ectivel+ recruitin$

coponents o! the %.R patha+6 This has "een success!ull+ achieved to an

etent in Aedes Aegypti here RBAi a$ainst B%#D coponent u<= resulted in

vastl+ iproved trans!oration eciencies :>asu et al6 2015;6 The e9ects o!

indel insertion could also "e counteracted "+ tar$etin$ an essential $ene ithin

the cell7 such that editin$ o! the tar$et site results in diinished cell ?tness:#svelt et al 2014;6 An unchan$ed seuence ould allo !or continued tar$etin$

o! the site :as lon$ as en&+e and $RBA are present; until eventual

incorporation o! the %.R seuence7 hich ould vastl+ iprove ecienc+6

(t is also possi"le to !oresee the use o! a d'as* odule7 attached to the 'as* in

order to e9ectivel+ locali&e the %.R seuence to the point o! interest6

2a,ard >5 Ine;ciencies in delivery 

(necienc+ in deliver+ can hold si$ni?cant risk o! har especiall+ i! considered

!or therapeutic applications such as the tar$etin$ o! virulence $enes or anti"ioticresistance $enes ithin "iolo$ical ori$in patho$ens6 The !ast $eneration tie o!

11 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 12/31

Leslie Tetteh Research Project and Report 13/04/15 2015

utations ithin these or$aniss eans that !or ever+ dela+ in e9ective

treatent7 or !or ever+ survivor !ro a round o! treatent there is an increased

risk o! the seuence utatin$ itsel! "e+ond reco$nition to the initial $RBA6 This

could ean a dela+ in havin$ to desi$n a ne $RBA to tar$et the ne seuence

hich could prove costl+6

Be vectors are "ein$ investi$ated !or use in the e9ective deliver+ o! 'as*6 (t

as !ound that a "acteriopha$e ('P1 as a"le to encode a !unctional 'R()PR'as

s+ste alon$ ith acuired spacers e9ectivel+ into its $enoe :)eed et al 2013;6

A "acteriopha$e ould "e a hi$hl+ e9ective choice due to speci?c tar$etin$ and

the a"ilit+ to replicate hundreds o! ties inside o! a "acterial cell7 as ell as

posin$ inial risk to eukar+otic cells :>ikard et al6 2014;

Gor use in aalian cell deliver+ AA vectors are $enerall+ pre!erred due to

reduced iuno$enicit+7 lo onco$enic potential and overall ecienc+ in

deliver+ :%su et al6 2014:2;;6 At the tie o! ritin$ a potential ne variant o!

'as* !ro Staphylococcus Aureus as !ound "+ Fhan$ and collea$ues :2015; to

edit $enoes ith siilar ecienc+ to )p'as*7 "ut to "e ore than 1k" in

len$th shorter alloin$ it to "e e9ectivel+ packa$ed into an AA vector7 hich

ould especiall+ help deliver+ to aalian cells6

An additional a+ o! iti$atin$ the risk o! the possi"le chan$e o! tar$et

seuence is "+ tar$etin$ ultiple seuences ithin the or$anis o! disease

ori$in6 This ould allo the continued tar$etin$ o! the species even i! a potential

tar$et seuence as utated :>ikard et al6 2014;6

2a,ard ?5 Interspecies and intraspecies trans!er 

A ?nal ha&ard hich could produce an unintended novel $enetic seuence !ro

use o! this technolo$+ is inter and intraspecie trans!er6 This has "een shon to

"e a possi"ilit+ "+ the eistence o! a 'R()PR'as s+ste capa"le o! acuirin$

ne spacer seuences and use in the ('P1 "acteriopha$e hich as ost

hoolo$ous to the t+pe 1G s+ste !ound in @ersinia Pestis7 and the discover+ o!

a ild canola $rain ith novel resistance to 2 di9erent her"icides o"tained "+

cross"reedin$ ith a I, $rain :)eed et al6 2013< ,annion and ,orse 2012;6 This

could result in a serious environental har7 as ell as therapeutic risks7 i! a

potential aalian/eukar+otic patho$en ere to acuire ne har!ul traits6

 This threat is iti$ated "+ the !act that 'as*7 as a sin$le protein !or the t+pe ((

'R()PR'as s+ste is una"le to acuire ne spacer seuences on its on and

reuires at least 2 other 'as proteins !or the acuisition o! ne spacers :%eler et

al6 2015;6 The threat o! viral or other unanted interspecies incorporation can

also "e iti$ated "+ separate packa$in$ o! the 'as* and $RBA to "e epressed

into di9erent cassettes such that incorporation o! one seuence ould not result

in a !unctional protein6

 The threat o! intraspecies trans!er or cross"reedin$ ill have to "e addressed "+

incorporatin$ $enoic editin$ "+ 'R()PR'as* under current I,H re$ulations7 asit appears that ithout necessaril+ introducin$ a trans$ene7 althou$h 'R()PR

12 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 13/31

Leslie Tetteh Research Project and Report 13/04/15 2015

'as* editin$ has "een knon to $enerate ne traits7 plants odi?ed "+ 'R()PR

'as* editin$ are not necessaril+ re$arded as Ieneticall+ ,odi?ed Hr$aniss or

I,Hs under current la :o+tas and Iao 2014;6 (t ould see that in order to

protect native and ild species a$ainst possi"le acuisition o! ne traits "+

cross"reedin$7 current las a+ need to "e epanded to incorporate or$aniss

o"tained via 'R()PR'as* $enoic en$ineerin$6

%ab!e 1: is& Assessment %ab!e 'or unintended ris&s arisin( 'rom Cas#

.iscussion

As can "e seen in ta"le 1 the pro"a"ilit+ o! a lot o! these events occurrin$ is

uite hi$h6 (ndels and o9tar$et "indin$ are "oth assued to "e alost certain6

 This is attested "+ the literature7 here s$RBAs t+picall+ have 262 other sites in a

cellular $enoe that sho per!ect coplientarit+ ith the initial 12"p NseedO

re$ion directl+ net to the PA,< an area hi$hl+ critical !or "indin$ :Ean$ et al

2014< Irat& et al 2014;6 Prior screens and assa+s such as the t endonuclease

13 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 14/31

Leslie Tetteh Research Project and Report 13/04/15 2015

assa+ and the )urve+or assa+ t+picall+ reveal ultiple o9tar$et cleava$e sites

!or an+ $iven tar$et site at hi$h :S2= ; rates o! induction :)ander and Doun$

2014< %su et al6 2013 :2;;6 #ven the use o! 'as* nickases and tru$RBAs does

not !ull+ eliinate o9tar$et "indin$ :Gu et al6 2014;6 The RBA$uided Gok(

Buclease and + theoretical RBA$uided Gok( nickase have "een descri"ed as

possi"le !urther enhanceents on speci?cit+7 hoever "ein$ relativel+ netechnolo$ies7 these ethods have not +et "een !ull+ characterised :Tsai et al

2014:2;;6

e+ to the developent o! enhanced speci?cit+ in tar$etin$ is the developent

o! an un"iased ethod o! detectin$ o9tar$et cleava$e sites !or speci?c RBAs6 (n

recent ties three ne technolo$ies have "een touted as potential $enoeide

detection ethods< IK(.#se7 .i$enoese and %TIT) all clai to provide

un"iased $enoescale ethods !or detectin$ .)>)s at eciencies onl+ sli$htl+

loer than the incorporation o! indels:Tsai et al 2014< i et al 2015< Grock et al

2014;6 The latest stud+ :.iIenoe)e; copared o9tar$et sites identi?ed "+each ethod and !ound that the+ had "road a$reeent on the eistence o! o9

tar$et sites6 Gro the data $athered "+ one project it as possi"le to create ne

rules !or predictin$ hi$hl+ speci?c RBAs7 and o9tar$et seuences6

 This provides a !urther la+er o! control and iti$ation7 here"+ seuences can

"e tested7 ith the !act that there are 3 separate ethods7 alloin$ !or a ore

coprehensive stud+7 and the possi"le ?netunin$ o! detection ethods in

!uture6

 The pro"a"ilit+ o! inecient activation o! the %.R patha+ as also !ound to "e

alost certain7 "ased on the in!oration contained in the literature6 (t as !oundthat the rate o! hoolo$+ driven reco"ination a!ter suppression o! a ke+

coponent o! the B%#D patha+ as around 23=7 hich althou$h lo is several

de$rees hi$her than the "asal rate o! 1 o! 10- C 10* !or a rando event7 shoin$

positive pro$ress :>asu et al6 2015;6 These ould alost certainl+ lead to

unintended $enetic e9ects such as indels and translocations7 and thus the risk

arisin$ !ro use o! this technolo$+ !or in vivo therapeutic or environental use

%.R as deeed to "e su"stantial6

.eliver+ ethods ould also have to "e su"stantiall+ iproved !or use in

therapeutic scenarios or ithin the environent6 A$ain it as !ound that thereas an alost certain pro"a"ilit+ o! unintended $enetic e9ects arisin$ !ro its

use i! used in a therapeutic contet or ithin the ider environent6 This arises

!ro inecient tar$etin$ to cells7 leavin$ open the possi"ilit+ o! utations6 (t

as !ound even ith in vitro tar$etin$ o! "acterial cells that the proportion o!

survivors as !ar hi$her than the epected value o! 165 10 * :#"ina et al6 2013;6

(t as !ound upon investi$ation that $enetic reco"ination as takin$ place

ithin the deliver+ vehicle7 hich !urther reduced the ecienc+ o! deliver+6 A$ain

this eant that the risk arisin$ !ro in vivo therapeutic use o! this technolo$+

as deeed to "e su"stantial6

All o! this is consistent ith the !act that 'R()PR'as* is a ver+ nel+ developedtechnolo$+ havin$ onl+ "een ?rst shon to "e capa"le o! in vivo editin$ in 20136

14 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 15/31

Leslie Tetteh Research Project and Report 13/04/15 2015

 The conseuence o! these !our events occurrin$ ran$ed !ro inor to oderate7

"ut the overall risk associated ith pro"a"ilit+ and eposure as $enerall+

deeed to "e su"stantial7 on the "asis o! its putative in vivo therapeutic and

ecolo$ical uses includin$ crop editin$ and $eneration o! trans$enic anials !or

drivin$ inherited traits into ild populations6 (n li$ht o! these risks it as deeed

that !urther iproveents have to "e ade on the 'R()PR'as technolo$+"e!ore it "ecoes accepta"le to use the 'R()PR technolo$+ !or in vivo

therapeutics and as !or use as an ecolo$ical a$ent6

Althou$h "ased on the literature availa"le the overall risk o! chroosoal

translocations occurrin$ as deeed to "e lo7 in ters o! its conseuences it

as deeed the ost severe7 "ecause o! the potential ha&ard it presents !or in

vivo use6 'hroosoal translocations are knon to "e o!ten c+totoic and

tuori$enic :Tsai et al6 2014;7 and hilst the ajorit+ o! indels that occur a+

not result in a deleterious e9ect on the cell "ein$ caused7 a chroosoal

translocation ill $enerall+ result in direct har and decreased via"ilit+ o! thecell7 i! not carcino$enesis and other a"noralities :Platt et al6 2014;6 Althou$h

the risk o! this occurrin$ is relativel+ lo the iproveent o! speci?cit+7 ould

reduce the overall risk cate$or+ o! this !actor6

 (nterspecies/(ntraspecies trans!er as de?ned as a oderate risk7 due to the

!act that it is knon to happen priaril+ "ased on interspecies trans!er "eteen

plants7 "ut also in li$ht o! the !act that viruses such as the %erpesvirus are

knon to incorporate $enoic seuences !ro huans7 and the ('P1

"acteriopha$e is knon to have incorporated an entire !unctional codin$

'R()PR/'as locus into its $enoe :,annion and ,orse 2012< %ol&erlandt 2002<

)eed et al6 2013;6 As has "een entioned risk is increased "+ a lack o!

re$ulations surroundin$ the su"ject6 .ue to the use o! rando uta$enesis in

conventional nonI, !arin$ techniues7 via eans such as ra+s the use o!

B%#D is not necessaril+ re$arded as I, :Araki et al6 2015;6 The lack o! e9ective

re$ulation on $enoic editin$ and the co"ined risk leads e to class the

potential har as oderate6

Intentional harm:

As ell as unintentional har arisin$ !ro potential ha&ards there is also the

possi"ilit+ o! intentional har arisin$ !ro the use o! 'R()PR'as* technolo$+6 This is ore dicult to accuratel+ classi!+ in ters o! pro"a"ilit+ o! occurrence

and likelihood o! huan/environental eposure7 "ut these ill "e discussed

"rie8+ in li$ht o! their potential conseuences6

 There are to ajor potential a+s in hich 'as* can "e used to cause

intentional har6 The ?rst is the direct deliver+ o! 'as* into a huan "ein$ in

order to disa"le $ene !unction7 and the second ould entail editin$ an or$anis

!or deliver+ o! a potential har!ul toin or patho$en6

Althou$h in the ?rst case7 serious har and possi"l+ death ould possi"l+ !ollo7

'as* could "e replaced in the euation "+ a hu$e variet+ o! cheical/"iolo$ical

15 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 16/31

Leslie Tetteh Research Project and Report 13/04/15 2015

ha&ards that ould have the sae e9ect7 and even the creation o! a potential

$ene drive ould onl+ pro$ress ver+ slol+6

(n ters o! editin$ the $enoe o! an anial or soe other "iolo$ical a$ent to

cause har7 this constitutes a ore serious ha&ard and conseuences could "e

severe6 A natural "arrier is that in $eneral an+ editin$ o! an or$aniss $enoeresults in the reduced ?tness o! that or$anis7 and the su"seuent utation o!

said edited $ene/$ene insert in order to restore the or$aniss ori$inal ?tness6

 This ould $enerall+ !unction in icro"ial populations6 An+ attept to alter the

$enoe o! a icro"e ould have to tar$et a seuence in a $ene responsi"le !or

?tness7 to prevent the icro"e utatin$ it "ack6

(n ters o! eukar+otic or$aniss hich reproduce "+ seual reproduction a $ene

drive can counteract this e9ect6 %oever the construction o! reversal $ene dries

hich can overrite the ?rst one can ver+ si!tl+ "e released7 in order to

iti$ate an+ risk that a+ arise !ro this6 iral $enoes cannot initiate

hoolo$+ driven repair7 so an+ editin$ o! a viral $enoe ould have to "e done

via the viral $enoe inserted into the $enoe o! another or$anis6

A sa!et+ easure a$ainst these possi"ilities is the a"ilit+ o! 'as* to overrite

an+ chan$es that have "een ade in a $enoe7 hoever in ters o! use a$ainst

a viral or icro"ial population7 inherent pro"les such as the deliver+ ethod

ould have to "e addressed6 Althou$h it is hard to $au$e the possi"ilit+ o! these

scenarios occurrin$7 the idespread availa"ilit+ o! 'as*7 increase the possi"ilit+

that these a+ occur6

'onclusion

(n conclusion it can "e seen that the su"stantial risks associated ith 'as* ean

it is currentl+ not suita"le !or in vivo therapeutic applications7 or lar$e scale

release into the natural environent6 %oever it is also shon that

iproveents in technolo$+ are helpin$ to iti$ate those risks7 an iprovin$

the sa!et+ o! the technolo$+ on a re$ular "asis6

e'erences

 Araki, Motoko, and Tetsuya Ishii. "Towards social acceptance of plant breeding by genome

editing." Trends in plant science 20, no. !20#$% &#'&(.

)assett, Andrew *., +harlotte Tibbit, +hris . onting, and -i'ong iu. "/ighly efficient targeted

mutagenesis of rosophila with the +*I1*+as( system."Cell reports &, no. !20$% 220'223.

)asu, 1an4ay, A5adeh Aryan, -ustin M. 67ercash, 8lady /a5itha 1amuel, Michelle A9 Anderson,

Timothy -. ahlem, :e7in M. Myles, and ;ach <. Adelman. "1ilencing of end'4oining repair for efficient

site'specific gene insertion after TA9<+*I1* mutagenesis in Aedes aegypti." Proceedings of the

National Academy of Sciences 2, no. !20#$% &03'&0&.

)ikard, a7id, +had =. 9uler, =enyan -iang, hilip M. <ussen5weig, 8regory =. 8oldberg, >a7ier

uportet, ?incent A. @ischetti, and uciano A. Marraffini. "9ploiting +*I1*'+as nucleases to

produce seBuence'specific antimicrobials." Nature biotechnology  2, no. !20&$% &C'#0.

16 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 17/31

Leslie Tetteh Research Project and Report 13/04/15 2015

)I61A@9TD, *6T6+6 6<. "@I<A *96*T 6@ T/9 A /6+ T9+/<I+A 9>9*T 8*6E

6< *I1: A11911M9<T A< *I1: MA<A89M9<T E<9* T/9 +A*TA89<A *6T6+6 6<

)I61A@9TD." !202$.

)olotin, Aleander, )enoit FuinBuis, Aleei 1orokin, and 1. usko 9hrlich. "+lustered regularly

interspaced short palindrome repeats !+*I1*s$ ha7e spacers of etrachromosomal

origin." Microbiology  #, no. 3 !200#$% 2##'2#C.

+apecchi, Mario *. "Altering the genome by homologous recombination."Science 2&&, no. &(0

!(3($% 233'2(2.

+hang, <annan, +hanghong 1un, u 8ao, an ;hu, >iufei >u, >iao4un ;hu, -ing'=ei >iong, and

-ian5hong -eff >i. "8enome editing with *<A'guided +as( nuclease in 5ebrafish embryos." Cell

research 2, no. & !20$% &C#'&G2.

+heng, Albert =., /aoyi =ang, /ui Dang, inyu 1hi, Darden :at5, Thorold =. Theunissen,

1udharshan *angara4an, +hikdu 1. 1hi7alila, aniel ). adon, and *udolf -aenisch. "Multipleed

acti7ation of endogenous genes by +*I1*'on, an *<A'guided transcriptional acti7ator system." Cell 

research 2, no. 0 !20$% C'G.

+itorik, *obert -., Mark Mimee, and Timothy :. u. "1eBuence'specific antimicrobials using efficiently

deli7ered *<A'guided nucleases." Nature biotechnology  2, no. !20&$% &'&#.

i+arlo, -ames 9., -ulie 9. <or7ille, rashant Mali, >a7ier *ios, -ohn Aach, and 8eorge M. +hurch.

"8enome engineering in 1accharomyces cere7isiae using +*I1*'+as systems." Nucleic acids

research !20$% gkt#.

i+arlo, -ames 9., Ale4andro +ha7e5, 17en . iet5, :e7in M. 9s7elt, and 8eorge M. +hurch. "*<A'

guided gene dri7es can efficiently bias inheritance in wild yeast." bioRxiv  !20#$% 03(C.

oudna, -ennifer A., and 9mmanuelle +harpentier. "The new frontier of genome engineering with

+*I1*'+as(." Science &C, no. C2 !20&$% 2#30(C.

9bina, /irotaka, <aoko Misawa, Duka :anemura, and Doshio :oyanagi. "/arnessing the

+*I1*+as( system to disrupt latent /I?' pro7irus."Scientific reports  !20$.

@rock, *ichard ., -ia5hi /u, *obin M. Meyers, Du'-ui /o, 9rina :ii, and @rederick =. Alt. "8enome'

wide detection of <A double'stranded breaks induced by engineered nucleases." Nature

biotechnology  !20&$.

@u, Danfang, -effry . 1ander, eepak *eyon, ?incent M. +ascio, and -. :eith -oung. "Impro7ing

+*I1*'+as nuclease specificity using truncated guide *<As." Nature biotechnology  2, no.

!20&$% 2G('23&.

8ant5, ?alentino M., and 9than )ier. "The mutagenic chain reaction% A method for con7erting

hetero5ygous to homo5ygous mutations." Science Express ( !20#$.8rat5, 1cott -., Aleander M.

+ummings, -ennifer <. <guyen, anielle +. /amm, aura :. onohue, Melissa M. /arrison, -ill

=ildonger, and :ate M. 6H+onnor'8iles. "8enome engineering of rosophila with the +*I1* *<A'

guided +as( nuclease." enetics (&, no. & !20$% 02('0#.

8he5raoui, /ind, Marion iganeau, )en4amin *enouf, -ean')aptiste *enaud, Annahita 1allmyr, )rian

*uis, 1ehyun 6h et al. "+hromosomal translocations in human cells are generated by canonical

nonhomologous end'4oining." Molecular cell  ##, no. C !20&$% 32('3&2.

17 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 18/31

Leslie Tetteh Research Project and Report 13/04/15 2015

8rat5, 1cott -., @iona . Ekken, +. ustin *ubinstein, 8ene Thiede, aura :. onohue, Aleander M.

+ummings, and :ate M. 6H+onnor'8iles. "/ighly specific and efficient +*I1*+as('cataly5ed

homology'directed repair in rosophila." enetics (C, no. & !20&$% (C'(G.

/ol5erlandt, *ia, +hristine 6rengo, aul :ellam, and M. Mar Alba. "Identification of new herpes7irus

gene homologs in the human genome."enome research 2, no. !2002$% G('G&3.

/su, atrick ., a7id A. 1cott, -oshua A. =einstein, @. Ann *an, 1il7ana :onermann, ?ineeta

 Agarwala, DinBing i et al. "<A targeting specificity of *<A'guided +as( nucleases." Nature

biotechnology  , no. ( !20$% 32G'32.

/su, atrick ., 9ric 1. ander, and @eng ;hang. "e7elopment and applications of +*I1*'+as( for 

genome engineering." Cell  #G, no. C !20&$% 2C2'2G3.

/u, =enhui, *afal :aminski, @an Dang, Donggang ;hang, aura +osentino, @ang i, )iao uo et al.

"*<A'directed gene editing specifically eradicates latent and pre7ents new /I?'

infection." Proceedings of the National Academy of Sciences , no. !20&$% &C'&CC.

Ishino, Doshi5umi, /ideo 1hinagawa, :o5o Makino, Mitsuko Amemura, and Atsuo <akata. "<ucleotideseBuence of the iap gene, responsible for alkaline phosphatase iso5yme con7ersion in 9scherichia

coli, and identification of the gene product." !ournal of bacteriology  C(, no. 2 !(3G$% #&2('#&.

-inek, Martin, :r5ys5tof +hylinski, Ines @onfara, Michael /auer, -ennifer A. oudna, and 9mmanuelle

+harpentier. "A programmable dual'*<Aguided <A endonuclease in adapti7e bacterial

immunity." Science G, no. C0(C !202$% 3C'32.

-inek, Martin, Aleandra 9ast, Aaron +heng, 1te7en in, 9nbo Ma, and -ennifer oudna. "*<A'

programmed genome editing in human cells." Elife 2 !20$% e00&G.

:im, 9un4i, 1o4ung :im, uk /young :im, )eom'1oon +hoi, Ik'Doung +hoi, and -in'1oo :im.

"recision genome engineering with programmable <A'nicking en5ymes." enome research 22, no.

G !202$% 2G'.

:onermann, 1il7ana, Mark . )righam, Aleandro 9. Tre7ino, -ulia -oung, 6mar 6. Abudayyeh, +lea

)arcena, atrick . /su et al. "8enome'scale transcriptional acti7ation by an engineered +*I1*'

+as( comple." Nature!20&$.

imitations, 8ene ri7e. "+oncerning *<A'8uided 8ene ri7es for the Alteration of =ild

opulations."

Ma, /anhui, Ardalan <aseri, ablo *eyes'8utierre5, 1cot A. =olfe, 1hao4ie ;hang, and Thoru

ederson. "Multicolor +*I1* labeling of chromosomal loci in human cells." Proceedings of the

National Academy of Sciences 2, no. 0 !20#$% 002'00G.

Mali, rashant, uhan Dang, :e7in M. 9s7elt, -ohn Aach, Marc 8uell, -ames 9. i+arlo, -ulie 9.

<or7ille, and 8eorge M. +hurch. "*<A'guided human genome engineering 7ia +as(." Science (,

no. C2 !20$% 32'32C.

Mali, rashant, -ohn Aach, . )en4amin 1tranges, :e7in M. 9s7elt, Mark Moosburner, 1riram :osuri,

uhan Dang, and 8eorge M. +hurch. "+A1( transcriptional acti7ators for target specificity screening

and paired nickases for cooperati7e genome engineering." Nature biotechnology  , no. ( !20$%

3'33.

Marraffini, uciano A., and 9rik -. 1ontheimer. "+*I1* interference limits hori5ontal gene transfer in

staphylococci by targeting <A." science 22, no. #(0( !2003$% 3&'3&#.

1" | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 19/31

Leslie Tetteh Research Project and Report 13/04/15 2015

Mo4ica, @rancisco -M, +esar Je5U?illaseKor, 9lena 1oria, and 8uadalupe -ue5. ")iological

significance of a family of regularly spaced repeats in the genomes of Archaea, )acteria and

mitochondria." Molecular microbiology  C, no. !2000$% 2&&'2&C.

Mo4ica, @rancisco -M, -esLs 8arcJa'MartJne5, and 9lena 1oria. "Inter7ening seBuences of regularly

spaced prokaryotic repeats deri7e from foreign genetic elements." !ournal of Molecular evolution C0,

no. 2 !200#$% G&'32.

6H+onnell, Mitchell *., )en4amin . 6akes, 1amuel /. 1ternberg, Aleandra 9ast'1eletsky, Matias

:aplan, and -ennifer A. oudna. "rogrammable *<A recognition and clea7age by

+*I1*+as(." Nature #C, no. G#0 !20&$% 2C'2CC.

ere5'inera, ablo, . ewran :ocak, +hristopher M. ?ockley, Andrew @. Adler, Ami M. :abadi,

auren *. olstein, ratiksha I. Thakore et al. "*<A'guided gene acti7ation by +*I1*'+as('based

transcription factors." Nature methods 0, no. 0 !20$% (G'(GC.

latt, *andall -., 1idi +hen, Dang ;hou, Michael -. Dim, ukas5 1wiech, /annah *. :empton, -ames

9. ahlman et al. "+*I1*'+as( knockin mice for genome editing and cancer modeling." Cell  #(,

no. 2 !20&$% &&0'&##.

ourcel, +., 8. 1al7ignol, and 8illes ?ergnaud. "+*I1* elements in Dersinia pestis acBuire new

repeats by preferential uptake of bacteriophage <A, and pro7ide additional tools for e7olutionary

studies." Microbiology  #, no. !200#$% C#'CC.

*an, @. Ann, e +ong, =inston >. Dan, a7id A. 1cott, -onathan 1. 8ootenberg, Andrea -. :ri5,

)ernd ;etsche et al. "In 7i7o genome editing using 1taphylococcus aureus +as(." Nature !20#$.

*othemund, aul =:. "@olding <A to create nanoscale shapes and patterns."Nature &&0, no. G032

!200C$% 2(G'02.

1ampson, Timothy *., 1unil . 1aro4, Anna +. lewellyn, Dih'ing T5eng, and a7id 1. =eiss. "A

+*I1*+as system mediates bacterial innate immune e7asion and 7irulence." Nature &(G, no. G&&3

!20$% 2#&'2#G.

1ander, -effry ., and -. :eith -oung. "+*I1*'+as systems for editing, regulating and targeting

genomes." Nature biotechnology  2, no. & !20&$% &G'##.

1hen, )in, -un ;hang, /ongya =u, -ianying =ang, :e Ma, ;heng i, >ueguang ;hang, umin

;hang, and >ingu /uang. "8eneration of gene'modified mice 7ia +as(*<A'mediated gene

targeting." Cell research 2, no. # !20$% G20'G2.

1ternberg, 1amuel /., 1y *edding, Martin -inek, 9ric +. 8reene, and -ennifer A. oudna. "<A

interrogation by the +*I1* *<A'guided endonuclease +as(." Nature #0G, no. G&(0 !20&$% C2'CG.

Tsai, 1hengdar F., ;ongli ;heng, <hu T. <guyen, Matthew iebers, ?ed ?. Topkar, ?ishal Thapar,

<icolas =y7ekens et al. "8EI9'seB enables genome'wide profiling of off'target clea7age by

+*I1*'+as nucleases." Nature biotechnology  !20&$.

E1 <ational Institutes of /ealth. "<I/ guidelines for research in7ol7ing recombinant or synthetic

nucleic acid molecules. 20."

?oytas, aniel @., and +aiia 8ao. "recision genome engineering and agriculture% opportunities and

regulatory challenges." P"oS biology  2, no. C !20&$% e003GG.

=ang, Tim, -enny -. =ei, a7id M. 1abatini, and 9ric 1. ander. "8enetic screens in human cellsusing the +*I1*'+as( system." Science &, no. CCC !20&$% 30'3&.

1# | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 20/31

Leslie Tetteh Research Project and Report 13/04/15 2015

Din, /ao, =en >ue, 1idi +hen, *oman . )ogorad, 9ric )enedetti, Markus 8rompe, ?ictor

:oteliansky, hillip A. 1harp, Tyler -acks, and aniel 8. Anderson. "8enome editing with +as( in

adult mice corrects a disease mutation and phenotype." Nature biotechnology  !20&$.

;hang, 8uo'+hang, In Iok :ong, /ee4in :im, -ing'-ing iu, -amie / +ate, and Dong'1u -in.

"+onstruction of a Fuadruple Auotrophic Mutant of an Industrial olyploid 1accharomyces cere7isiae

1train by Esing *<A'8uided +as( <uclease." Applied and environmental microbiology  30, no. 2&

!20&$% GC(&'GG0.

Appendix I

Gi$htin$ the 'olorado potato "eetle ith RBA inter!erence

httpJ//ph+s6or$/nes/201502coloradopotato"eetlerna6htl

Gull crop protection !ro an insect pest "+ epression o! lon$ dou"lestranded

RBAs in plastids

Re$ulatin$ $enoeedited crops that arenVt I,Hs httpJ//ph+s6or$/nes/2015

02$enoeeditedcrops$os6htl

 Toards social acceptance o! plant "reedin$ "+ $enoe editin$

httpJ//6sciencedirect6co/science/article/pii/)13-0135150002*1

>etter $enes !or "etter "eans httpJ//ph+s6or$/nes/201502$enes"eans6htl

Iene a+ help reduce I, containation httpJ//ph+s6or$/nes/201502$ene

$containation6htl

A sta"le DAF protein !ro peach ediates the transition !ro outcrossin$ to sel!

pollination httpJ//6"ioedcentral6co/content/pd!/s12*150150124-6pd! 

(nvasive eedVs resistance to ellknon her"icide stes !ro increase in $ene

copies httpJ//ph+s6or$/nes/201502invasiveeedresistanceellknon

her"icide6htl

 Tande Apli?cation o! a 'hroosoal )e$ent %ar"orin$ 5

#nolp+ruv+lshikiate3Phosphate )+nthase Locus 'on!ers Il+phosateResistance in ochia scoparia

httpJ//6plantph+siol6or$/content/1--/3/12006!ull6pd! 

)+nthetic "iolo$+ +ields ne approach to $ene therap+

httpJ//ph+s6or$/nes/201502s+nthetic"iolo$++ieldsapproach$ene6htl

'R()PR"ased sel!cleavin$ echanis !or controlla"le $ene deliver+ in huan

cells httpJ//nar6o!ordjournals6or$/content/43/2/12*6!ull

Apples odi?ed to resist "ronin$ receive !ederal approval

httpJ//ph+s6or$/nes/201502applesresist!ederal6htl

)tructure"ased desi$n used as tool !or en$ineerin$ deiuni&ed

2$ | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 21/31

Leslie Tetteh Research Project and Report 13/04/15 2015

"iotherapeutics httpJ//ph+s6or$/nes/201502structure"asedtool

deiuni&ed"iotherapeutics6htl

Protein deiuni&ation via structure"ased desi$n ena"les ecient epitope

deletion at hi$h utational loads

httpJ//onlineli"rar+6ile+6co/doi/1061002/"it625554/epd! 

Ioo$lest+le rankin$ used to descri"e $ene connectivit+

httpJ//ph+s6or$/nes/201502$oo$lest+le$ene6htl

,icroarra+ enriched $ene rank httpJ//6"iodatainin$6or$/content//1/2

)cientists develop hi$her +ieldin$ sor$hu plants httpJ//ph+s6or$/nes/2015

02scientistshi$her+ieldin$sor$hu6htl

Researchers repro$ra plants !or drou$ht tolerance httpJ//ph+s6or$/nes/2015

02repro$radrou$httolerance6htl

A$rocheical control o! plant ater use usin$ en$ineered a"scisic acid receptors

'oplete $enoes !ro sin$le cells still elusive httpJ//ph+s6or$/nes/201502

$enoescellselusive6htl

Reconstructin$ each cellVs $enoe ithin cople icro"ial counities C

drea or realit+WX

httpJ//journal6!rontiersin6or$/article/10633*/!ic"620146001/!ull

'ulture shockJ Are la"$ron cells a !aith!ul odel !or huan diseaseW

httpJ//ph+s6or$/nes/201502culturela"$roncells!aith!ulhuan6htl

Rapid repro$rain$ o! epi$enetic and transcriptional pro?les in aalian

culture s+stes httpJ//$enoe"iolo$+6co/2015/1-/1/11

Iettin$ +east to pup up the protein production httpJ//ph+s6or$/nes/201502

+eastproteinproduction6htl

>lockin$ endoc+totic echaniss to iprove heterolo$ous protein titers in

)accharo+ces cerevisiae

>ioen$ineers develop tool !or repro$rain$ $enetic code

httpJ//ph+s6or$/nes/201501"ioen$ineerstoolrepro$rain$$enetic

code6htl

#n$ineerin$ 'ople )+nthetic Transcriptional Pro$ras ith 'R()PR RBA

)ca9olds)+nthetic Transcriptional 'ontrol #leents and ,ethods o! Ieneratin$ and Ksin$

)uch #leents (nventorsJ 'han$ Liu7 Ada P Arkin7 Lei ) Qi

Ienetic sa!et+ sitches could help cur" potential "ioterror risks

httpJ//ph+s6or$/nes/201501$eneticsa!et+cur"potential"ioterror6htl

(ntrinsic "iocontainentJ ,ultiple $enoe sa!e$uards co"ine transcriptional

and reco"inational control o! essential +east $enes

httpJ//6pnas6or$/content/112/-/1036!ull

,illions o! I,H insects could "e set loose in Glorida e+s

httpJ//ph+s6or$/nes/201501illions$oinsectsloose8orida6htl

21 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 22/31

Leslie Tetteh Research Project and Report 13/04/15 2015

Be coputation ethod helps identi!+ !unctional .BA

httpJ//ph+s6or$/nes/201501ethod!unctionaldna6htl

A ethod !or calculatin$ pro"a"ilities o! ?tness conseuences !or point

utations across the huan $enoe

,appin$ the ai&e $enoe httpJ//ph+s6or$/nes/201501ai&e$enoe6htl

Positional clonin$ in ai&e :Fea a+s su"sp6 a+s7 Poaceae;

httpJ//6"ioone6or$/doi/!ull/106332/apps614000*2

 Trans$enic cropsJ ,ultiple toins not a panacea !or pest control

httpJ//ph+s6or$/nes/201501trans$eniccropsultipletoinspanacea6htl

Hptii&in$ p+raided trans$enic >t crops !or sustaina"le pest ana$eent

V,olecular scissorsV could prevent $enetic diseases "e!ore conception

httpJ//ph+s6or$/nes/201501olecularscissors$eneticdiseases

conception6htlAs+etric parental $enoe en$ineerin$ "+ 'as* durin$ ouse eiotic eit

httpJ//6nature6co/srep/2014/141223/srep0-21/!ull/srep0-216htl

 Toard itochondrial plant cell !actories httpJ//ph+s6or$/nes/201501

itochondrialcell!actories6htl

Iene introduction into the itochondria o! Ara"idopsis thaliana via peptide

"ased carriers

httpJ//6nature6co/srep/2015/150113/srep051/!ull/srep0516htl

Girst contractin$ huan uscle $ron in la"orator+ httpJ//ph+s6or$/nes/2015

01huanuscle$ronla"orator+6htl

>ioen$ineered huan +o"undles iic clinical responses o! skeletal uscle to

dru$s httpJ//eli!esciences6or$/content/4/e045

I,Hs ith health "ene?ts have a lar$e arket potential

httpJ//ph+s6or$/nes/201501$oshealth"ene?tslar$epotential6htl

)tatus and arket potential o! trans$enic "io!orti?ed crops

#K laakers pass controversial I,H !ood la httpJ//ph+s6or$/nes/201501

euparliaent$crops6htl

%+"rid Vsuper osuitoV resistant to insecticidetreated "ed nets

httpJ//ph+s6or$/nes/201501 h+"ridsuperosuitoresistantinsecticide

treated6htl

Adaptive intro$ression in an A!rican alaria osuito coincident ith the

increased usa$e o! insecticidetreated "ed nets

httpJ//6pnas6or$/content/112/3/156!ull

Hne pipeline that co"ines an+ $ene?ndin$ tools httpJ//ph+s6or$/nes/2015

01pipelineco"ines$ene?ndin$tools6htl

Iene prediction and annotation in Pensteon :Planta$inaceae;J A ork8o !or

22 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 23/31

Leslie Tetteh Research Project and Report 13/04/15 2015

arker developent !ro etreel+ locovera$e $enoe seuencin$6

httpJ//6"ioone6or$/doi/!ull/106332/apps61400044

Patho$en strains copetin$ !or the sae host plant chan$e disease d+naics

httpJ//ph+s6or$/nes/201501patho$enstrainshostdiseased+naics6htl

'oin!ection alters population d+naics o! in!ectious disease

:,eta"olic en$ineerin$ suar+ paper;

Addition o! a heat $ene increases the "li$ht resistance o! Aerican chestnut

trees httpJ//ph+s6or$/nes/201412additionheat$ene"li$htresistance6htl

 Trans$enic Aerican chestnuts sho enhanced "li$ht resistance and transit

the trait to T1 pro$en+

httpJ//6sciencedirect6co/science/article/pii/)01-*45214000*

Quest to unravel +steries o! our $ene netork httpJ//ph+s6or$/nes/201412

uestunravel+steries$enenetork6htl#K court clears ste cell patentin$ httpJ//ph+s6or$/nes/201412eucourt

stecellpatentin$6htl

Be ethod identi?es $enoeide o9tar$et cleava$e sites o! 'R()PR'as

nucleases httpJ//ph+s6or$/nes/201412ethod$enoeideo9tar$et

cleava$esites6htl

IK(.#se ena"les $enoeide pro?lin$ o! o9tar$et cleava$e "+ 'R()PR'as

nucleases httpJ//6nature6co/n"t/journal/v33/n2/!ull/n"t63116htl

:product e"site; httpJ//6$enoa+6co/services/crisprcas*odels/crispr

cas*o9er6htWutYsourceZ$oo$le[utYediuZcpc[utYcapai$nZdispla+

selecti! 

Ancient isdo "oosts sustaina"ilit+ o! "iotech cotton

httpJ//ph+s6or$/nes/201412ancientisdo"oostssustaina"ilit+"iotech6htl

Lar$escale test o! the natural re!u$e strate$+ !or dela+in$ insect resistance to

trans$enic >t crops

Research reveals structure o! ke+ 'R()PR cople httpJ//ph+s6or$/nes/2014

12revealske+crisprcople6htl

Ienoescale transcriptional activation "+ an en$ineered 'R()PR'as* cople

Fhan$ la" :,(T;J httpJ//&la"6it6edu/pu"lications6htl

Re$ions in the $enoe cause in!ertilit+ in soe h+"rids o! house ouse

su"species httpJ//ph+s6or$/nes/201412re$ions$enoein!ertilit+h+"rids

house6htl

IenoeEide ,appin$ (n A %ouse ,ouse %+"rid Fone Reveals %+"rid )terilit+

Loci And .o"&hansk+,uller (nteractions6

Be research could revolutioni&e $enoic seuencin$ o! dru$resistant "acteria

httpJ//ph+s6or$/nes/201412revolutioni&e$enoicseuencin$dru$resistant

"acteria6htl

,in(HB nanopore seuencin$ identi?es the position and structure o! a "acterialanti"iotic resistance island

23 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 24/31

Leslie Tetteh Research Project and Report 13/04/15 2015

Iene critical to the developent o! lo arsenic plants identi?ed "+ scientists

httpJ//ph+s6or$/nes/201412$enecriticalarsenicscientists6htl

Ienoeide Association ,appin$ (denti?es a Be Arsenate Reductase #n&+e

'ritical !or Liitin$ Arsenic Accuulation in Plants

httpJ//journals6plos6or$/plos"iolo$+/articleWidZ106131/journal6p"io6100200*

.ru$s in the environent a9ect plant $roth httpJ//ph+s6or$/nes/201412

dru$senvironenta9ect$roth6htl

#valuation o! "iolo$ical endpoints in crop plants a!ter eposure to nonsteroidal

antiin8aator+ dru$s :B)A(.s;J (plications !or ph+totoicolo$ical assessent

o! novel containants

)cientists uncover !ourstranded eleents o! ai&e .BA

httpJ//ph+s6or$/nes/201412scientistsuncover!ourstrandedeleents

ai&e6htlIQuadruple :I4; ,oti!s in the ,ai&e :Fea a+s L6; Ienoe Are

#nriched at )peci?c Locations in Thousands o! Ienes 'oupled to #ner$+ )tatus7

%+poia7 Lo )u$ar7 and Butrient .eprivation

httpJ//6sciencedirect6co/science/article/pii/)1-35214001--

Relia"le RBA anal+sis no easier ith B()T Vdash"oardV tool

httpJ//ph+s6or$/nes/201412relia"lernaanal+siseasiernist6htAssessin$

 Technical Per!orance in .i9erential Iene #pression #perients ith #ternal

)pikein RBA 'ontrol Ratio ,itures6

'oputer odel ena"les desi$n o! cople .BA shapes

httpJ//ph+s6or$/nes/201412ena"lescopledna6htl

Lattice!ree prediction o! threediensional structure o! pro$raed .BAasse"lies

httpJ//6nature6co/ncos/2014/141203/ncos-5/!ull/ncos-56ht

l

Butrition7 sa!et+ ke+ to consuer acceptance o! nanotech7 $enetic odi?cation

in !oods httpJ//ph+s6or$/nes/201412nutritionsa!et+ke+consuer

nanotech6htl

%etero$eneous 'onsuer Pre!erences !or Banotechnolo$+ and Ienetic

odi?cation Technolo$+ in Good Products

Appendi) II

Pro$raa"le .BA scissors !ound !or "acterial iune s+ste

httpJ//ph+s6or$/nes/20120-pro$raa"lednascissors"acterialiune6htl

A Pro$raa"le .ualRBACIuided .BA #ndonuclease in Adaptive >acterial

(unit+

Eho Hns the >i$$est >iotech .iscover+ o! the 'entur+W

httpJ//6technolo$+revie6co/!eaturedstor+/532*-/hoonsthe"i$$est

"iotechdiscover+o!thecentur+/

24 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 25/31

Leslie Tetteh Research Project and Report 13/04/15 2015

#ditin$ the $enoeJ Be ethod allos scientists to insert ultiple $enes in

speci?c locations7 delete de!ective $enes httpJ//ph+s6or$/nes/201301

$enoeethodscientistsinsertultiple6htl

Pro$raa"le repression and activation o! "acterial $ene epression usin$ an

en$ineered 'R()PR'as s+ste

httpJ//&la"6it6edu/assets/reprints/>ikardY.YBucleicYAcidsYResY20136pd! 

'heap and eas+ techniue to snip .BA could revolutioni&e $ene therap+

httpJ//ph+s6or$/nes/201301cheapeas+techniuesnipdna6htl

RBAIuided %uan Ienoe #n$ineerin$ via 'as*

,ultiple Ienoe #n$ineerin$ Ksin$ 'R()PR/'as )+stes

RBApro$raed $enoe editin$ in huan cells

httpJ//rna6"erkele+6edu/?les/jinekYetalYeli!eY20136pd! 

.isease knoled$e a+ advance !aster ith 'R()PR $ene pro"in$ tool

httpJ//ph+s6or$/nes/201303diseaseknoled$eadvance!astercrispr6htl

Repurposin$ 'R()PR as an RBAIuided Plat!or !or )euence)peci?c 'ontrol o!

Iene #pression

httpJ//6sciencedirect6co/science/article/pii/)00*2-413002110

>acterial securit+ a$ents $o ro$ue httpJ//ph+s6or$/nes/201304"acterial

a$entsro$ue6htl

A 'R()PR/'as s+ste ediates "acterial innate iune evasion and virulence

Poer!ul $eneeditin$ tool appears to cause o9tar$et utations in huan cells

httpJ//ph+s6or$/nes/20130-poer!ul$eneeditin$toolo9tar$et

utations6htl%i$h!reuenc+ o9tar$et uta$enesis induced "+ 'R()PR'as nucleases in

huan cells

Research updateJ Ienoe editin$ "ecoes ore accurate

httpJ//ph+s6or$/nes/20130$enoeaccurate6htl

.BA tar$etin$ speci?cit+ o! RBA$uided 'as* nucleases

httpJ//&la"6it6edu/assets/reprints/%suYP.YBatY>iotechnolY20136pd! 

>roadscale $enoe tinkerin$ ith help o! an RBA $uide

httpJ//ph+s6or$/nes/20130"roadscale$enoetinkerin$rna6htl

RBA$uided $ene activation "+ 'R()PR'as*C"ased transcription !actors

Knprecedented control o! $enoe editin$ in 8ies proises insi$ht into huan

developent7 disease httpJ//ph+s6or$/nes/20130unprecedented$enoe

8iesinsi$hthuan6htl

'R()PR/'as*ediated $enoe en$ineerin$ and the proise o! desi$ner 8ies on

deand httpJ//6tand!online6co/doi/!ull/10641-1/8+62-5--\a"stract

Bovel approach to $ene re$ulation can activate ultiple $enes siultaneousl+

httpJ//ph+s6or$/nes/20130approach$eneultiple$enessiultaneousl+6htl

,ultipleed activation o! endo$enous $enes "+ 'R()PRon7 an RBA$uided

transcriptional activator s+ste httpJ//6nature6co/cr/journal/v23/n10/!ull/cr2013122a6htl

25 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 26/31

Leslie Tetteh Research Project and Report 13/04/15 2015

Be ethod !or turnin$ $enes on and o9 could ena"le ore cople s+nthetic

"iolo$+ circuits httpJ//ph+s6or$/nes/20130*ethod$enesena"lecople

s+nthetic6htl

 Tuna"le and ,ulti!unctional #ukar+otic Transcription Gactors >ased on

'R()PR/'as

A !oundation !or !uture researchJ )tud+ tests precision o! protein VachineV

httpJ//ph+s6or$/nes/20130*!oundation!utureprecisionproteinachine6htl

%i$hthrou$hput pro?lin$ o! o9tar$et .BA cleava$e reveals RBApro$raed

'as* nuclease speci?cit+

Poer!ul tool !or $enetic en$ineerin$J Researchers descri"e ne possi"ilities o!

the 'R()PR'ass+ste httpJ//ph+s6or$/nes/201311poer!ultool$enetic

possi"ilitiescrisprcass+ste6htl

Ph+lo$en+ o! 'as* deterines !unctional echan$ea"ilit+ o! dualRBA and 'as*

aon$ ortholo$ous t+pe (( 'R()PR'as s+stes

httpJ//nar6o!ordjournals6or$/content/42/4/256!ull

)peedin$ up $ene discover+ httpJ//ph+s6or$/nes/201312$enediscover+6htl

Ienetic )creens in %uan 'ells Ksin$ the 'R()PR'as* )+ste

Ienoe)cale 'R()PR'as* nockout )creenin$ in %uan 'ells

)hortenin$ $uide RBA arkedl+ iproves speci?cit+ o! 'R()PR'as nucleases

httpJ//ph+s6or$/nes/201401shortenin$rnaarkedl+speci?cit+crisprcas6htl

(provin$ 'R()PR'as nuclease speci?cit+ usin$ truncated $uide RBAs

Pu&&lin$ uestion in "acterial iune s+ste ansered

httpJ//ph+s6or$/nes/201401pu&&lin$"acterialiune6htl

.BA interro$ation "+ the 'R()PR RBA$uided endonuclease 'as*

Researchers reverse a liver disorder in ice "+ correctin$ a utated $ene

httpJ//ph+s6or$/nes/201403reverseliverdisordericeutated6htl

Ienoe editin$ ith 'as* in adult ice corrects a disease utation and

phenot+pe

Iene reoval could have iplications "e+ond plant science

httpJ//ph+s6or$/nes/201404$eneiplicationsscience6htl

Be $enoeeditin$ plat!or si$ni?cantl+ increases accurac+ o! 'R()PR"ased

s+stes httpJ//ph+s6or$/nes/201404$enoeeditin$plat!orsi$ni?cantl+

accurac+crispr"ased6htl

.ieric 'R()PR RBA$uided Gok( nucleases !or hi$hl+ speci?c $enoe editin$

>reakthrou$h shos ho .BA is VeditedV to correct $enetic diseases

httpJ//ph+s6or$/nes/201405"reakthrou$hdna$eneticdiseases6htl

.irect o"servation o! Rloop !oration "+ sin$le RBA$uided 'as* and 'ascade

e9ector coplees

'R()PR s+ste can proote anti"iotic resistance httpJ//ph+s6or$/nes/20140crispranti"ioticresistance6htl

26 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 27/31

Leslie Tetteh Research Project and Report 13/04/15 2015

A 'R()PR'as s+ste enhances envelope inte$rit+ ediatin$ anti"iotic

resistance and in8aasoe evasion

httpJ//6pnas6or$/content/111/30/111-36!ull

Political scientist discusses re$ulator+ $aps in assessin$ the ipact o! X$ene

drivesX httpJ//ph+s6or$/nes/20140politicalscientistdiscussesre$ulator+$aps6htl

Re$ulatin$ $ene drives httpJ//6sciencea$6or$/content/345/-1*/-2-6!ullW

sidZ51d00132c44c551"0a05!e0!4!-

Iene drives raise dualuse concerns]Response

An easier a+ to anipulate alaria $enes httpJ//ph+s6or$/nes/20140

easieralaria$enes6htl

#cient 'R()PR'as*Cediated $enoe editin$ in Plasodiu !alciparu

'rop iproveent and resistance to patho$ens "ene?ts !ro noncodin$ RBA

studies httpJ//ph+s6or$/nes/20140*cropresistancepatho$ens"ene?tsnon

codin$6htl

Boncodin$ RBAs as potent tools !or crop iproveent

httpJ//nsr6o!ordjournals6or$/content/1/2/1-6!ull

Banocontainers !or nanocar$oJ .eliverin$ $enes and proteins !or cellular

ia$in$7 $enetic edicine and cancer therap+ httpJ//ph+s6or$/nes/20140*

nanocontainersnanocar$o$enesproteinscellular6htl

iral nanoparticleencapsidated en&+e and restructured .BA !or cell deliver+

and $ene epression httpJ//6pnas6or$/content/111/3/1331*6!ull

>attlin$ super"u$s ith $eneeditin$ s+ste httpJ//ph+s6or$/nes/20140*

super"u$s$eneeditin$6htl

)euencespeci?c antiicro"ials usin$ ecientl+ delivered RBA$uided

nucleases

R'as*J A pro$raa"le RBA editin$ tool httpJ//ph+s6or$/nes/201410rcas*

pro$raa"lernatool6htl

Pro$raa"le RBA reco$nition and cleava$e "+ 'R()PR/'as*

Precise control over $enes results !ro $aechan$in$ research

httpJ//ph+s6or$/nes/201410precise$enesresults$aechan$in$video6htlIenoe)cale 'R()PR,ediated 'ontrol o! Iene Repression and Activation

.esi$ner viruses could "e the ne anti"iotics httpJ//ph+s6or$/nes/201410

virusesanti"iotics6htl

#ploitin$ 'R()PR'as nucleases to produce seuencespeci?c antiicro"ials

)euencespeci?c antiicro"ials usin$ ecientl+ delivered RBA$uided

nucleases

27 | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 28/31

Leslie Tetteh Research Project and Report 13/04/15 2015

VPro$raa"leV anti"iotic harnesses an en&+e to attack dru$resistant

icro"es httpJ//ph+s6or$/nes/201410pro$raa"leanti"ioticharnesses

en&+edru$resistant6htl

Revolutioni&in$ $enoe en$ineerin$J Revie on histor+ and !uture o! the 'R()PR

'as* s+ste pu"lished httpJ//ph+s6or$/nes/201412revolutioni&in$$enoehistor+!uturecrisprcas*6htl

 The ne !rontier o! $enoe en$ineerin$ ith 'R()PR'as*

>ioen$ineers develop tool !or repro$rain$ $enetic code

httpJ//ph+s6or$/nes/201501"ioen$ineerstoolrepro$rain$$enetic

code6htl

#n$ineerin$ 'ople )+nthetic Transcriptional Pro$ras ith 'R()PR RBA

)ca9olds

)+nthetic Transcriptional 'ontrol #leents and ,ethods o! Ieneratin$ and Ksin$

)uch #leents (nventorsJ 'han$ Liu7 Ada P Arkin7 Lei ) Qi

V,olecular scissorsV could prevent $enetic diseases "e!ore conception

httpJ//ph+s6or$/nes/201501olecularscissors$eneticdiseases

conception6htl

As+etric parental $enoe en$ineerin$ "+ 'as* durin$ ouse eiotic eit

httpJ//6nature6co/srep/2014/141223/srep0-21/!ull/srep0-216htl

 Tea en$ineers V'as*V anial odels to stud+ disease and in!or dru$ discover+

httpJ//ph+s6or$/nes/20140*teacas*anialdiseasedru$6htl

'R()PR'as* knockin ice !or $enoe editin$ and cancer odelin$

)tructure o! ke+ 'R()PR cople revealed httpJ//ph+s6or$/nes/201402ke+

crisprcoplerevealed6htl

'r+stal structure o! 'as* in cople ith $uide RBA and tar$et .BA

httpJ//6sciencedirect6co/science/article/pii/)00*2-4140015-1

Be insi$ht into an eer$in$ $enoeeditin$ tool httpJ//ph+s6or$/nes/2014

02insi$hteer$in$$enoeeditin$tool6htl

)tructures o! 'as* endonucleases reveal RBAediated con!orational

activation

Be ethod identi?es $enoeide o9tar$et cleava$e sites o! 'R()PR'asnucleases httpJ//ph+s6or$/nes/201412ethod$enoeideo9tar$et

cleava$esites6htl

IK(.#se ena"les $enoeide pro?lin$ o! o9tar$et cleava$e "+ 'R()PR'as

nucleases

Research reveals structure o! ke+ 'R()PR cople httpJ//ph+s6or$/nes/2014

12revealske+crisprcople6htl

Ienoescale transcriptional activation "+ an en$ineered 'R()PR'as* cople

Li$htactivated $enes i$ht "e precisel+ controlled and tar$eted

httpJ//ph+s6or$/nes/201502li$htactivated$enesprecisel+6htlA li$htinduci"le 'R()PR'as* s+ste !or control o! endo$enous $ene activation

2" | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 29/31

Leslie Tetteh Research Project and Report 13/04/15 2015

)+nthetic "iolo$+ +ields ne approach to $ene therap+

httpJ//ph+s6or$/nes/201502s+nthetic"iolo$++ieldsapproach$ene6htl

'R()PR"ased sel!cleavin$ echanis !or controlla"le $ene deliver+ in huan

cells httpJ//nar6o!ordjournals6or$/content/43/2/12*6!ull

Research e9orts +ieldin$ ajor advances in understandin$ iunolo$icaleor+ httpJ//ph+s6or$/nes/201502e9orts+ieldin$ajoradvances

iunolo$ical6htl

Paper 1J (nte$raseediated spacer acuisition durin$ 'R()PRC'as adaptive

iunit+7 Bature :2015;

Paper 2J 'as* speci?es !unctional viral tar$ets durin$ 'R()PRC'as adaptation7

Bature :2015;

Bes and iesJ ,icro"iolo$+J %o "acteria $et spacers !ro invaders7 Bature

:2015;

#nd o! 'R()PR'A)* controvers+ httpJ//ph+s6or$/nes/201502crisprcas*

controvers+6htl

.i$enoeseJ $enoeide pro?lin$ o! 'R()PR'as* o9tar$et e9ects in huan

cells

Re$ulatin$ $enoeedited crops that arenVt I,Hs httpJ//ph+s6or$/nes/2015

02$enoeeditedcrops$os6htl

 Toards social acceptance o! plant "reedin$ "+ $enoe editin$

httpJ//6sciencedirect6co/science/article/pii/)13-0135150002*1

Link identi?ed "eteen virus reco$nition7 destruction in "acterial iune

s+ste httpJ//ph+s6or$/nes/201503linkvirusreco$nitiondestruction"acterial6htl

'as* !unction and host $enoe saplin$ in t+pe ((A 'R()PR'as adaptation

iruscuttin$ en&+e helps "acteria ree"er a threat

httpJ//ph+s6or$/nes/201502viruscuttin$en&+e"acteriathreat6htl

'as* speci?es !unctional viral tar$ets durin$ 'R()PRC'as adaptation

A sin$le tar$et !or icroRBA re$ulation httpJ//ph+s6or$/nes/201503

icrorna6htl

 The let icroRBA directs vulval developent throu$h a sin$le tar$et

Activatin$ $enes on deand httpJ//ph+s6or$/nes/201503$enesdeand6htl

%i$hl+ ecient 'as*ediated transcriptional pro$rain$

Researcher develops novel strate$+ to iprove crops and treat diseases

httpJ//ph+s6or$/nes/201503strate$+cropsdiseases6htl

>oostin$ 'R()PR/'as* ultiple editin$ capa"ilit+ ith the endo$enous tRBA

processin$ s+ste6

'olorcoadin$ $ene seuences in huan cells httpJ//ph+s6or$/nes/201503

colorcoadin$$eneseuenceshuancells6htl

,ulticolored 'R()PR/'as* la"elin$ s+ste precisel+ easures distance "eteenchroosoes7 $enetic eleents httpJ//ph+s6or$/nes/201503ulticolored

2# | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 30/31

Leslie Tetteh Research Project and Report 13/04/15 2015

crisprcas*precisel+distancechroosoes6htl

,ulticolor 'R()PR la"elin$ o! chroosoal loci in huan cells

httpJ//6pnas6or$/content/112/10/30026!ull

Li!e scientists strealine cuttin$ed$e techniue to edit osuito $enoe

httpJ//ph+s6or$/nes/201503li!escientistscuttin$ed$etechniueosuito6htl

)ilencin$ o! endjoinin$ repair !or ecient sitespeci?c $ene insertion a!ter

 TAL#B/'R()PR uta$enesis in Aedes ae$+pti

 Dail"reakin$ +east could ap up ineVs health "ene?ts7 reduce ornin$a!ter

headaches httpJ//ph+s6or$/nes/201503jail"reakin$+eastapine

health6htl

'onstruction o! a Quadruple Auotrophic ,utant o! an (ndustrial Pol+ploid

)accharo+ces cerevisiae )train "+ Ksin$ RBAIuided 'as* Buclease

Be $enetic ethod proises to advance $ene research and control insect pests httpJ//ph+s6or$/nes/201503$eneticethodadvance$eneinsect6htl

 The uta$enic chain reactionJ A ethod !or convertin$ hetero&+$ous to

hoo&+$ous utations

Appendi) III

.evelopent and applications o! 'R()PR'as* !or $enoe en$ineerin$

'as* as a versatile tool !or en$ineerin$ "iolo$+

'R()PR'as s+stes !or editin$7 re$ulatin$ and tar$etin$ $enoes

%arnessin$ the 'R()PR/'as* s+ste to disrupt latent %(1 provirus

RBAdirected $ene editin$ speci?call+ eradicates latent and prevents ne %(1

in!ection

*ptimi+ed CI,-.Cas too!s 'or e/cient (erm!ine and somatic (enome

en(ineerin( in 0rosophi!a

http:..bior)ior(.content.ear!y.2$15.$1.16.$13"#6

Concernin( A(uided (ene dries 'or the a!teration o' i!dpopu!ations

 :#cient $enoe editin$ in &e"ra?sh usin$ a 'R()PR'as s+ste;

:Repurposin$ 'R()PR as an RBA$uided plat!or !or seuencespeci?c control o!

$ene epression;

:A "acteriopha$e encodes its on 'R()PR/'as adaptive response to evade host

innate iunit+6;

:Ienoeide detection o! .BA dou"lestranded "reaks induced

3$ | P a $ e

7/23/2019 3BIO7A2 12980356 Leslie Tetteh

http://slidepdf.com/reader/full/3bio7a2-12980356-leslie-tetteh 31/31

Leslie Tetteh Research Project and Report 13/04/15 2015

>iosa!et+ and risk assessent in the use o! $eneticall+ odi?ed osuitoes !ordisease control

Iuidance !or contained ?eld trials o! vector osuitoes en$ineered to contain a$ene drive s+steJ recoendations o! a scienti?c orkin$ $roup

 The e9ect o! $ene drive on containent o! trans$enic osuitoes6

,ethodolo$ies and special considerations !or environental risk anal+sis o!$eneticall+ odi?ed auatic "iocontrol or$aniss

'reatin$ a Research A$enda !or the #colo$ical (plications o! )+nthetic >iolo$+6

Precision $enoe en$ineerin$ ith pro$raa"le .BAnickin$ en&+es6

Appendi) I

Qualitative risk assessent o! ultipleuse arine park e9ectiveness

#valuatin$ the 'ontrol >andin$ Banotool a ualitative risk rsk assessent

Qualitative Risk Assessent o! the Acuisition o! ,R)A in .o$s

A ualitative ecolo$ical risk assessent o! the invasive Bile tilapia7 Hreochrois

niloticus in a su"tropical A!rican river s+ste :Lipopo River7 )outh A!rica;

Qualitative Assessent o! the 'oodit+ Risk !or )pread o! Gootand,outh

.isease Associated ith (nternational Trade in .e"oned >ee! 

A ualitative risk"ased assessent o! ipacts on arine ha"itats and harvested

species !or a data de?cient ild capture ?sher+

A Qualitative Risk Assessent Approach !or )iss .air+ ProductsHpportunities

and Liitations