CRISPR/Cas9
Liu, Bin; Saber, Ali; Haisma, Hidde J
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Drug Discovery Today
DOI:
10.1016/j.drudis.2019.02.011
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Publication date:
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Citation for published version (APA):
Liu, B., Saber, A., & Haisma, H. J. (2019). CRISPR/Cas9: A powerful tool for identification of new targets
for cancer treatment. Drug Discovery Today, 24(4), 955-970. https://doi.org/10.1016/j.drudis.2019.02.011
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Teaser
This
review
provides
the
latest
findings
regarding
the
application
of
CRISPR/Cas9
for
the
identification
of
new
therapeutic
targets
and
associated
major
challenges
in
cancer
treatment.
CRISPR/Cas9:
a
powerful
tool
for
identification
of
new
targets
for
cancer
treatment
Bin
Liu
z,
Ali
Saber
zand
Hidde
J.
Haisma
DepartmentofChemicalandPharmaceuticalBiology,GroningenResearchInstituteofPharmacy,Universityof Groningen,TheNetherlands
Clustered
regularly
interspaced
short
palindromic
repeats
(CRISPR)/
CRISPR
associated
nuclease
9
(Cas9),
as
a
powerful
genome-editing
tool,
has
revolutionized
genetic
engineering.
It
is
widely
used
to
investigate
the
molecular
basis
of
different
cancer
types.
In
this
review,
we
present
an
overview
of
recent
studies
in
which
CRISPR/Cas9
has
been
used
for
the
identification
of
potential
molecular
targets.
Based
on
the
collected
data,
we
suggest
here
that
CRISPR/Cas9
is
an
effective
system
to
distinguish
between
mutant
and
wild-type
alleles
in
cancer.
We
show
that
several
new
potential
therapeutic
targets,
such
as
CD38,
CXCR2,
MASTL,
and
RBX2,
as
well
as
several
noncoding
(nc)RNAs
have
been
identified
using
CRISPR/
Cas9
technology.
We
also
discuss
the
obstacles
and
challenges
that
we
face
for
using
CRISPR/Cas9
as
a
therapeutic.
Introduction
Theaccumulationofgeneticmutationsincellsovertimeleadstocancer.Inaddition,certain
genechanges,suchasdrivermutationsinTP53,EGFR,KRAS,BRAF,HER2,andMET,canmakea
cellcancerous.Treatmentstrategiesareconventionallybasedonhistologicalsubtypes.However,
in addition to the conventional histological classifications, each cancer type can now be
subdividedintovariousmolecularsubtypesthathaveacrucialroleinthetreatment
decision-makingprocess.Eachmolecular subtypeis treateddifferentlyand clinicianscanalso predict
treatmentoutcomes andpatient survival.Forinstance, patientswithlungcancer with
EGFR-activatingmutationsaretreatedwithdifferenttypesoftyrosinekinaseinhibitor(TKI),suchas
gifitinib,erlotinib,orafatinib,dependingonthemutation.Yet,resistancetotheTKIsinevitably
emergeseitherbyDNAmutationsor/andmetabolicchanges.Asaresult,treatmentstrategiesare
modifiedbasedonthenewmolecularsignature.However,eventually,the tumorcellsdonot
respondto anytreatment[1].Therefore,identificationofnewtherapeutictargets toimprove
patientsurvivalandclinicaloutcomesiscrucial[2,3].
Inrecentyears,CRISPR/Cas9hassignificantlyinfluencedthefieldofmolecularbiologyand
genetherapy.Solidtumorsarethemostcommontypeoftumors,butlessprogresshasbeenmade
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BinLiuisaPhDstudentin theDepartmentof Chemicaland PharmaceuticalBiology, UniversityofGroningen, TheNetherlands.His researchfocusesoncancer moleculartherapybased onadenovirus,CRISPR/
Cas9,andtargeteddrugs.HeisamemberofDutch SocietyofGeneandCellTherapy.
AliSaberdidhisPhDon tumorheterogeneityand drugresistanceatthe UniversityofGroningen, TheNetherlands,focusing ontheroleofgenomic aberrationsindrug resistance.Currently,heis apostdoctoralresearcher
attheUniversityofGroningen.Heisinterestedinthe identificationofnoveltherapeutictargetsto overcomedrugresistanceandimproveclinical outcomesinpatientswithcancer.
HiddeHaismawas awardedanMScinmedical biologyattheUniversityof Utrecht,TheNetherlands in1983;hethenworkedas ascientistatCentocorInc. Malvern,PA,USA(1985˘ 1986),DanaFarberCancer Institute,Brighamand
Women’sHospitalBostonMA,USA(1986˘1987),and asaninstructoratHarvardMedicalSchool,Boston MA,USA(1986˘1987).In1987,hewasawardedhis PhDbytheUniversityofUtrecht,TheNetherlands forhisthesisentitled‘Monoclonalantibodiesand cancer’.From1987to1997,heworkedasascientistin theSectionforExperimentalTherapyofthe DepartmentofMedicalOncology,UniversityHospital ofAmsterdam,TheNetherlands,where,in1997,he wasappointedassistantprofessor/sectionleaderof theDivisionofGeneTherapy.In2000,hebecamea fullprofessorattheUniversityofGroningen,The Netherlands.Haisma’sresearchisdevotedtothe developmentofgeneticmedicinesfortherapeutic interventionofgeneexpressioninpatients.Heis memberoftheeditorialboardofseveraljournals,has co-authoredover150publications,andholdsseveral patents.Haismaistheformerpresidentandmember oftheboardofTheDutchSocietyofGeneandCell Therapy.Inaddition,heismemberoftheGeneand CellDopingExpertGroupoftheWorldAnti-Doping Agency(WADA).
Correspondingauthor.
zTheseauthorscontributedequally.
for gene therapy-based treatment compared with nonsolid
tumors, such as leukemia. However, this situation is rapidly
changingwithdevelopmentsinCRISPR/Cas9.Thereisagrowing
amountofpromisingpreclinicaldatashowingCRISPR/Cas9tobe
an effectivetool tospecifically target cancer cellsand suppress
tumor growth [4–6].This could lead to the discoveryof novel
moleculartargetsforcancertreatment.
ThereisanincreasingnumberofclinicaltrialsutilizingCRISPR/
Cas9technologytotreatcancersofdifferentorigin(Table1).Most
ofthesetrialsarebasedongeneticallyengineeredTcellsforcancer
immunotherapy,ratherthantargetingaspecificgeneinthetumor
cellsthemselves.Oneofthe mainproblemsassociatedwiththe
directtargetingofcanceristhelackofaneffectiveandsafedelivery
method that can be used in patients. Tumor heterogeneity is
anotherissuethatmightbeachallengebecausetumorsusually
comprisedifferentsubclones(i.e.,intratumorheterogeneity)[7–
10].Thus, evenwith the rightdelivery system, outgrowthof a
minorsubclonecanemergeandthetreatmentwouldnolongerbe
effective.Nonetheless,identificationofdifferentmajorsubclones
beforetreatmentandrecruitmentofmultipleCas9/guide(g)RNA
mightbeanoptiontominimizerelapseinpatients.
Here,weprovideanoverviewofstudiesinwhichCRISPR/Cas9
has been utilized forthe identification ofpotential therapeutic
targetsinsomeofthemostfrequentsolidtumors,includinglung,
breast, brain,liver, and colorectal cancer. Wediscuss potential
candidatesfortherapythatareeitherhighlyexpressedoractivated
in different cancer types,becausethey are moreconvenient to
inhibitordisrupt.Weendbypresentingrecentadvancesin,and
differentdeliverymethodsfor,CRISPR/Cas9.
CRISPR/Cas9
gene-editing
technology
CRISPR/Cas9isarecentlydiscovered,powerfulgene-editingtool
derivedfromaprokaryoticdefensesystem[11–14].This
technol-ogyhasenabledresearcherstoeditthegenomeofeukaryoticcells
morepreciselyandefficientlycomparedwithpreviousmethods,
suchaszinc-fingernucleases(ZFNs)andtranscription
activator-likeeffectornucleases(TALENs)[15].
The
structure
of
CRISPR/Cas9
ThestructureofCRISPR/Cas9hascomprehensivelybeendescribed
elsewhere [15–17]. The CRISPR/Cas9 system has three
compo-nents;a singleguideRNA(sgRNA),whichisspecifictoatarget
sequenceofDNA;Cas9proteinwithDNAendonucleaseactivity;
andatracrRNAthatinteractswithCas9(Fig.1).ThegRNA
(ap-proximately20basepairsinlength)bindstothetargetsiteinthe
genomeanddirectstheCas9protein.TheCas9proteinisa
RNA-guidednucleasethatwasdiscoveredintheCRISPRtypeIIadaptive
immunitysystemofStreptococcuspyogenesanditisresponsiblefor
cleavingdouble-strandDNA[17].
gRNAsandtheirspecificity
Severalfactors,suchassequence,length,andsecondarystructure,
ofgRNAscaninfluencetheirefficiencyandspecificity[18,19].In
addition,theefficiencyoftheCRISPR/Cas9complexcanbe
influ-encedbyotherfactors,includingthegenomiclocusofthetarget,
chromatin accessibility, nucleosomes, and other components
aroundgRNA-bindingsites[19].ThegRNAsequencehasacrucial
roleinthe efficiency, specificity,and accuracyof
CRISPR/Cas9-mediatedgenomeediting.Thefirst10–12nucleotidesatthe30end
ofgRNA,immediatelyadjacent toa protospaceradjacent motif
(PAM),calledthe‘seedsequence’,bindtothetargetsequenceand
determinethe specificity[18,20].TruncatedgRNAswithshorter
complementarynucleotides(<20)canreduceoff-targeteffectsby
5000-foldwithoutsacrificingon-targetefficiency[21].Moreover,
extending the gRNA duplex by 5 base pairs can significantly
improvetheknockoutefficiency[22].
CRISPR-associatednucleases
Different versions of CRISPR-associated nucleases are currently
underdevelopment,greatlyexpandingtheCRISPR-basedtoolbox
forgenomeediting(Table2).Cpf1isanRNA-guidedendonuclease
thatbelongstotheclass2CRISPR-Cassystem,thesameasCas9
TABLE1
PhaseI/IIclinicaltrialsthatuseCRISPR/Cas9genome-editingtechnologies
Condition Target Interventions Phase Status ClinicalTrialsGov
Identifier Solidtumor,adult PD-1andTCR Anti-mesothelinCAR-T
cells
I Active NCT03545815
Melanoma;synovial sarcoma;liposarcoma
TCRendoandPD-1 NY-ESO-1;drug: cyclophosphamide, fludarabine
I Active NCT03399448
Gastrointestinalcancer CISH,inactivatedTIL Drug:
cyclophosphamide, fludarabine,aldesleukin
I – NCT03538613
Human papillomavirus-relatedmalignantneoplasm
HPV-relatedcervical intraepithelialneoplasia
Biological:TALEN, CRISPR/Cas9
I – NCT03057912
Gastrointestinalinfection Hostfactorsofnorovirus Duodenalbiopsy;saliva – Active NCT03342547 Bcellleukemia;Bcell
lymphoma CD19andCD20orCD22 CAR-T Universaldual-specificity CD19andCD20orCD22 CAR-TCells I/II Active NCT03398967
Leukemia;lymphoma RelapsedorRefractory CD191
UCART019 I/II Active NCT03166878
StageIVgastriccarcinoma; StageIVnasopharyngeal carcinoma;StageIVTcell lymphoma – Drug:fludarabine, cyclophosphamide, interleukin-2 I/II Active NCT03044743 Reviews FOUNDA TION REVIEW
[23].However,Cpf1hasdifferentfeaturescomparedwithCas9.For
example,ithasonlyone singlenucleasedomainand a shorter
gRNA.Creating staggeredcutsis oneofthe maincharacteristic
features ofCpf1. This typeof cut isimportant forintroducing
exogenousDNAintothegenomebythehomologydirectedrepair
(HDR)pathway.Inaddition,Cpf1hasbothendoribonucleaseand
endonucleaseactivities,whichisuniqueforanuclease[24].These
propertiesmakeCpf1bothacomplexandeffective
genome-edit-ingtoolforgenetargeting andgenesilencing.C2C2isanother
memberofclass2typeVI-ACRISPR-Casandwasfoundin
Lepto-trichiashahii,whereitprotectsthebacteriumagainstRNAphages.
C2C2canbeusedtotargetandregulateRNAs.IthastwoRNase
catalyticpocketswithdualRNaseactivities,whichcanberecruited
fortheidentificationofcellulartranscripts[25].Thestructureand
function ofC2C2 isuniqueand provides a noveltool forRNA
manipulation[25–27].
Preciselyeditingasinglebaseinthegenomewithout
introduc-ingdouble-strandedbreaks(DSBs)wasalongstandinggoalthat,
with engineered Cas9 base editors, is now possible. The ‘base
editors’comprisefusionsofa deadCas9domainandacytidine
deaminase enzyme that is able to convert GC to AT without
introducingDSBs[28].Recently,researcherscreatedaCas9fused
withatransferRNAadenosinedeaminasethatcanmediate
con-versionofATtoGC[29].Thesebaseeditorsarevaluabletoolsfor
repairingdisease-relatedmutations.
Advantages
of
CRISPR/Cas9
over
ZFN
and
TALENs
The CRISPR/Cas9 system has several advantagesover ZFN and
TALENsintermsofitssimplicity,flexibility,andaffordability.The
most important difference is that the CRISPR system relieson
RNA–DNArecognition,ratherthanontheprotein–DNA-binding
mechanism [11,17,30]. Thus, it is more ‘doable’ and easier to
constructacustomizedCRISPR/Cas9complexbyonlychanging
the gRNAsequence instead of engineeringa newprotein. The
target sequence needs to be immediately upstream of a PAM
sequence(50-NGG-30)[17],becausethelatterisessentialfortarget
recognitionby Cas9. Thisshortsequence occursapproximately
onceeveryeightbasepairsinthehumangenome,whichmakesit
possibletodesignseveralgRNAsforonespecifictargetgene[31].
Targeting
cancer-related
genes
and
identification
of
potential
therapeutic
targets
in
solid
tumors
CRISPR/Cas9isroutinelyusedinresearchlaboratoriesbecauseof
its simplicityand efficiency. Inaddition, the costofthis
gene-editing tool is reducing daily. The CRISPR/Cas9 gene-editing
technology helped researchers to identify the role of different
genesincancer;forinstancewhethertheyfunctionasoncogenes
ortumorgenes[32–36].Severalgroupsgeneratedinvitroandinvivo
knockoutmodelstostudythemolecularbasisofdifferentcancer
types[37–39].CRISPR/Cas9isalsowidelyusedforinducing
spe-cificmutationsincertaingenestoexplorethepotentialcausative
Cas9 nuclease gRNA HDR NHEJ Target DNA Indel
Knockout
Knock-in
HDR template DSB DSBDrug Discovery Today
FIGURE1
Schematicpictureofgenomeeditingmediatedbyclusteredregularlyinterspacedshortpalindromicrepeats(CRISPR)/CRISPRassociatednuclease9(Cas9),and DNArepairing.TheCas9protein,whichisguidedbyadesiredsingle-strandguideRNA(gRNA),cutsthedouble-strandedDNAandmakesadoublestrandbreak (DSB).Subsequently,DNArepairoccursthrougheitherthenonhomologousendjoining(NHEJ)orthehomology-directedrepair(HDR)pathways.
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role of these mutations in disease development. In addition,
CRISPRbarcoding technologycanbeusedto investigatetumor
heterogeneity [40,41]. Moreover, genome-wide CRISPR/Cas9
screening is frequently used to identify potential therapeutic
targetsindifferentcancers.Here,wemainlyfocusonnew
poten-tialmoleculartargetsthathavebeenidentifiedbyusingCRISPR/
Cas9insomeofthemostfrequentsolidtumors,includinglung,
breast,brain,liver,andcolorectalcancers.
Lung
cancer
Lungcanceristheleadingcauseofcancer-relateddeathworldwide.
Non-smallcelllungcancer(NSCLC)subtypesaccountfor85%of
alllungcancers[42].Theidentificationofspecificgenetic
aberra-tions isimportanttochoosethe appropriatetreatmentstrategy,
particularly in patientswithadenocarcinoma. Currently,several
moleculartargets,suchasEGFR,BRAF,ALK-EML4,andcMET,are
clinicallyavailableforthetreatmentofNSCLC.However,treatment
optionsare limited by the number ofmoleculartargets and by
emergingdrugresistance[1,42].Inrecentyears,CRISPR/Cas9-based
in vitro and in vivo studies of lung cancer have identified new
treatmentstrategiesandpotentialtherapeutictargets.
TargetingthemutantversionofcertaingenesusingtheCRISPR/
Cas9 gene-editing system can specifically target mutant cancer
cells, butnotnormalcells. Targetingthe mutantversionofthe
EGFRgene(L858R)resultedintheselectiveeliminationofmutant
cellsandreducedcellproliferationbothinvitroandinvivo[43].In
anotherstudy,Kooetal.selectivelyabrogatedEGFRmutantalleles
(L858R)inaNSCLCcellline(H1975)usinganadenovirus(AdV)
vectorthatresultedincancercelldeathandsignificantlyreduced
tumorsizeinvivo[4].Thesefindingsunderscorethepotentialof
CRISPR-basedtherapeuticsintumor-specifictargetedtherapyand
indistinguishingnormalfrommutanttumorcells.
Approximately30%ofpatientswithlungcancerhavesomatic
activatingKRASmutations.Currently,thereisnoeffective
treat-ment for KRAS mutant lung tumors, which are considered as
undruggable.CRISPR-baseddeletionofmurineKrasintwo
differ-entKrasG12D/1/p53/lungcancercelllinesresultedinasignificant
reductionincellproliferation,butthecellswerestillviableand
sustained their ability to form tumors in vivo. Transcriptome
sequencingrevealeda substantially higherexpressionofFas
re-ceptorin theknockoutcells. Interestingly,anactivatingFas
re-ceptor antibody selectively induced apoptosis in these Kras/
lung cancer cells [44]. Oncogenic Ras can inhibit Fas
ligand-mediatedapoptosisthroughdownregulationofFas[45];therefore,
simultaneousinhibitionofKRASandactivationofFASmightbe
aneffectivetherapeuticapproachagainstKRAS-drivenlungcancer
tumors.
CD38isaglycoproteinthatfunctionsasbothanADP-ribosyl
cyclaseandaNADglycohydrolase.Itsexpressionlevelis
negative-lyassociatedwithpoorprognosisinpatients withchronic
lym-phocyticleukemiaanditisusedasatherapeutictargetinmultiple
myeloma[46,47].However,itsroleissolidtumors,suchaslung
cancer, is not clear. CRISPR-based deletion of CD38 in a lung
adenocarcinomacellline(A549)resultedinsubstantial
suppres-sionofcellgrowthandinvasioninvitroandinxenograftsinmice,
suggesting CD38 as a potential target in lung cancer. Further
investigationsunraveled anelevatedlevelofCD38in 93%(27/
29)oflungcancercelllinesand40%(11/27)ofNSCLCprimary
tumors.Hence,directdisruptionofthistargetthrough
monoclo-nal antibodies, such as daratumumab, might be effective in
patientswithNSCLCwithCD38overexpression[48].
Disruptionoffocaladhesionkinase(FAK),anonreceptor
tyro-sinekinasethatisfrequentlyamplifiedinlungcancercelllines,
resultsin DNAdamageandsensitivity to ionizingradiation.In
addition,usingCRISPR/Cas9approaches,thepresenceofFAKwas
showntobecrucialfortheoncogenicandclonogenicabilitiesof
KRASmutants in tumor xenografts[49,50].Inaddition, FAK is
significantlyoverexpressedinpatientswithNSCLCandis
associ-ated with poorer clinical outcomes [51–53], which make it an
attractivetargettotreatNSCLCandpreventdistantmetastasis.
TAZisacoactivatoroftheHippopathwayandisupregulatedin
lungcancer.DualinactivationofTAZandYAP(atranscriptional
TABLE2
CRISPR-associatednucleases
Class Name Advantages Applications Refs
NaturallyCas9 SpCas9 Mostcommonused Geneediting [17,23,200,201]
SaCas9 1kbsmallerthanSpCas9 StCas9 DifferinPAMsequence CjCas9
FnCas9
CasX MostcompactnaturallyCRISPRvariant Potentiallyuseful untappednucleases CasY
Cas12a(Cpf1) SmallerthanSpCas9;gRNAshorterthanSpCas9 Moreeconomicalto produce
EngineeredCas9 variants
Cas9nickases VariantsnickasingleDNAstrandratherthanDSB Highfidelity [202,203]
eSpCas9 Enhancednuclease Highspecificityandlow
off-targetrate Baseeditors BE1/2/3;ABEs Completerangeofnucleotideexchangeswithout
DSBs
Genewriting [28,29]
RNAeditors Cas13a/Cas13b/Cas13d TargetingRNAratherthanDNA RNAediting [26,200,204]
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activator)suppressedcellproliferationaswellascancerstemcell
(CSC)sphereformationinlungcancer,suggestingthemaspotential
molecular targets [54]. CRISPR-based disruption of oncogenic
MUC1-ChinderedthegrowthofKRAS-dependentlung
adenocarci-nomacells(i.e.,A549andA460)[55].Inaddition,overexpressionof
MUC1hasbeenshownin>80%ofNSCLCsandisassociatedwith
poorprognosis[56,57].Italsohasaroleinepithelial-mesenchymal
transition(EMT)andself-renewalability,eachofwhicharedrug
resistance mechanisms in cancer [58,59]. Thus, suppression of
MUC1-Cmightdelayresistanceorevenpreventtumorrecurrence.
Likewise, it has been reported that overexpression of TNC, an
extracellularmatrix(ECM)protein,isassociatedwithlungcancer
recurrence[60].CRISPR-basedtranscriptionalactivationofTncled
tometastaticdisseminationoflungadenocarcinomacellsinvivo.
ThishighlightsthecentralroleofEMC-relatedproteinsinmetastasis
andtheirpotentialuseasrecurrenceandmetastasisbiomarkersas
wellastherapeutictargetsinNSCLCs[61].
Chromatin-remodelinggenes arefrequently mutated,mostly
inactivatingmutations,inlungadenocarcinoma[62–64].Arecent
study usedCRISPRtechnologyto knockoutSmarca4, Arid1a,or
Setd2toinvestigatetheirroleinlungtumorigenesis.LossofArid1a
andSetd2resultedinthedevelopmentofhigher-gradetumorsand
strongtumor progression in both early- and late-stage lesions,
respectively.Bycontrast,ablationofSmarca4ledtotumor
devel-opment, whereas itattenuated disease progression in vivo over
time[34].Previously,itwasshownthat SMARCA4inactivation
promotesNSCLC aggressiveness[65].Nevertheless,
loss-of-func-tionmutationsinSMARCA4havebeenreportedtoincreasetumor
cellsensitivitytotheAurorakinaseAinhibitorVX-680bothinvitro
andinxenograftmousemodels[66].Thesediscrepanciesmakeit
challengingtodecidewhetherSMARCA4isanappropriate
molec-ularcandidatefortherapy.However,geneticdisruptionor
chemi-cal-basedinhibitionofSMARCA4couldbeofbenefitforpatients
withmoreadvancedNSCLC.
miRNAshaveimportant rolesincellsandtheirdysregulation
hasbeen shownin varioustypesofcancer [67].Arecentstudy
exploitedCRISPR/Cas9-basedgeneactivationtechnologyto
inves-tigatetheroleofmiRNAslocatedon14q32inlungcancercells.
OverexpressionofthosemiRNAssignificantlyelevatedcell
migra-tionandinvasion.Moreover,higherexpressionlevelsofmir-323b,
mir-487a,andmir-539wereassociatedwithmetastasisandpoorer
prognosis in patients with lung adenocarcinoma, especially in
those who had never smoked[68]. Thus,these 14q32 miRNAs
mightbepotentialtargetstopreventtumorcelldisseminationand
distantmetastasisinpatientswithlungadenocarcinoma.
Overall,applicationoftheCRISPR/Cas9genome-editingsystem
inlung cancerhas ledto theidentification ofseveral potential
therapeutictargets.Bothinvitroandinvivostudieshaveshown
promisingresults,especiallyinthesuppressionofdistant
metas-tasis,whichisthemaincauseofdeathofpatients.Inaddition,
CRISPR/Cas9cantargetspecificoncogenicallelesofcertaingenes,
suchasEGFR,which isanimportant step towardscancer gene
therapy.
Breast
cancer
Breastcanceristhemostcommontypeofcancerandtheleading
cause of cancer-related death in women worldwide [69]. It is
dividedintovarioussubtypeswithdistinctmorphologies.Based
ontheexpressionofestrogenreceptor(ER),progesteronereceptor
(PR),ERBB2(HER2),p53,andKi-67,itcanbeclassifiedintofour
main molecular subtypes: triple-negative/basal-like; the
Her2-enriched; luminal A; and luminal B [70]. ER-positive luminal
subtypes arethe most common types of breast cancer (almost
70%) and resistanceto endocrine therapies occursin 30%of
thesepatients [71].Therefore,findingnewtreatmentoptionsis
crucial,especiallyinthecaseofrecurrence.Recent
CRISPR-medi-atedstudieshaveledtotheidentificationofpotentialtherapeutic
targetsindifferentbreastcancersubtypes.
Distantmetastasisisoneofthemaincharacteristicsoflate-stage
cancersandthemainreasonforcancermortality.Identificationof
newtherapeutictargetscouldhelptoprolongpatientsurvivaland
improvetheirlifequality.MLK3isamemberoftheMAP3Kfamily,
which is involved in signaltransduction and activation of the
MAPK pathway. Abrogation of Mlk3 in murine triple negative
breastcancer(TNBC)4T1cells,whicharehighlymetastatic,led
to suppressionof cell invasionand migration [72]. Inanother
study,CRISPR-mediateddepletionofCX3CR1,aproteininvolved
inthe disseminationoftumorcellsintobloodvessels,inbreast
cancercellsimpairedlodgingofthecancercellstoboneandledto
areductioninthenumberofcancerouslesionsinmice[73].
Ina recentstudy by Liaoand colleagues, deletionofUbr5, a
member of the E3 ligase family, in a murine mammary TNBC
model resulted in the inhibition of tumor growth and distant
metastasis in vivo as well as the promotion of apoptosis and
necrosisthroughimpairment of angiogenesis. The authorsalso
showed highexpressionlevelsofUBR5in patients withTNBC,
whichmakethisproteininterestingforfurtherinvestigationfor
targetedtherapy[74].CRISPR-mediatedknockoutofCXCR2(IL-8
receptor)inbreastcancercells,showedasignificantreductionin
cellmigrationinvitroaswellasalowerrateoflungmetastasisin
vivo[75].CXCR2isastem-likecellmarkerforTNBCandshows
significantlylowerexpressioninthissubtypecomparedwith
non-TNBC[76].ItisalsoknownthattargetingCXCR2improvesthe
chemotherapeuticresponseinlungcancer[77].Thus,treatmentof
patientswithadvancednon-TNBCwithanti-CXCR2drugsmight
bebeneficial.Inaddition,MARK4andFERMT2areotherpotential
targetsrelatedtobreastcancercellmigrationandmetastasisthat
havebeenidentifiedbyCRISPR/Cas9[78–80].
Several research groups have functionally studied different
proteinswithoncogeniceffectsthatmightbesuitableforfurther
investigationastherapeutictargetsinbreastcancer.Forinstance,
microtubule-associated serine/threonine kinase-like (MASTL) is
involved in the DNA damage response and its overexpression
correlateswithpoorclinicaloutcomesinER-positivebreastcancer
[81,82].CRISPR-based disruptionof MASTL kinase reduced cell
proliferationin breastcancer cell linesand showed therapeutic
effectsinvivo.FurtherMASTLexpressionanalysisinhumanbreast
primary tumors showed higherexpression in tumor cells
com-paredwithnormaltissue.Inaddition,higherMASTLexpression
wassignificantlyassociatedwithpoorerprognosisandits
abun-dancewasassociatedwithhigherhistologicalgrades,suggesting
its crucialrole in the progression of breastcancer [83].Hence,
inhibitionofMASTLmightsuppresstumorgrowthinhigh-grade
breasttumors.
Onestudyshowedthat theSRCfamilykinase (SFK)FYNand
proteintyrosinephosphatase N23(PTPN23)have animportant
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roleinbreastcancer.DoubleknockoutofPTPN23andFYNusing
CRISPR/Cas9significantlyattenuatedcellgrowthinbothCal-51
cells(aTNBCcellline)andxenograftmousemodels.Theauthors
also foundthat expressionofPTPN23was positivelyassociated
withabetterclinicaloutcome[84].Moreover,FYNisimplicatedin
drug resistance and is highly expressed in resistant cell lines
comparedwithparentalcells[85,86].Thus,inhibitionofFYNin
resistantpatientswithPTPN23lossmightbeasuitableoptionto
improveclinicaloutcomes.
Lossofcyclin-dependentkinase7(CDK7)indifferentTNBCcell
linessubstantiallyreducedcancercellgrowthinthesecells,but
notinnon-TNBCcells[87].However,elevatedexpressionofCDK7
hasbeenshowninprimarybreastcancertissuesanditsexpression
isnegatively associatedwithtumorgradeand size.Inaddition,
higherexpression ofCDK7wasassociated withbetteroutcome
[88].Therearesome discrepanciesbetweeninvivo and
patient-basedstudies,whichmightbetheresultofdifferencesinbreast
cancersubtypes.Nevertheless,CDK7mightbeasuitabletargetat
least for a subset of patients with breast cancer. Disruption of
SHCBP1, a member of the Src homolog and collagenhomolog
family,inbreastcancercelllinesinhibitedcellproliferationand
promoted apoptosis. The authors also showed that SHCBP1 is
overexpressedin>60%ofbreastprimarytumorsandisassociated
with poorer survival [89]. Thus, suppression of these proteins
mightresult inthe inhibitionoftumorgrowth inpatients and
improvetheirsurvival.
Zhengetal.performedCRISPRscreeningforRNA-binding
pro-teins(RBP)involvedin breasttumorigenesis.They showedthat
PHD finger protein5A (PHF5A) is a key splicing factor and its
knockoutleadstosuppressionofcellproliferation,tumor
forma-tion,andcellmigrationinbreastcancercells.Theyalsoidentified
anelevated expressionlevelofPHF5Ain primarybreast cancer
samplesandshoweditsinversecorrelationwithpatientsurvival
[69].Thisissimilartoarecentreportinprimarylung
adenocarci-noma[90].Interestingly,inhibitionofPHF5Aledtotumorgrowth
suppression in glioblastoma xenograft mice [91]. Thus, PHF5A
might be a suitable therapeutic candidate not only in breast
tumors,butalsoothertypesofcancer.
ncRNAsarealso importantincarcinogenesisand their
dysre-gulationcanleadtocancer.Singhetal.revealedhigherexpression
ofBC200,alongncRNA(lncRNA)thatregulatesproteinsynthesis,
in ER-positivebreastcancerprimarytumors comparedwith
ER-negative ones.Genetic abrogationofBC200 invitro(MCF7 cell
line)andinvivoledtoreducedcellgrowththroughexpressionof
Bcl-x,aproapoptoticprotein[92].Inaddition,ablationofmiR-10b
significantlysuppressedcellmigrationinmetastaticbreastcancer
cells (i.e., MDA-MB-231) [93]. These data suggest that genetic
suppressionofspecificncRNAswitharoleinimportantsignaling
pathways in tumor cells is a good therapeutic option for the
treatmentofbreastcancer.
In general, breast cancer preclinical studies have led to the
identification of several genes and proteins, the inhibition of
whichmightsignificantlyimprovetreatmentoutcomes.
Gliomas
Malignant glioma is the most frequent form of brain primary
tumors.BasedonWHOguidance,itisclassifiedintoastrocytomas,
ependymomas,oligoastrocytomas,andoligodendrogliomas.The
grade of malignancy varies from one subtype to another. For
instance,oligodendrogliomasand oligoastrocytomasare
catego-rizedasgradeIIandIII,respectively,whereasastrocytomas
com-prisefourdifferentgrades(I–IV)[94].AstrocytomasgradeIVare
alsocalledglioblastomamultiforme(GBM),andaccountsfor60–
70%of allmalignant gliomas [94,95].GBM isone ofthe most
heterogeneousandaggressiveformsofbraintumor,withapoor
prognosis [96]. These characteristics might increase chance of
recurrenceinpatientswithGBM.Currently,surgery,
radiothera-py, and chemotherapy are the common treatment options for
GBM[97].However,differentsignaltransductionsand TKIsare
nowbeingevaluatedinclinicaltrials.
Thereareseveralpreclinicalstudiesinvestigatingdifferent
pu-tativetherapeutictargets inglioma.For example,patients with
GBMwithEGFRmutationsandamplificationshavepoorer
prog-nosiscomparedwithpatientswithwild-typeEGFR.Knockoutof
eitherwild-typeormutant(EGFRvIII)EGFRresultedinthe
inhibi-tionoftumorgrowthinhumangliomaU87andLN229cellsas
wellasinmice[98].Thus,completeinhibitionofEGFRcouldbea
promising therapeuticoptionforpatients withGMB. However,
moredetailedstudiesandconfirmationsareneeded.
Targeting key playersofany activatedpathwayin cancers is
predictedtohaveinhibitoryeffectsontumorcells.Basedonthe
pathway,itmightinfluencecellproliferation,invasion,and
mi-grationabilities.STAT3activation,rangingfrom9%to83%,has
beenreportedinpatientswithglioma,probablybecauseof
differ-ences in tumor grade [99,100]. Apositive correlationhas been
shownbetweenSTAT3activation levelsand gliomagrades,
sug-gestingacrucialroleforthisproteinintumorinvasivenessand
possiblydistantmetastasis[101].InducingCRISPR-mediated
loss-of-functionmutationsinSTAT3stronglyinhibitedGBM
tumori-genesisinvivo inmice. Moreover, glioma-initiating cells(GICs)
werehighlyaddictedtoSTAT3andwerenotviableuponSTAT3
depletion[102].Arecentpreclinicalstudyalsoshowedthatthe
AK2/STAT3inhibitorpacritinibstronglyinhibitedpatient-derived
GBMcells[103].Basedontheseresults,inactivationorinhibition
ofSTAT3by eithergenemanipulationorchemicalcompounds,
especially during early disease stages, could be beneficial for
patientswithGBM.
Liuetal.investigatedtheroleofER
b
isoformsintheprogressionofGBMusingCRISPR-mediatedknockoutinpatient-derivedGBM
cells.DisruptionofER
b
increasedmigratoryandinvasiveproper-tiesof the gliomacells. The authorsrevealed tumorsuppressor
activityofER
b
1inGBMcells,whereasERb
5hadmoreoncogeniceffectsanditsrestorationincreasedcellviability.Theyalso
identi-fied significantly higher expression of ER
b
5 in glioma tumorscomparedwithnormalbraintissues,withthehighestexpression
in GBM [104]. Thus, anti-ER
b
5 drugs could be prescribedas atherapeutic strategy in patients with high-grade glioma in the
future.
TheCRISPR/Cas9knockoutsystemhasfrequentlybeenusedto
elucidatethe role ofdifferent proteinsin glioma pathogenesis.
Pengandcolleaguesinvestigatedtheroleofchromatinassembly
factor 1 subunit A (CHAF1A) in glioma cell survival. Loss of
CHAF1Asignificantly increased apoptosisand causedcell cycle
arrestintwoGBMcelllines(U251andU87).Theyalsoobserved
thatexpressionofCHAF1Awasnegativelyassociatedwithoverall
survivalofpatientswithGBM[105],similartoapreviousstudyon
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coloncancer[106].Therefore,suppressionofCHAF1Ainpatients
might prolongtheir overall survival. Disruptionof brain
lipid-bindingprotein(BLBP)resultedintheinhibitionofcell
prolifera-tioninU251GBMcells.Furthermore,higherexpressionofBLBP
wasassociatedwithpoorerclinicaloutcomeinpatientswithGBM
[107].Inanotherstudy,depletionofpolo-likekinase1(PLK1)in
mousemodelbrain tumorsimproved survival and significantly
inhibitedtumorgrowth[108].Interestingly,severalPLK1
inhibi-tors,includingBI-2536,GSK461364,andvolasertib,arecurrently
inclinicaltrialsforthetreatmentofothersolidtumors,butnot
gliomas[109].Inhibitionofallthegenesandproteinsdiscussed
couldbebeneficial forpatients andimprove clinical outcomes.
However, further preclinical and clinical studies are warranted
beforesuchapproachesentertheclinic.
Thereare alsosomereports showingthatloss-of-functionchanges
in several ncRNAs can be apromising treatmentapproach in patients
with GBM. CRISPR-mediated knockout of the lncRNA MANTIS
resultedinan50%reductioninangiogeniccapacityofthe
endo-thelial cells isolated fromGBM and attenuated tube formation.
Thesedatashowedthattheangiogeniccapacityofendothelialcells
isdependentonMANTIS[110],whichcouldbeexploitedtoimpair
angiogenesisintumors.Inaddition,disruptionofmiR-10binGBM
cellsrevealedtheirstrongaddictiontothismiRNA.Itimpairedcell
viabilityand resultedintheupregulationofmiR-10bverifiedtargets,
including cell-cycle inhibitor P21 andthe mediator of apoptosis BIM.
DisruptionofmiR-10bwaslethalbothinvitroandinvivo.
Interest-ingly,miR-10bwasoverexpressedin patients withGBM,which
makesitasuitabletargetingliomas[93].Thus,suppressionofsuch
ncRNAswithoncogenicactivitycouldsignificantlyinhibittumor
growthinpatientswithglioma.
As discussed above, CRISPR-based preclinical studies have
shown promising results in the treatment of gliomas. Several
genes, including those encoding kinases, have been shown to
haveoncogenicrolesinglioma,whichcouldbeexploitedtotarget
tumorcellsinasubsetofpatients.However,furtherclinicalstudies
areneededtovalidatethesecandidatetargets.
Liver
cancer
Primarylivercanceristhesecondcauseofcancer-relateddeathin
theworldand ismorecommoninmen. Primarylivercancer is
dividedintotwomainsubtypes:hepatocellularcarcinoma(HCC)
and intrahepatic cholangiocarcinoma (ICC). HCC and ICC
ac-countfor80%and 15%ofthe totalcases,respectively. The
remaining 5%arethe raresubtypes of livercancer [111].Most
patientswithlivercancerhaveapoorprognosis,frequent
recur-rences, and limited treatment options.Therefore, CRISPR/Cas9
technologycouldbeapromisingapproachtoidentifynew
thera-peutictargetsinthiscancer[112].
CXC chemokine receptor 4 (CXCR4) expression levels have
significantnegativecorrelationwithclinicaloutcomesinpatients
withHCC[113].CRISPR-mediatedablationofCXCR4inHepG2
cellsresultedinsuppressedcellproliferationandmigration.
More-over,CXCR4attenuationresultedinasmallertumorsizeinvivo
[114].Thus,CXCR4hasanoncogenicroleinHCCandcouldbe
usedasatreatmenttargettocontrolgrowthordistantmetastasis.
SeveralotherpotentialtargetsinHCChavebeeninvestigatedby
using CRISPR/Cas9. For example, simultaneous CRISPR-based
knockoutofPtenandoverexpressionofNrasledtoHCCformation
invivo,whereassinglegenemanipulationdidnotresultin
tumor-igenesis.AberrationintheexpressionofPtenorNrasisnecessary
forlivertumorigenesis,butitisnotsufficientifthesechangesare
notpresentsimultaneously [115].Therefore,inhibitionof
over-expressedNRASinpatientswithPTENlossmightsuppresstumor
growth andimprove patientsurvivala subsetofHCCs.Nuclear
receptorcoactivator5(NCOA5)encodesacoregulatorforERsand
is dysregulated in several typesof cancer. Geneticknockout of
NCOA5significantlyinhibitedcellgrowthandmigrationinHCC
cells. Inaddition, lossofthe NCOA5proteinsubstantially
sup-pressedEMT,whichisacommondrugresistancemechanismin
different cancer types [116]. Nogo-B is a negative regulator of
apoptosis[117] and itsCRISPR-baseddeletionled tosignificant
inhibitionof cellproliferation invitroaswell assuppressionof
tumorgrowthanddistantmetastasisinvivo[118].Allthe
afore-mentionedgenescouldbeconsideredasappropriatetherapeutic
candidatesforfurtherinvivostudiesorclinicaltrialsasinhibitors
aredeveloped.
HepatitisBvirus(HBV)isoneofthepathogensforHCC,and
HBVinfectioncanleadtoHCC.Oneofthepotentialtherapeutic
applicationsofCRISR/Cas9systemistopreventHBV-derivedliver
cancer by destroyingviral DNA. HBV particles containrelaxed
circular DNA (rcDNA), whichis converted to covalentlyclosed
circularDNA(cccDNA)uponenteringhepatocytes.cccDNAserves
astemplateforallHBVtranscripts.Thus,eliminationofcccDNA,
for instance by cleaving, could cure HBV. Several preclinical
studiestargetingHBVhaveshown promisingresultsthatmight
bebeneficialtoeradicateHBVasoneofthemainriskfactorsfor
liver cancer. Lin and colleagues used CRISPR/Cas9 technology
againstHBV.UsingagRNAtargetingaconservedHBVgenomic
region,productionofEBVsurfaceandcoreproteinswas
success-fully suppressedbothin vitroand invivo. Theauthors also
effi-ciently reduced rcDNA and cccDNA levels in the cells [119].
AnotherresearchgroupshowedthatHBV-specificgRNA
combina-tionsreducedHBVDNAandcccDNAupto1000-andtenfoldin
vitro,respectively[120].Lietal.usedtheCRISPR/Cas9systemto
successfully eradicate HBV infection from HBV-positive HepG2
cellsbydeletingtheentireintegratedHBVgenomeanddisruption
of cccDNA [121].Similarresults werereported by othergroups
[122–124].Inaddition,CRISPR-basedablation offlap
structure-specificendonuclease1 (FEN1)in Hep38.7-Tet,anin vitroHBV
model,resultedinreducedcccDNAlevels,suggestingacrucialrole
forthehostFEN1proteinincccDNAproduction[125].
Colorectal
cancer
Colorectalcancerisoneofthemostfrequenttypesofcancerand
thefourthcauseofcancer-relateddeathworldwide[126].
Adeno-carcinoma(ADC)accountsfor>90%ofallcolorectalcarcinomas.
Theremaining10%areraresubtypes,includingadenosquamous,
squamouscell,neuroendocrine,spindlecell,andundifferentiated
carcinomas.Approximately 95%ofcolorectalcasesaresporadic
and5%areinherited.Colorectaltumorscanbetriggeredbydriver
mutationsinwell-knowncancergenes,suchasKRASandBRAF,in
40%and10%ofpatients,respectively.Treatmentofpatientsis
based onhistology and moleculartesting, but,similar toother
cancers, tumorseventuallybecomeresistanttotreatment[127].
Therefore, overcoming resistance by the identification of new
therapeutictargetsiscrucial.
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Yauetal.usedgenome-wideCRISPR/Cas9screeningofKRASmut
andKRASwtcolorectalcancerxenograftstoidentifygenesthat
pro-moteorsuppresstumorgrowthinvivo.Theyshowedthatdisruption
ofNADkinase(NADK)orketohexokinase(KHK)significantly
inhib-itedKRASmuttumorxenograftscomparedwithKRASwtxenografts,
suggesting them aspotential therapeutic targets in KRAS-driven
colorectaltumors [128].SeveralNADKandNHKinhibitorshave
beensynthesized[129,130].However,preclinicalandclinicalstudies
areneededtoevaluatetheirefficacyandtoxicityinhumancells.
Caspase-3(CASP3)isinvolvedinapoptosisanditshigher
activ-ityisassociatedwithahigherriskoftumorrelapseincoloncancer
[131].Itisoverexpressedincolorectalcancerprimarytissuesand
itshigherexpressionissignificantlyassociatedwithshorteroverall
survival [132]. CRISPR-mediatedknockout of CASP3 in a colon
cancercellline(HCT116)significantlyattenuatedcell
tumorigen-esis, invasion, and migration. Interestingly, deletion of CASP3
resultedinthehighersensitivityofthecellstoradiotherapyboth
invitroandinvivo[133].Together,thesedatasuggestthat
inhibi-tionofcaspase-3could beusedtoimprove clinicaloutcome in
patientswithcoloncancer.
NAT1 is a member of the N-acetyltransferases family and is
associated with cancer cell proliferation [134]. In addition,
NAT1canprotectcoloncancercellsduringnutrientdeprivation,
anditsabsenceislethal[135,136].GeneticablationofNAT1in
HT-29coloncancercellssuppressedcellgrowthandpromoted
apo-ptosisunderglucosestarvationbyenhancedproductionof
reac-tive oxygen species (ROS) [134]. Thus, inhibition of NAT1 in
combinationwithspecificdietscouldbeaneffectivemethodto
treatcoloncancer.AstudybyNishietal.revealedthatproduction
ofROSsignificantlyreducedexpressionofaproteincalled
NHL-repeat-containing protein 2 (NHLRC2), resulting in increased
levelsof apoptosisin HCT116colon cancercells. They showed
thatgeneticallyengineeredNHLRC2/cellsweremore
suscepti-bletoROS-inducedapoptosis[137],whichmighthavepotential
therapeuticbenefitsforpatientswithcolorectalcancer.
Dysregulationinautophagyisknowntobeinvolvedincancer
pathogenesis [138]. For instance, MARCH2 is involved in the
regulationofautophagy[139].CRISPR/Cas9-baseddisruptionof
MARCH2attenuatedcellproliferation,whilepromotingapoptosis
andcellcyclearrestincoloncancercellsbothinvitroandinvivo.
Furtherinvestigationsonprimarytumorsamplesrevealedhigher
expressionofMARCH2comparedwithnormaltissue.Moreover,
higherexpressionofthisproteinwassignificantlyassociatedwith
pooreroverallsurvivalinpatients[140].Therefore,eithergenetic
disruption or chemical inhibition of MARCH2 could improve
clinicaloutcomesinasubsetofpatientswithcoloncancer.
ApplicationofCRISPR/Cas9genome-editingtechnologyhasled
totheidentificationofseveralothercandidatetargetsincolorectal
cancer.Forexample,deletionoftheRNA-bindingproteinELAVL1
(HuR)incolorectalcancercellsresultedinasignificantreductionin
tumorgrowthcomparedwiththecontrolgroupinvivo.Theauthors
alsorevealedthatELAVL1issubstantiallyoverexpressedincolon
primarytumors,whichmakesita suitabletherapeuticcandidate
[141].Wuetal.knockedoutRBX2,acomponentofE3ubiquitin
ligases, in two colon cancer cell linesto investigate its role in
tumorigenesis.RBX2/cellsshowedsignificantlyreducedcolony
formationandmigrationcapacityinvitro.Inaddition,abrogationof
RBX2significantlyreducedtumorsizeandlungmetastasisinvivo.
Furtherinvestigationsusingcolorectalprimarytumorsrevealeda
higher expressionofthis proteincompared withnormal tissues
[142].Thus,RBX2appearstobeasuitabletargetinpatientswith
overexpressedRBX2toprevent,oratleastdelay,distantmetastasis,
whichisthemaincauseofdeathinallcancertypes.
Similartoothertypesofcancer,ncRNAshaveanimportantrolein
colontumorigenesis.Ithasbeendemonstratedthatasmall
nucleo-larRNA,called SNORA21,hasanoncogenicroleincolorectalcancer.
CRISPR-basedablationofSNORA21resultedinsubstantial
suppres-sionofcellproliferation, invasion,andtumorgrowthcapacities
bothinvitroandinvivo.Theauthorsalsorevealedasignificantly
higherexpressionofSNORA21inprimarycolontumorscompared
withnormal tissues. In addition, they showed that higherexpression
ofSNORA21wasnegativelycorrelatedwithoverallsurvival,disease
stage and distance metastasis [143].Thus,SNORA21could bea
potentialtherapeutictargetanditsinhibitioncouldbebeneficial
forpatientswithhigh-stagecolorectaltumors.
CRISPR/Cas9
delivery
to
cancer
cells
Deliveryofnucleases,suchasCas9,ZFN,andTALENs,remainsone
ofthemostsubstantialchallengestotheuseofsuchtherapyinthe
clinic.WithrapiddevelopmentsofCRISPR-basedtechniques,itis
crucialtodevelopmoreefficient,precise, and accuratedelivery
methods.Allnucleases,includingthemeganucleases,Cas9,ZFN,
andTALENs,arebiomacromoleculesandhavesimilarchallenges
totheirdeliveryintohumancells.Despitedifferentcomponents
andcharacteristics,allnucleasescanbegenerallydeliveredinthe
format of DNA, mRNA, or protein. The only difference in the
CRISPR/Cas9systemisthatthegRNAshouldbedeliveredasDNA
orRNAmoleculescomparedwiththeothernucleases[144–146].
Therearetwotypesofgenedeliverysystem:viralandnonviral
vectors.Forbasicresearch,forinstance,theidentificationofgene
functionand identificationofnoveltherapeutictargets,diverse
categories of viral or nonviral delivery methods can be used.
Recently,nonviraldeliverytechniques,suchaselectroporation,
hydrodynamic injection, microinjection, and self-assembled
nanoparticles (NPs), were used in ex vivo gene-editing using
nucleases[147,148].Yet,exvivogeneeditingwithNPsand
inte-gration ofa viral system cannot be used in most tissues types
becauseoflowefficiencyandsafetyconcerns[149].
Viralvectorsaremoreeffectiveandattractiveforthedeliveryof
CRISPR/Cas9invivo. Comparedwithtraditional non-integrated
viral-basedgenetherapy,inwhichcontinuoustransgene
expres-sionisneededbyrepeateddose,permanentgenomeeditingcanbe
achieved by transient expression of CRISPR/Cas9 with a single
administration.GiventhatCRISPR/Cas9hasshownhighlevelsof
efficiency, specificity, and stability, requirements of the viral
vectors for delivery of CRISPR/Cas9 might not be as strict as
previously required. The preferred viruses for the delivery of
nucleasesare AdVs, integrase-defective lentiviruses (IDLV), and
adeno-associatedviruses (AAVs)[150–152].Here,we providean
overviewof appropriate viraland nonviralvectors usedforthe
deliveryofnucleasesincancercells.
Viral
vectors
AdVsandAAVvectors
AdVandAAVvectorshavebeenextensivelystudiedforthedelivery
ofdifferentnucleasesinvitro,exvivo,andinvivobecauseoftheirhigh
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titer,relativelymildimmuneresponse,abroadrangeofcell
infec-tivity,andsafety.OnestudyshowedthatZFNscanbeexpressed
moreefficientlyusingAdVvectorscomparedwithIDLVin
geneti-callyengineeredprimaryTcells[153].Moreimportantly,modified
AdVvectorsshowedmoreaccuracyandspecificitythanIDLVfor
donorDNAdelivery[153].However,hostinflammatoryresponses,
includingcytotoxicTcells,limitsAdVvectorapplication[154].The
other drawback is the expression level of coxsackie-adenovirus
receptor(CAR)onthetumorcell surface,whichdeterminesthe
efficiencyofdeliveryusingAdVvectors.TheCARexpressionlevel
has a negative correlation with tumor grade.Thus, AdVis not
efficientforthedeliveryofnucleasesdirectlytohigh-gradetumors.
RecombinantAAVs(rAAVs)arealsoattractiveoptionsforthe
deliveryofnucleases.Theyarecommonlyusedinthedeliveryof
ZFNs,TALENs,andCRISPR/Cas9,becauseAAVvectorshavethe
abilitytoavoidrandomintegrationandshowlowimmunogenic
characteristics [152,155–158]. The low packaging capacity
(4.7kbp)isthemainlimitationofAAVvectors.Therefore,the
recombinantsequenceneedstobecarefullydesignedtomatchthe
AAVcapacity.
LVandIDLVvectors
Self-inactivating lentiviral vectors (LVs) are commonly used as
vehicles for gene therapy in dividing and nondividing cells
[159–161]. Afterdecades ofpreclinical and clinical testing,LVs
arebeingused totreat a variety ofgeneticdiseases, suchas
X-linked adrenoleukodystophy and Wiskott–Aldrich syndrome
[160,162–164]. Currently, several studies have shown that LVs
canbeusedtoefficientlydeliverCRISPR/Cas9tomammaliancells
and mice for cancer gene therapy [165–167]. However, unlike
usingLVsforgenereplacementtherapy,thecontinuous
expres-sionofCas9isunnecessaryandmightbeaproblem.
ComparedwithintegratingLVs,IDLVsminimizeproviral
inte-grationbecauseofmutationsintheintegraseprotein,whichmake
itmoresuitableforthedeliveryofCRISPR/Cas9[151,166].IDLVs
havebeensuccessfullyusedtodeliverCRISPR/Cas9anditsdonor
template in vitro. Approximately 6–11% gene knock-in was
achieved in hematopoietic stem and progenitor cells (HSPCs)
[168].IDLVshavealsobeenusedtocodeliverZFNs/TALENsand
HDRdonortemplatesintoavarietyofcelltypes,including
prima-ryandstemcells,witharelativelyhighefficiency(>20%).
How-ever, IDLV-mediated gene editing can cause undesired gene
modifications,suchasepigeneticsilencingandtheinductionof
genomicrearrangements,particularlyinrepetitivesequenceloci.
AnotherdisadvantageisthatIDLV-mediatedCas9deliveryleadsto
ahigheroff-targeteffectinquiescentorslowlydividingcells,such
ashepatocytesandneurons[169].
Nonviral
vectors
Nonviralvectorsarebeingrapidlydevelopedandseveralnonviral
vectors,includinglipid-basedvectorsandpolymer-basedvectors,
arewidelyusedin genetherapy [170].Unlikeviralvectors,the
capacityofnonviralvectorsisflexible.Inaddition,itappearsthat
many disadvantages of viral vectors, such as mutagenesis and
immunogenicity, can be addressed by using nonviral vectors
[170–172].Lowerimmunityorabsenceofpre-existingimmunity
inpatientsisanimportantadvantageofnonviralvectorsoverviral
vectors. However, compared with viral vectors, some nonviral
vectors are not able to penetrate cells efficiently, resulting in
low levelsof gene transfer efficiencyand highin vivo toxicity,
whichcouldlimittheiruseofinclinicaltrials[16].
Inrecentyears,nonviralvectorshave beenused forCRISPR/
Cas9deliveryinvitroandinvivo.Forinstance,onestudyshowed
thatupto80%genemodificationcanbeachievedinvitrousing
cationic liposomes. In addition, cationic liposomes have been
successfullyusedtodeliverCRISP/Cas9totheinnerearinmouse
models[172].Anotherstudysuccessfullyusedpolyethylenimine
(PEI)forthedeliveryofCRISPR/Cas9tomousebrain[173].
Combined
viral
and
nonviral
delivery
Arecentstudyshowedthatthecombineduseofviralandnonviral
vectors(lipid-basedvectorsandAAVs)achieved>6%generepair
(HDR)inhepaticcells[174].Theyalsoappliedthisapproachtoa
mousemodelandsuccessfullyrepairedthedisease-causinggene.
This method might be an efficient option for the delivery of
CRISPR/Cas9topatients.
Tissue-
or
tumor-specific
delivery
of
CRISPR/Cas9
SeveralstudieshaveshownthatCRISPR/Cas9iscapableofediting
geneswithhighaccuracyand efficiency[19,175,176].However,
forcancergenetherapy,howtospecificallydeliverCRISPR/Cas9to
cancercellsisstillamajorobstacle.Herein,webrieflyexplainsome
ofthesedeliverymethods,includingtheuseofappropriateviral
serotypes,specificpromoters,andbispecificconjugates.
More than 200 AAV serotypes have been identified so far.
Packaging CRISPR/Cas9 into the appropriate AAV serotype for
tissue-specific delivery is a promising strategy for cancer gene
therapy. Several rAAV vectorshave been developedfor clinical
trials and, recently, the firstgene therapy for treatment of an
inheritedretinaldiseaseusingAAVwasapprovedbytheUSFood
and Drug Administration(FDA) [177]. One study showed that
CRISPR/Cas9 deliveryusing AAVserotype9 (AAV9)was highly
efficientfortissue-specificdeliveryanddidnotcausesubstantial
cellulardamageinvivo[178].
Utilizingapromoter-based‘AND’logicgatecancontrolCRISPR/
Cas9tobespecificallyexpressedinbladdercancercells[179].The
strategyofpromoter-basedlogicgates,including‘AND’,‘OR’,and
‘NOT’, have the potential to create more complex and precise
regulatorypatternsthatcanbeconditionallyexpressed.Delivery
ofCRISPR/Cas9totumorcellswithsuchprecisioncouldaccelerate
theuseofCRISPR-basedcancergenetherapyfrombenchtobedside.
LowexpressionofCARonthe tumorcellsurfaceand lackof
specificreceptorssignificantlylimitstheclinicaluseofAdVvectors
in cancer gene therapy. However, redirecting AdV vectors to
tumor-specificcellsurfacetargetreceptors,suchasEGFR,EpCAM,
CD40, PDGF-Rbeta, and VEGFR, can selectively target tumors,
whichmayimprovethespecificityandefficiencyofAdV
simulta-neously[180–183].
CRISPR/Cas9;
hopes
and
challenges
in
cancer
treatment
CRISPR/Cas9 genome-editing technologyhas shown promising
results in preclinical studies. It is a convenient tool that has
enabled researchers to perform gene manipulation at a single
base-pairresolutioninarelativelyefficientway.Preclinicalstudies
have led to the identification of several potential therapeutic
targetsbyusingCRISPR-basedmethods(Table3).Thishasbrought
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TABLE3
Listof(potential) therapeutictargetsindifferentsolidtumorsidentifiedbyusingCRISPR/Cas9genome-editingtechnologiesa
Target invitro Cellline invivo CRISPR Vector Effect Refs
Lungcancer
EGFR + H1975 + Knockoutofmutantallele
(L858R)
AdV Promotedcelldeathandreducedtumor size
[4]
EGFR + H1975 + Knockoutofmutantallele
(L858R)
LV Reducedcellproliferationandtumorsize [43]
KRAS + A549andKP-KrasG12D/1 (mouselungcancercells)
+ Bothallelesknockout LV Reducedcellproliferation [44]
CD38 + A549 + Bothallelesknockout Nonviral Inhibitedcellgrowthandinvasion [48]
FAK + H460 + Bothallelesknockout Nonviral Reducedcellproliferationandtumorsize [50]
TAZandYAP + A549 + Bothallelesknockout LV Dualknockoutinhibitedcellproliferation, cancerstemcellsphereformation,and tumorformation
[54]
MUC1 + A549andH460 Bothallelesknockout LV SuppressedMYCexpressionandinhibited cellgrowth
[55]
Tnc + Collectionofcelllines isolatedfrommicewith primarylungtumors(KP mice)
+ CRISPR-mediatedgene activation
LV Enhancedmetastaticpotentialofcells [61]
Smarca4 – + Bothallelesknockout LV Inducedtumordevelopment,but
attenuateddiseaseprogressioninvivoover time [34] mir-323b,mir-487a andmir-539 + H2009 CRISPR-mediatedgene activation
LV Promotedcellmigrationandinvasion [68]
Breastcancer
Mlk3 + 4T1(murinebreast cancercells)
+ Bothallelesknockout Nonviral Suppressedcellinvasionandmigration [72]
CX3CR1 + MDA-MB-231 + CRISPR-basedtranscriptional suppression(CRISPR interference)
LV Impairedlodgingofcancercellstomouse boneandreducednumberofcancerous lesionsineachanimal
[73]
Ubr5 + 4T1(murinebreast cancercells)
+ Bothallelesknockout Nonviral Promotedapoptosisandinhibitedtumor growthanddistantmetastasis
[74]
CXCR2 + MDA-MB-231 + Bothallelesknockout Nonviral Inhibitedcellproliferation,migration, tumorsize,andrateoflungmetastasis
[75]
MARK4 + MDA-MB-231 Bothallelesknockout Nonviral Inhibitedcellproliferationandmigration [79]
FERMT2 + MDA-MB-231and4T1 (murinebreastcancer cells)
+ Bothallelesknockout LV Inhibitedcellmigration,invasion,and tumorgrowth
[80]
MASTL + MDA-MB-231,MCF7, BT549,andBT-483
+ Bothallelesknockout LV Impairedcellproliferationandtumor growth
[83]
PTPN23andFYN + Cal-51 + Bothallelesknockout LV Dualknockoutinhibitedcellproliferation andtumorgrowth
[84]
CDK7 + MDA-MB-231,
MDA-MB-468,HCC38,BT549,and SUM149
+ Bothallelesknockout LV Inhibitedcellproliferationandimpaired cellviability
[87]
SHCBP1 + MDA-MB-231andMCF7 Bothallelesknockout LV Inhibitedcellproliferationandpromoted apoptosis
[89]
PHF5A + CA1aandDCIS + CRISPRscreentargeting RNA-bindingproteins
LV Suppressedcellproliferation,migration, andtumorformation
[69]
lncBC200 + MCF7 + Bothallelesknockout Nonviral Inhibitedcellproliferationandtumor growth
[92]
miR-10b + MDA-MB-231 Bothallelesknockout LV Suppressedcellmigration [93]
Glioma
EGFR + U87andLN229 + Knockoutofbothwild-type andmutant(EGFRvIII)EGFR
LV Inhibitedcellproliferationandtumor growthandimprovedsurvival
[98] Reviews FOUNDA TION REVIEW
hopeforthetherapeuticapplicationofthistechnologyincancer
treatment.However,therearesomeconcernsregardingits
appli-cationintheclinicalsetting.Here,wehavemainlydiscussedgenes
withoncogenicactivitiesbecausegeneknockoutismore
conve-nientcomparedwithgeneknock-inusingCRISPR/Cas9(Fig.2).In
addition,oncogenesareusuallyoverexpressedinprimarytumors
andaremoreamendabletopharmaceuticallyinhibition.
Althoughmalignanttumorsaregeneticallyheterogeneous,
tu-morbulkismainlytheresultofoutgrowthofoneortwodominant
clones[184]causedbydrivermutationsincertaingenes.sgRNAscan
distinguishbetweenmutantandwild-typeallelesintumorcells,
reducing off-target effects and improving the specificity [185].
Therefore,hotspotdrivermutationsingenessuchasEGFR,KRAS,
BAP1,BRAF,BRCA1,andBRCA2canbeexploitedasatherapeutic
TABLE3(Continued)
Target invitro Cellline invivo CRISPR Vector Effect Refs
STAT3 + MT330 + Bothallelesknockout LV Inhibitedtumorigenesisinvivo,butlimited effectoncellproliferationinvitro
[102]
ERb + U87andU251 + BothallelesknockoutofER
b
followedbyreintroduction ofERb
1andERb
5 separatelyNonviral/LV ER
b
knockoutelevatedmigratoryand invasiveproperties.Overexpressionof ERb
5enhancedcellmigrationand invasion[104]
CHAF1A + U87andU251 Bothallelesknockout LV Inhibitedcellproliferation,increased apoptosis,andcausedcellcyclearrest
[105]
BLBP + U251 Bothallelesknockout LV Inhibitedcellproliferationandcausedcell cyclearrest
[107]
PLK1 NA NA + Bothallelesknockout Nonviral Inhibitedtumorgrowthandimproved survival
[108]
lncRNAMANTIS Exvivo Endothelialcellsisolated fromGBM
+ Bothallelesknockout Nonviral Reducedangiogeniccapacityofcellsand suppressedtubeformation
[110]
miR-10b + U251,LN229,andA172 + Bothallelesknockout LV Impairedcellviability,enhancedexpression ofmiR-10btargets,andsuppressedtumor growth
[93]
Livercancer
CXCR4 + HepG2 + Bothallelesknockout Nonviral Inhibitedcellproliferationandmigration andreducedtumorsize
[114]
PtenandNras – + Bothallelesknockoutand
CRISPR-mediatedgene activation
Nonviral SimultaneousknockoutofPtenand activationofNrasledtolivertumorigenesis
[115]
NCOA5 + LM3 Bothallelesknockout LV Inhibitedcellproliferationandmigration, suppressedtumorformationandEMT
[116]
Nogo-B + SMMC-7721and QGY-7703
+ Bothallelesknockout Nonviral Inhibitedcellproliferationandsuppressed tumorgrowthanddistantmetastasis
[118]
Colorectalcancer
NADKandNHK + HCT116KRASWT/, HCT116KRASWT/mut, DLD-1KRASWT/,and DLD-1KRASWT/mut
+ Genome-wideCRISPR screeningknockout
LV InhibitedKRASmuttumorgrowth [128]
CASP3 + HCT116 + Bothallelesknockout NA SuppressedEMTandattenuatedcell tumorigenic,invasion,andmigration abilities
[133]
NAT1 + HT-29 Bothallelesknockout Nonviral Inhibitedcellgrowthandpromoted apoptosisunderglucosestarvation
[134]
NHLRC2 + HCT116 Bothallelesknockout LV Inhibitedcellproliferationandpromoted apoptosis
[137]
MARCH2 + HCT116 + Bothallelesknockout NA Inhibitedcellproliferation,promoted apoptosisandcellcyclearrest,reduced tumorsize
[140]
ELAVL1 + HCT116 + Bothallelesknockout Nonviral Suppressedtumorgrowth [141]
RBX2 + HCT116andSW480 + Bothallelesknockout Nonviral Inhibitedcolonyformationandcell migrationcapacity,reducedtumorsizeand lungmetastasis
[142]
SNORA21 + HCT116andSW48 + Bothallelesknockout LV Suppressedcellproliferation,invasionand tumorgrowth
[143]
a
Abbreviation:NA:notavailable.
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approachatleastinasubsetofpatients.Oneofthemainadvantages
of this strategyis thatnormal cells,do notcontain themutant alleles,
arenottargetedand,thus,remainintact.Inaddition,mutationsin
oncogenessuchasKRASareindicatorsofdrugresistanceandpoor
prognosisinasubgroupofpatientswithNSCLC,includingAsiansor
womenwithlow-gradetumors[186].Bycontrast,CRISPR/Cas9can
preciselytargetaspecificlocusinthegenome.Therefore,
substitut-ingKRASmutantversions(p.G12Vandp.G12D)withthewild-type
allelecouldpositivelyimprovetreatmentresponsein
KRAS-depen-dentcancers.
SeriesofCRISPR-associatednucleaseshavebeendiscoveredover
the past few years that can greatly facilitate genome editing.
However, there are still some major problems that need to be
solved before CRISPR/Cas9 enters the clinic, such as off-target
effects[187],continuousactivityofCas9[187],lowefficiencyof
current delivery systems [187], low efficiency of
CRISPR/Cas9-mediated gene knock-in [168], pre-existing adaptive immunity
[187],anduncontrollableDNArepair [187].Inaddition,recent
studieshaveshownthatp53caninhibitCRISPR/Cas9gene-editing
efficiency [188,189]. According to these studies, CRISPR/Cas9
tendstotargetcellswithintactp53,leavingbehindp53-deficient
cells,whichhavethepotentialtobecomecancerous.Thus,
evalu-ationofp53proteinbeforeandaftergeneeditingforthetreatment
ofthepatientsiscrucial.However,CRISPR-inducedp53activation
appearstooccuronlyduringgeneeditingbyHDR.Thebaseeditors
probably willnottriggerp53 and,thus,aresafer inthis regard
comparedwithCRISPR/Cas9technology.
Selectionoftherighttargetisanimportantsteptowards
devel-opinganewtreatmentstrategy.Acandidateproteinand/or
mole-culecannotbeconsideredasanappropriatetargetbasedonlyon
itshigherexpressioninprimarytumortissues.Extensive
function-alinvitroandinvivostudiesarerequiredbeforeproceedingtoa
clinicaltrial.Forexample,FOXC,atranscriptionfactor,is
consid-eredasprognosticbiomarkerinbreastcancerandhasbeen
sug-gestedasatherapeutictarget[190,191].However,arecentstudyby
Mott and colleagues showed no difference in tumor size and
metastasis between FOXC1/ and parental tumor cells in vivo
[192].Similarly,onerecentthought-provokingstudyshowedthat
maternalembryonicleucinezipperkinase(MELK)isnotacancer
target,whereasmultipleongoingclinicaltrialsaretryingtoinhibit
MELKtotreatcancer[193].Theseresultshighlighttheimportance
ofinvivostudiesusingCRISPR/Cas9inthevalidationof
biomark-ersandtherapeutictargetsandtheirreliability.
Overall,therearefourstepstobringaproteinormoleculeasa
therapeuticapproachfromthebenchtotheclinic:(i)
identifica-tionofkeymoleculesindifferentdiseases;(ii)validationofthose
moleculesbyinvitroandinvivostudies;(iii)developmentofan
efficientmethodto inhibitthatspecificmolecule;and (iv)
suc-cessfulPhaseI–III clinicaltrials.If anyofthesesteps fails,new
therapeuticapproacheswillnotbeabletoenterintoclinics.
Thelowrateofall-allelesknockoutin cancercellsisanother
important challengethat has to betaken intoaccount. This is
causedbythehighamountofaneuploidyincancercells,which
can result in an unpredictable outcome [194]. Therefore, the
application of multiple gRNAs for a certain gene can increase
thechanceofall-allelesknockout incancercells.However,this
strategymightleadtoasubpopulationofcellswithactivealleles
resultingfromanin-framerepairofDSBinthetargetsiteinduced
by several gRNAs. In addition, not all gRNAs have the same
efficiency;somearemoreactivethanothers.Onesolutionisto
TP53 TP53 TP53 TP53 TP53 APC KRAS TGFBR2 CDH1 GATA3 ESR1 TERT TERT ARID1A ARID2 IDH1 ERBB2 PTEN PTEN ATRX H3F3A CDKN2A CDKN2A CTNNB1 CIC EGFR ARID1A TBX3 EGFR LRP1B LRP1B LRP1B SMAD4 AXIN1 KRAS KEAP1 FAT4 FAT4 FAT4 KMT2C KMT2C KMT2C CDKN2A FAT1 STK11 PIK3CA PIK3CA ACVR2A BRAF PIK3CA Disruption Knockout Knock-in Repair
Activating mutations Loss-of-function mutations
38% 27% 27% 27% 20% 20% 27% 34% 40% 15% 15% 13% 13% 27% 21% 16% 11% 11% 11% 10% 10% 10% 8% 8% 8% 8% 8% 8% 7% 7% 7% 4% 4% 4% 4% 12% 14% 15% 13% 17% 18% 20% 31% 48% 50% 4% 4% 5% 6% 6%
Drug Discovery Today
FIGURE2
Potentialtherapeuticclusteredregularlyinterspacedshortpalindromicrepeats(CRISPR)-mediatedtargetingstrategiesforcancergenetherapy.Thepriority optionforcancergenetherapyisthedisruptionofoncogenesratherthanrepairinginactivatedtumorsuppressorgenes.Thebarchartsshowthetop-ten mutatedgenesindifferentcancersbasedontheCOSMICdatabase(v87).Mutationfrequencieshavebeencalculatedusingaweightedaveragemutation frequencybasedonsamplesizeacrossallstudies.ThisfigurecontainsmodifiedelementsfromServierMedicalArt(http://smart.servier.com).
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