Crispr and cancer

Mini-review

Application of CRISPR-mediated genome engineering in cancerresearchVolkan I. Sayin, Thales Papagiannakopoulos *Department of Pathology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA

A R T I C L E I N F O

Keywords:CRISPRCas9Genome engineeringCancerMouse models

A B S T R A C T

Cancer is a multistep process that arises from a series of genetic and epigenetic events. With recent tech-nological advances there has been a burst in genome sequencing and epigenetic studies revealing a plethoraof alterations that may contribute to cancer. However, the great challenge for the cancer research com-munity is the systematic functional characterization of these genetic and epigenetic events to assess theirrole in cancer initiation and progression. Recent advances in genome engineering using CRISPR/Cas9, anancient bacterial immune-like system, have revolutionized cancer genetics. Here we highlight the break-throughs in the effective use of these novel genome-editing techniques, and we discuss the challengesand potential applications of these tools for cancer biology.

© 2016 Elsevier Ireland Ltd. All rights reserved.

Introduction

Cancer is a genetically complex disease that can arise from a se-quence of genetic and epigenetic alterations that drive cellulartransformation, tumor growth, invasion and metastasis by regu-lating various hallmarks of tumorigenesis [1,2]. Genome sequencingstudies from multiple cancer types have revealed a myriad of pointmutations, copy number alterations, and chromosomal rearrange-ments [3–6]. Epigenetic differences at the level of both DNAmethylation and histone marks present an additional layer of com-plexity [4–7]. These altered genes can play a functional “driver” ornon-functional “passenger” role during tumorigenesis. Despiteseveral improved in silico methods for the identification of puta-tive “driver” events [8], the functional characterization of thesegenetically or epigenetically altered genes remains elusive.

Traditional approaches to functionally characterize cancergenes

Historically, researchers have employed various in vitro and invivo based approaches to functionally validate cancer genes, in-cluding primary cells [9–11], established cancer cell lines [12] andgenetically engineered mouse models (GEMMs) [13–15]. Using thesemodel systems, researchers typically disrupt gene function to de-termine the effects on hallmarks of cancer. A cancer researcher’stoolbox has consisted of methods to alter gene expression, such asRNA-interference (RNAi) for loss-of-function (LOF) or overexpressionof cDNA for gain-of-function (GOF) of genes of interest [13,16,17].

Genome editing in mouse models and human cells has led tomany important discoveries, but these traditional approaches havebeen technically challenging and time consuming [18–22]. Since theoriginal breakthrough of gene targeting by homologous recombi-nation in mouse embryonic stem cells in the mid-1980s [23–25],little progress had been made to improve gene-targeting methodsuntil the emergence of novel genome-engineering technologiesalmost two decades later. These approaches capitalize on the factthat double-strand breaks (DSB) at a locus of interest can improvegene-targeting efficiency [25–28]. By engineering and repurpos-ing proteins that have evolved to recognize specific DNA sequences,researchers initially developed programmable nucleases such as zinc-finger nucleases (ZFNs) and transcription-activator-like effectornucleases (TALENs) to induce DSB at loci of interest [29–34]. Al-though these programmable nucleases have been used with somesuccess [29,30,33,34], they can be costly, difficult to construct andhave variable targeting efficiency.

CRISPR/Cas9 system

While multiple efforts were being undertaken in genome engi-neering, microbiology researchers studying bacteria and archaea wereuncovering an ancient form of adaptive immunity termed clus-tered regularly-interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9 system [35–37]. Cas9 is a nucleasethat forms a complex with a single guide RNA (sgRNA) that recog-nizes a 20-nucleotide complementary genomic sequence containinga downstream protospacer-adjacent motif (PAM) (Fig. 1). Cas9enzymes from different bacterial species have evolved to recog-nize different PAM sequences. The most commonly used of theseis the Streptococcus pyogenes Cas9 (spCas9), which recognizes an NGG

* Corresponding author. Tel.: 646 501 0042; fax: 646 501 0226.E-mail address: [email protected] (T. Papagiannakopoulos).

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PAM motif with weaker affinity to NAG PAM [36,38–40]. This Cas9is predominantly discussed in this review.

Guided by sgRNAs, Cas9 cuts DNA to induce a DSB approximate-ly three nucleotides upstream of the PAM sequence. The Cas9-induced DSB will activate DNA repair machinery to repair the DSBby either the error-prone non-homologous end-joining (NHEJ)pathway that gives rise to insertions or deletions (indels) or thehomologous-directed repair (HDR) pathway, which can lead to preciseDNA modification in the presence of a donor DNA template (Fig. 1).

Use of the CRISPR/Cas9 system in mammalian cells has openedup a new era in genome engineering by providing a rapid, effi-cient and inexpensive method to characterize gene function. Herewe provide an overview of the breakthroughs in both in vitro and

in vivo modeling of cancer using CRISPR/Cas9 genome engineer-ing technologies.

In vitro modeling of genetic variants using CRISPR/Cas9

Modeling LOF or GOF of cancer genes in vitro has mostly beenaccomplished by use of RNA interference or cDNA expression, whichhas led to many important discoveries [14,41]. However, there aremany caveats to these approaches that can be overcome using theCRISPR/Cas9 system. Knockdown of mRNA levels of a gene by RNAiis incomplete and therefore, the levels of the remaining mRNA maystill play a functional role. In addition, despite recent improve-ments in the efficiency and specificity of RNAi [16,42–44], there are

Fig. 1. CRISPR/Cas9-based genome engineering. In the bottom, CRISPR-based approaches enable gene editing at a locus of interest. sgRNA guided Cas9 can be directed to acoding exon of a gene of interest. Once localized to a specific site (green), Cas9 will cut DNA to generate a double-strand break (DSB – indicated by two red arrows) threenucleotides upstream of the protospacer adjacent motif (PAM – marked in red). When cells encounter a DSB, DNA repair pathways will repair the DSB break by either, mostfrequently, the error-prone non-homologous end joining (NHEJ) pathway, which can lead to insertions or deletions (indels), or homology directed repair (HDR) pathway,which allows for precise DNA modifications in the presence of an exogenous template DNA. In the top panel, in CRISPR/Cas9-effector systems, Cas9 can be guided, for example,to promoters or enhancers of genes of interest. Chimeric sgRNAs containing aptamers can bind to RNA-binding domains fused to effector domains such as transcriptionalactivators/repressors, chromatin modifiers or fluorescent proteins.

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often off-target effects, which pose a problem to obtaining consis-tent results [45].

CRISPR/Cas9 technology allows for permanent genetic modifi-cation of single or multiple genes simultaneously by transient orstable viral (retrovirus or lentivirus) delivery of the CRISPR/Cas9 com-ponents into cells [46–48]. Taking advantage of the indels generatedin response to DSB and subsequent NHEJ, sgRNAs can be designedto target Cas9 to bind the initial protein-coding exons of a gene,which can generate LOF frameshift indel mutations upon DSB (Fig. 1).This approach has been commonly used to generate LOF of genesof interest in the germline, somatically and in cell lines [49]. Re-cently, the Church group successfully edited 62 copies of the porcineendogenous retrovirus (PERV) in porcine cells, which opens up theroad for safer porcine-to-human xenotransplantation by prevent-ing PERV transmission to human cells [50]. Alternatively, to introducepoint mutations or complex knock-in alleles using precise genomeediting can be achieved by designing sgRNAs against the locus ofinterest and co-delivering the sgRNAs with a template DNA usedfor HDR (Fig. 1).

The CRISPR/Cas9 system has been successfully used in estab-lished cell lines, organoids and patient-derived xenografts to engineerLOF mutations by NHEJ, GOF mutations by HDR and chromosomalre-arrangements by cutting at two distant loci. Several groups haveused the CRISPR system to study hematological malignancies by exvivo LOF CRISPR/Cas9-editing of genes in hematopoietic cells andsubsequent transplantation back into animals to assess tumorige-nicity [51–54]. Matano et al. used CRISPR/Cas9 to model both LOF(APC, SMAD4, TP53) and GOF mutations (KRAS, PIK3CA) in primaryhuman intestinal organoids in vitro, followed by non-orthotopictransplantation in vivo [55]. The Clevers group used the same in-testinal organoid system to sequentially mutate colon cancerassociated mutations using CRISPR/Cas9 [56]. Antal et al. used theCRISPR/Cas9 system to correct a PKCβ LOF mutation in patient-derived colon cancer cell lines, which led to both in vitro and in vivogrowth changes [57]. The above described approaches can be appliedto all malignancies where primary cells can be isolated and grownin organoid cultures in order to functionally test putative ‘driver’variants and perform sequential mutation of candidate cancer genes.

Complex genomic rearrangements in cancer have previously beenchallenging to model, but several studies have now utilized CRISPR/cas9 genome editing to solve this problem. Frequently observedrearrangements in lung cancer, including CD74-ROS1, EML4-ALK andKIF5B-RET, have been generated by CRISPR/Cas9 technology [58,59].In addition, chromosomal translocations commonly identified inEwing’s sarcoma and acute myeloid leukemia (AML) between theEWSR1 and FLI1 loci and between the AML1 and ETO loci have beengenerated by CRISPR in human cell lines [60].

Moreover, genome sequencing studies have identified splice sitemutations that can lead to exon skipping [61]. For example, recur-rent mutations in the splice site of exon 14 of the oncogenic receptortyrosine kinase MET lead to loss of this exon, which is often ob-served in lung cancer [62]. Togashi et al. used a dual sgRNA systemto delete exon 14 of MET in HEK293 cells and demonstrated thatexon 14 deletion promoted cell growth [63]. Modeling MET exon14 deletion in primary lung epithelial cells, in established lung cancercell lines or in vivo would greatly enhance our understanding of howMET drives lung cancer initiation or progression.

Genome-wide association studies have identified several non-coding single nucleotide polymorphisms (SNPs) associated withdiseases, including cancer [64,65]. These SNPs can play an impor-tant functional cis- or trans- acting regulatory role, but modelingthese events has been difficult [66]. Recently, the Kellis group usedCRISPR/Cas9 in primary adipocytes to edit a SNP that is associatedwith obesity [67]. Similarly, Yao and colleagues deleted a colorectalcancer risk-associated SNP in an enhancer [68]. Editing of this SNPrestored the ability of transcriptional repressors to bind to the en-

hancer. Finally, the Cech group performed single base-pairmodifications in the TERT promoter, reverting a recurrent cancer-associated TERT promoter mutation. This resulted in decreasedtelomerase activity, indicating that this non-coding SNP may becausal for telomerase reactivation in multiple cancers [69].

Modeling of genetic variants in vivo

Traditionally, mouse models of cancer have relied on germline-based conditional or constitutive mutations. The Cre/LoxP systemhas been the major method of modeling various types of cancersin mice, allowing for temporal and tissue specific control of cancergene mutations [70]. In addition, given the complexity of geneticevents that are found in cancer, it has been challenging to func-tionally interrogate the role of each mutation or the combinatorialeffect of multiple genes on tumorigenesis with traditional methods.However, with the advances in CRISPR/Cas9-based genome engi-neering, many of the challenges in generating germline or somaticmutations have become trivial.

Several groups have successfully used CRISPR/Cas9-editing to in-troduce LOF indels or precise mutations via HDR in both ES cellsand zygotes in order to generate new GEMMs. To accelerate gen-eration of genetically modified mice, Cas9-based editing can beachieved in a one-cell-stage embryo [38,71]. Early studies by theJaenisch lab achieved rapid fluorescent reporter tagging ofpluripotency factors and conditional mutation of Mecp2 by co-injection of Cas9, sgRNAs and donor DNA [72]. The same groupdemonstrated LOF of multiple members of the Tet family of epi-genetic regulators in the germline [40,47]. Similar approaches havenow been utilized in other species, including rats, zebra fish,Caenorhabditis elegans, pigs and cynomologus monkeys, wheregermline targeting used to be a great challenge [73–77]. This ap-proach allows for rapid editing of multiple genes in order to testgenetic compensation or epistatic relationships of genes. Similar ap-proaches can now be applied to generate germline and conditionalmutant alleles in existing or new GEMMs of cancer.

Somatic genome engineering with CRISPR/Cas9

Somatic mutations, rearrangements and copy number changesoccur at different stages of cancer progression and can contributeto various aspects of tumorigenesis. Using novel CRISPR/Cas9 genomeediting approaches it is now possible to systematically interrogatethe function of these mutations in the context of novel, as well as,established GEMMs of cancer. Several groups have achieved somaticgenome editing using different methods to deliver Cas9 and sgRNAsagainst genes of interest to specific cell types and tissues. Here wewill summarize different techniques for delivering the CRISPR/Cas9 system ranging from delivery of plasmid DNA and viral deliveryto germline knock-in alleles.

Platt et al. were the first to generate a conditional Cre-dependentallele of Cas9, which allows for tissue-specific temporal expres-sion of Cas9 [78]. Using an adeno-associated virus (AAV) theydelivered sgRNAs against Trp53 and Lkb1 for LOF editing. The sameAAV contained an sgRNA against Kras and a template to mediatean HDR KrasG12D mutant upon Cas9 cutting at exon1 of Kras. Al-though no detectable KrasG12D mutations were found, animalsinfected with this AAV developed lung adenocarcinomas, with mu-tations in both Trp53 and Lkb1.

The Jacks group was the first to demonstrate successful somaticediting in the liver by hydrodynamic injection of plasmid DNA ex-pressing Cas9 and sgRNAs against Pten and p53 [79]. CRISPR-based LOF editing of both Trp53 and Pten led to hepatocellularcarcinoma formation, which pheno-copied Cre/LoxP based LOF ofboth genes. Furthermore, the same study generated a constitu tively

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active point mutant β-catenin by delivering a single-stranded DNAoligonucleotide donor [79].

The same group utilized an existing Cre/LoxP-based GEMM ofmutant K-ras driven lung adenocarcinoma that is initiated by intra-tracheal delivery of Cre-recombinase along with Cas9 and sgRNAsagainst Pten, Nkx2.1 and Apc in a single lentivirus termed pSECC [80].They demonstrated efficient editing and LOF of Pten, Nkx2.1 or Apcin approximately 70% of all tumors. Furthermore, loss of these tumorsuppressor genes led to dramatic differences in the differentiationstatus and growth of these tumors. The same pSECC-based lentiviralapproach was also successfully used in a K-ras driven pancreatic ad-enocarcinoma model where CRISPR/Cas9-based LOF of p57 was shownto abrogate the therapeutic efficacy of a combination therapy [81].

Also, Chiou et al. generated a Cre-dependent conditional Cas9 knock-in allele, which was crossed to a conditional K-ras mutant allele(KrasLSL-G12D/+). Through pancreatic retrograde delivery of a lentivirusthat expressed Cre-recombinase and an sgRNA against the tumor sup-pressor Lkb1, they demonstrated that LOF of Lkb1 through CRISPR/Cas9-editing accelerates pancreatic tumorigenesis [82].

In contrast, modeling chromosomal re-arrangements in vivo hasbeen challenging. Recently, the Ventura group generated a newmodel of Eml4-Alk-driven lung cancer by intra-tracheal delivery ofan adenovirus carrying Cas9 and sgRNAs against the Eml4 and the

Alk4 locus in wild-type animals. This resulted in efficient 11megabase translocation/inversion in lung epithelial cells similar towhat had previously been seen in vitro [83]. Furthermore, theseEML4-Alk driven tumors were sensitive to Crizotinib, a therapy usedto treat human patients with tumors containing this re-arrangement[84]. Additionally, a different group was also able to generate a similarlung cancer model using two lentiviruses separately expressingsgRNAs targeting Eml4 and Alk [85].

Finally, the Lowe laboratory generated a doxycycline inducibleallele of Cas9 or the nickase mutant Cas9D10A. They generated a seriesof alleles also expressing 1 to 6 different sgRNAs and demon-strated efficient inducible editing of multiple alleles in vivo in theintestinal epithelium and in vitro both in ES cells and intestinal or-ganoid cultures [86].

CRISPR/Cas9-based effectors

Several groups have utilized a catalytically dead Cas9 withoutnuclease activity that can be guided by an sgRNA to genomic loci ofinterest (dCas9) [87] which can be coupled with different tran-scriptional activators [88–97] or repressors [90,91,93] to modulatelocus specific endogenous gene expression (Fig. 2). Additional ap-proaches like these can be multiplexed with the use of scaffold RNAs

Fig. 2. Functional applications of CRISPR/Cas9-based genome engineering in cancer biology. Upper panel, CRISPR-based approaches in genome engineering. (Left) Loss-of-function mutations caused by single cut followed by repair by non-homologous end joining (NHEJ). (Middle) Genomic rearrangements in a locus between two cuts afterNHEJ. (Right) Precise genome editing dependent on a donor-oligo (template DNA) and homology directed repair (HDR). Lower panel, directed-Cas9 (dCas9) with effectorscausing transcriptional activation (left), transcriptional repression (right) or epigenetic regulation of genes (middle).

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consisting of locus-specific sgRNA fused to an RNA aptamer that re-cruits an activator or repressor effector [98]. These CRISPR/Cas9-based effector systems can be used to model gene expression changesand copy number variations that are often observed in human cancers.The potential to modify endogenous gene expression provides aunique advantage, particularly when studying cancer-associated cisor trans acting regulatory non-coding RNAs [99–103]. Further-more, it is widely accepted that the deposition of epigenetic marksand RNA-splicing can be coupled to transcription [104–107], there-fore endogenous regulation of transcription with the use of CRISPR/Cas9-based effectors may be more physiologic than traditional cDNAor RNAi based methods for modulating gene expression.

Enzymatically dead Cas9 (dCas9) can also be used to imagegenomic loci [108] or single protein molecules [89] in living cells.Live imaging of genomic loci can be used to monitor the dynamicsof copy number variations or genomic rearrangements during tumorprogression or in the context of positive or negative selection. Thedetection of single proteins can be utilized to assess differences inthe dynamic subcellular localization of proteins such as transcrip-tion factors shuttling from the cytoplasm to the nucleus or themembrane localization of proteins involved in cancer cell motilityand invasion.

CRISPR/Cas9-based genetic screens

With the emergence of the CRISPR technology, several groupshave used CRISPR/Cas9-based editing to perform both positive andnegative selection genetic screens. Wang et al. used a pooled lentivirallibrary (~73,000 sgRNAs) to perform a positive selection screen intwo human cell lines. They identified DNA mismatch repair pathwaygenes, TOP2A and CDK6 whose loss confers resistance to6-thioguanine or DNA topoisomerase II poison etoposide respec-tively [109]. Shalem et al. performed a positive selection screen usinga pooled lentiviral library (~65,000 sgRNAs) in BRAF-V600E mutantmelanoma cell lines to identify genes whose loss confers resis-tance to the BRAF-V600E inhibitor vemurafinib [110]. In addition,focused genetic screens against particular pathways have provedvery fruitful in identifying cancer cell dependencies. The Vakoc groupperformed a screen targeting 192 chromatin regulatory domains inmouse acute myeloid leukemia cells and identified known drugtargets and additional dependencies [111]. Interestingly, this studyobserved that targeting functional chromatin domains leads to moreefficient LOF because in addition to frameshift indels, even in-frame variants produced upon cas9-mediated DSB lead to nullmutations. In a recent study, Birsoy et al. performed a focused geneticscreen with ~30,000 sgRNAs targeting ~3000 metabolic pathwaygenes to identify genes whose loss sensitizes human cells to the mi-tochondrial complex I inhibitor phenformin. They observed that lossof GOT1, the cytosolic aspartate aminotransferase, sensitizes cellsto mitochondrial complex I inhibition [112]. A recent report fromthe Sabatini group used a genome-wide sgRNA library to screen forgenes required for proliferation and survival in a human cancer cellline. They identified a set of cell-essential genes, which were vali-dated by an orthogonal gene-trap-based screen and comparison withyeast gene knockouts [113]. The Shendure group has successfullyperformed multiplexed CRISPR/Cas9 HDR-based saturation muta-genesis screens to identify gene variants with functional impact [114].Going beyond the LOF screens with the Cas9 nuclease, dCas9 hasbeen utilized to perform parallel genome-wide activator and re-pressor screens [90,96].

Looking forward: caveats and future applications of theCRISPR/Cas9 system in cancer biology

All the approaches reviewed here can facilitate functionaldissection of gene variants, and of cis- and trans-acting regulatory

elements whose significance in cancer remain unknown. However,as with every emerging technology, there are certain disadvan-tages and technical difficulties that need to be overcome, particularlyfor in vivo CRISPR/Cas9-based gene editing. The nature of CRISPR/Cas9-based gene editing by NHEJ-mediated indels leads to a lot ofvariation in the mutant alleles generated. This is a challenge for invivo somatic-editing where in-frame or out-of-frame mutationscan undergo selection in the context of tumor progression andone must therefore be careful in systematically characterizingthese somatic events. Furthermore, efficient somatic HDR in vivousing CRISPR/Cas9 has been a major challenge. Such approachesare particularly important when trying to model specific LOF orGOF mutations in tumor suppressor and oncogenes, respectively.The efficiency of HDR may be improved by better delivery oftemplate DNA and/or with the use of the Class 2 CRISPR system[115].

Future use of CRISPR/Cas9 for both clinical and pre-clinical ap-plications will require the comprehensive characterization of possibleoff-target effects of both enzymatically active and dead Cas9. Severalstudies so far have demonstrated minimal off-target effects, but suchefforts have mostly focused on indels in the top predicted off-target sites [46,47,50,79,80,110]. However, there are several unbiasedhigh-throughput genome-wide sequencing platforms that allow forthe identification of off-target effects [116,117] and future studieswill likely use this method to demonstrate off-target activity of theirCRISPR/Cas9-editing.

Many studies highlighted in this review have used individualCRISPR/Cas9 tools to either edit genes or modulate gene expres-sion. However, orthogonal approaches for using dCas9 effectorsystems in parallel to editing with Cas9 nuclease are starting tobecome well established either by varying the sgRNAs when usingthe same species Cas9 [118,119] or by using different Cas9 enzymeswith differential PAM recognition [120–122]. Recently, using di-rected evolution, the Joung group engineered new forms of Cas9enzymes that recognize alternative PAM sites [120,121]. Further-more, the development of novel knock-in alleles in mice with botheffector and editing capabilities will allow for the creation of moresophisticated mouse models of human cancers. Sequential muta-genesis of cancer genes in GEMMs has been challenging until now,but the use of multiple CRISPR/Cas9 systems in vivo and in vitro willallow for more complex modeling of both LOF and GOF events withthe potential of sequential modifications in cancer cell lines, primarycells and patient-derived xenografts.

In addition, there is great need to simultaneously perform LOFediting and genomic rearrangement of multiple loci in the samecell in order to model complex genetic variations identified in humancancer genomes. This can be achieved with the use of a bacterial,RNaseIII endoribonucleases, Csy4, which catalyzes the maturationof individual sgRNAs expressed from a single-transcript [123].Csy4 has been adapted to mammalian systems to enable multi-plexed expression of sgRNAs from a single polI-driven promoter[124,125]. This approach will enable cancer researchers to studythe genetic cooperation of multiple putative cancer genes intumorigenesis.

The extensive efforts to utilize the CRISPR/Cas9 system in mam-malian cells have already paved the way to some incrediblediscoveries in cancer biology. In the era of personalized medicine,CRISPR/Cas9-based approaches arm cancer researchers with im-proved tools to tackle the complexity of the various cancers (Fig. 3).The extensive information recorded from an individual patient tumorcan be functionally validated using CRIPSR/Cas9 approaches in invitro and in vivo models. Once important functional variants are iden-tified, genetic and drug screens can be performed to identifygenotype-specific vulnerabilities that may pave the road to new per-sonalized genotype-based therapies, which is the ultimate goal ofprecision medicine.

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Conflict of interest

The authors declare that they have no conflicts of interest.

Acknowledgements

We would like to thank Sarah LeBoeuf, Francisco Sánchez-Rivera and David G. McFadden for critical reading of the manuscriptand Tyler Jacks for insightful scientific discussions. Volkan I. Sayinis the recipient of an EMBO Long Term Fellowship (ALTF 1451-2015) co-funded by the European Commission (LTCOFUND2013,GA-2013-609409) with support from Marie Curie Actions. Weprovide a general overview of the field; we apologize for the omis-sion of any pertinent work related to this review due to spaceconstraints.

References

[1] D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (2000) 57–70.[2] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell 144

(2011) 646–674.[3] M.S. Lawrence, P. Stojanov, C.H. Mermel, J.T. Robinson, L.A. Garraway, T.R. Golub,

et al., Discovery and saturation analysis of cancer genes across 21 tumourtypes, Nature 505 (2014) 495–501.

[4] Cancer Genome Atlas Research Network, Comprehensive genomiccharacterization of squamous cell lung cancers, Nature 489 (2012) 519–525.

[5] Cancer Genome Atlas Research Network, Comprehensive molecularcharacterization of gastric adenocarcinoma, Nature 513 (2014) 202–209.

[6] Cancer Genome Atlas Research Network, Comprehensive molecular profilingof lung adenocarcinoma, Nature 511 (2014) 543–550.

[7] ENCODE Project Consortium, An integrated encyclopedia of DNA elements inthe human genome, Nature 489 (2012) 57–74.

[8] K. Cibulskis, M.S. Lawrence, S.L. Carter, A. Sivachenko, D. Jaffe, C. Sougnez, et al.,Sensitive detection of somatic point mutations in impure and heterogeneouscancer samples, Nat. Biotechnol. 31 (2013) 213–219.

[9] M. Hidalgo, F. Amant, A.V. Biankin, E. Budinska, A.T. Byrne, C. Caldas, et al.,Patient-derived xenograft models: an emerging platform for translationalcancer research, Cancer Discov. 4 (2014) 998–1013.

[10] X. Li, L. Nadauld, A. Ootani, D.C. Corney, R.K. Pai, O. Gevaert, et al., Oncogenictransformation of diverse gastrointestinal tissues in primary organoid culture,Nat. Med. 20 (2014) 769–777.

[11] S.F. Boj, C.I. Hwang, L.A. Baker, I.I. Chio, D.D. Engle, V. Corbo, et al., Organoidmodels of human and mouse ductal pancreatic cancer, Cell 160 (2015) 324–338.

[12] C.J. Torrance, V. Agrawal, B. Vogelstein, K.W. Kinzler, Use of isogenic humancancer cells for high-throughput screening and drug discovery, Nat. Biotechnol.19 (2001) 940–945.

[13] T. Van Dyke, T. Jacks, Cancer modeling in the modern era: progress andchallenges, Cell 108 (2002) 135–144.

[14] C.P. Day, G. Merlino, T. Van Dyke, Preclinical mouse cancer models: a mazeof opportunities and challenges, Cell 163 (2015) 39–53.

[15] J. Jonkers, A. Berns, Conditional mouse models of sporadic cancer, Nat. Rev.Cancer 2 (2002) 251–265.

[16] L. Flintoft, Animal models: mastering RNAi in mice, Nat. Rev. Genet. 12 (2011)380.

[17] L.E. Dow, S.W. Lowe, Life in the fast lane: mammalian disease models in thegenomics era, Cell 148 (2012) 1099–1109.

[18] S. Shirasawa, M. Furuse, N. Yokoyama, T. Sasazuki, Altered growth of humancolon cancer cell lines disrupted at activated Ki-ras, Science 260 (1993) 85–88.

[19] T. Waldman, K.W. Kinzler, B. Vogelstein, p21 is necessary for the p53-mediatedG1 arrest in human cancer cells, Cancer Res. 55 (1995) 5187–5190.

[20] F. Bunz, A. Dutriaux, C. Lengauer, T. Waldman, S. Zhou, J.P. Brown, et al.,Requirement for p53 and p21 to sustain G2 arrest after DNA damage, Science282 (1998) 1497–1501.

[21] F. Bunz, P.M. Hwang, C. Torrance, T. Waldman, Y. Zhang, L. Dillehay, et al.,Disruption of p53 in human cancer cells alters the responses to therapeuticagents, J. Clin. Invest. 104 (1999) 263–269.

[22] A. Berns, Cancer. Improved mouse models, Nature 410 (2001) 1043–1044.[23] O. Smithies, R.G. Gregg, S.S. Boggs, M.A. Koralewski, R.S. Kucherlapati, Insertion

of DNA sequences into the human chromosomal beta-globin locus byhomologous recombination, Nature 317 (1985) 230–234.

[24] K.R. Thomas, K.R. Folger, M.R. Capecchi, High frequency targeting of genes tospecific sites in the mammalian genome, Cell 44 (1986) 419–428.

[25] S.L. Mansour, K.R. Thomas, M.R. Capecchi, Disruption of the proto-oncogeneint-2 in mouse embryo-derived stem cells: a general strategy for targetingmutations to non-selectable genes, Nature 336 (1988) 348–352.

[26] A. Plessis, A. Perrin, J.E. Haber, B. Dujon, Site-specific recombination determinedby I-SceI, a mitochondrial group I intron-encoded endonuclease expressed inthe yeast nucleus, Genetics 130 (1992) 451–460.

[27] S.D. Priebe, J. Westmoreland, T. Nilsson-Tillgren, M.A. Resnick, Induction ofrecombination between homologous and diverged DNAs by double-strand gapsand breaks and role of mismatch repair, Mol. Cell. Biol. 14 (1994) 4802–4814.

[28] H. Puchta, B. Dujon, B. Hohn, Homologous recombination in plant cells isenhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease, Nucleic Acids Res. 21 (1993) 5034–5040.

[29] M. Bibikova, K. Beumer, J.K. Trautman, D. Carroll, Enhancing gene targetingwith designed zinc finger nucleases, Science 300 (2003) 764.

[30] J.K. Joung, J.D. Sander, TALENs: a widely applicable technology for targetedgenome editing, Nat. Rev. Mol. Cell Biol. 14 (2013) 49–55.

[31] T. Li, S. Huang, W.Z. Jiang, D. Wright, M.H. Spalding, D.P. Weeks, et al., TALnucleases (TALNs): hybrid proteins composed of TAL effectors and FokIDNA-cleavage domain, Nucleic Acids Res. 39 (2011) 359–372.

[32] T. Li, S. Huang, X. Zhao, D.A. Wright, S. Carpenter, M.H. Spalding, et al.,Modularly assembled designer TAL effector nucleases for targeted geneknockout and gene replacement in eukaryotes, Nucleic Acids Res. 39 (2011)6315–6325.

[33] J.C. Miller, S. Tan, G. Qiao, K.A. Barlow, J. Wang, D.F. Xia, et al., A TALE nucleasearchitecture for efficient genome editing, Nat. Biotechnol. 29 (2011) 143–148.

[34] F.D. Urnov, E.J. Rebar, M.C. Holmes, H.S. Zhang, P.D. Gregory, Genome editingwith engineered zinc finger nucleases, Nat. Rev. Genet. 11 (2010) 636–646.

[35] G. Gasiunas, R. Barrangou, P. Horvath, V. Siksnys, Cas9-crRNA ribonucleoproteincomplex mediates specific DNA cleavage for adaptive immunity in bacteria,Proc. Natl. Acad. Sci. U.S.A. 109 (2012) E2579–E2586.

[36] J.A. Doudna, E. Charpentier, Genome editing. The new frontier of genomeengineering with CRISPR-Cas9, Science 346 (2014) 1258096.

[37] P. Mali, K.M. Esvelt, G.M. Church, Cas9 as a versatile tool for engineeringbiology, Nat. Methods 10 (2013) 957–963.

[38] H. Yang, H. Wang, R. Jaenisch, Generating genetically modified mice usingCRISPR/Cas-mediated genome engineering, Nat. Protoc. 9 (2014) 1956–1968.

[39] I. Lokody, Genetic therapies: correcting genetic defects with CRISPR-Cas9, Nat.Rev. Genet. 15 (2014) 63.

[40] H. Wang, H. Yang, C.S. Shivalila, M.M. Dawlaty, A.W. Cheng, F. Zhang, et al.,One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering, Cell 153 (2013) 910–918.

[41] K.K. Frese, D.A. Tuveson, Maximizing mouse cancer models, Nat. Rev. Cancer7 (2007) 645–658.

In vitro systems: Primary cell culture/organoids Cancer cell lines

Patient tumor data

In vivo pre-clinical models: GEMMs Orthotopic or xenograft Transplant

CRISPR-based modeling of genetic and epigenetic variants

in vitro

Germline or somatic in vivomodeling of variants using CRISPR-based methods

Genetic and Drug screens to identify

vulnerabilities

Fig. 3. Personalized approaches to cancer biology using CRISPR/Cas9-system. Thegreat complexity of genetic and epigenetic variants identified in patient tumors canbe functionally validated using CRIPSR/Cas9 approaches in in vitro systems such asprimary cell cultures/organoids and cancer cell lines. Following the in vitro valida-tion of such events, in vivo validation can be performed in genetically-engineeredmouse models (GEMMs) and patient-derived xenografts. Once important function-al variants are identified, genetic and drug screens can be performed to identifygenotype-specific vulnerabilities that may pave the road to new personalizedgenotype-based therapeutic strategies.

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[42] L. Zender, W. Xue, J. Zuber, C.P. Semighini, A. Krasnitz, B. Ma, et al., Anoncogenomics-based in vivo RNAi screen identifies tumor suppressors in livercancer, Cell 135 (2008) 852–864.

[43] J. Zuber, K. McJunkin, C. Fellmann, L.E. Dow, M.J. Taylor, G.J. Hannon, et al.,Toolkit for evaluating genes required for proliferation and survival usingtetracycline-regulated RNAi, Nat. Biotechnol. 29 (2011) 79–83.

[44] G. Livshits, S.W. Lowe, Accelerating cancer modeling with RNAi andnongermline genetically engineered mouse models, Cold Spring Harb. Protoc.2013 (2013).

[45] C.E. Shamu, S. Wiemann, M. Boutros, On target: a public repository forlarge-scale RNAi experiments, Nat. Cell Biol. 14 (2012) 115.

[46] L. Cong, F.A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, et al., Multiplex genomeengineering using CRISPR/Cas systems, Science 339 (2013) 819–823.

[47] P. Mali, L. Yang, K.M. Esvelt, J. Aach, M. Guell, J.E. DiCarlo, et al., RNA-guidedhuman genome engineering via Cas9, Science 339 (2013) 823–826.

[48] M. Jinek, A. East, A. Cheng, S. Lin, E. Ma, J. Doudna, RNA-programmed genomeediting in human cells, Elife 2 (2013) e00471.

[49] F.J. Sanchez-Rivera, T. Jacks, Applications of the CRISPR-Cas9 system in cancerbiology, Nat. Rev. Cancer 15 (2015) 387–395.

[50] L. Yang, M. Guell, D. Niu, H. George, E. Lesha, D. Grishin, et al., Genome-wideinactivation of porcine endogenous retroviruses (PERVs), Science 350 (2015)1101–1104.

[51] B.J. Aubrey, G.L. Kelly, A.J. Kueh, M.S. Brennan, L. O’Connor, L. Milla, et al., Aninducible lentiviral guide RNA platform enables the identification of tumor-essential genes and tumor-promoting mutations in vivo, Cell Rep. 10 (2015)1422–1432.

[52] D. Heckl, M.S. Kowalczyk, D. Yudovich, R. Belizaire, R.V. Puram, M.E. McConkey,et al., Generation of mouse models of myeloid malignancy with combinatorialgenetic lesions using CRISPR-Cas9 genome editing, Nat. Biotechnol. 32 (2014)941–946.

[53] A. Malina, J.R. Mills, R. Cencic, Y. Yan, J. Fraser, L.M. Schippers, et al., RepurposingCRISPR/Cas9 for in situ functional assays, Genes Dev. 27 (2013) 2602–2614.

[54] C. Chen, Y. Liu, A.R. Rappaport, T. Kitzing, N. Schultz, Z. Zhao, et al., MLL3 is ahaploinsufficient 7q tumor suppressor in acute myeloid leukemia, Cancer Cell25 (2014) 652–665.

[55] M. Matano, S. Date, M. Shimokawa, A. Takano, M. Fujii, Y. Ohta, et al., Modelingcolorectal cancer using CRISPR-Cas9-mediated engineering of human intestinalorganoids, Nat. Med. 21 (2015) 256–262.

[56] J. Drost, R.H. van Jaarsveld, B. Ponsioen, C. Zimberlin, R. van Boxtel, A. Buijs,et al., Sequential cancer mutations in cultured human intestinal stem cells,Nature 521 (2015) 43–47.

[57] C.E. Antal, A.M. Hudson, E. Kang, C. Zanca, C. Wirth, N.L. Stephenson, et al.,Cancer-associated protein kinase C mutations reveal kinase’s role as tumorsuppressor, Cell 160 (2015) 489–502.

[58] P.S. Choi, M. Meyerson, Targeted genomic rearrangements using CRISPR/Castechnology, Nat. Commun. 5 (2014) 3728.

[59] H. Ghezraoui, M. Piganeau, B. Renouf, J.B. Renaud, A. Sallmyr, B. Ruis, et al.,Chromosomal translocations in human cells are generated by canonicalnonhomologous end-joining, Mol. Cell 55 (2014) 829–842.

[60] R. Torres, M.C. Martin, A. Garcia, J.C. Cigudosa, J.C. Ramirez, S. Rodriguez-Perales,Engineering human tumour-associated chromosomal translocations with theRNA-guided CRISPR-Cas9 system, Nat. Commun. 5 (2014) 3964.

[61] S.N. Dorman, C. Viner, P.K. Rogan, Splicing mutation analysis reveals previouslyunrecognized pathways in lymph node-invasive breast cancer, Sci. Rep. 4(2014) 7063.

[62] G.M. Frampton, S.M. Ali, M. Rosenzweig, J. Chmielecki, X. Lu, T.M. Bauer, et al.,Activation of MET via diverse exon 14 splicing alterations occurs in multipletumor types and confers clinical sensitivity to MET inhibitors, Cancer Discov.5 (2015) 850–859.

[63] Y. Togashi, H. Mizuuchi, S. Tomida, M. Terashima, H. Hayashi, K. Nishio, et al.,MET gene exon 14 deletion created using the CRISPR/Cas9 system enhancescellular growth and sensitivity to a MET inhibitor, Lung Cancer 90 (2015)590–597.

[64] S.F. Kingsmore, I.E. Lindquist, J. Mudge, D.D. Gessler, W.D. Beavis, Genome-wideassociation studies: progress and potential for drug discovery anddevelopment, Nat. Rev. Drug Discov. 7 (2008) 221–230.

[65] M.I. McCarthy, G.R. Abecasis, L.R. Cardon, D.B. Goldstein, J. Little, J.P. Ioannidis,et al., Genome-wide association studies for complex traits: consensus,uncertainty and challenges, Nat. Rev. Genet. 9 (2008) 356–369.

[66] I.K. Sur, O. Hallikas, A. Vaharautio, J. Yan, M. Turunen, M. Enge, et al., Micelacking a Myc enhancer that includes human SNP rs6983267 are resistant tointestinal tumors, Science 338 (2012) 1360–1363.

[67] M. Claussnitzer, S.N. Dankel, K.H. Kim, G. Quon, W. Meuleman, C. Haugen, et al.,FTO obesity variant circuitry and adipocyte browning in humans, N. Engl. J.Med. 373 (2015) 895–907.

[68] L. Yao, Y.G. Tak, B.P. Berman, P.J. Farnham, Functional annotation of colon cancerrisk SNPs, Nat. Commun. 5 (2014) 5114.

[69] L. Xi, J.C. Schmidt, A.J. Zaug, D.R. Ascarrunz, T.R. Cech, A novel two-step genomeediting strategy with CRISPR-Cas9 provides new insights into telomerase actionand TERT gene expression, Genome Biol. 16 (2015) 231.

[70] B. Sauer, N. Henderson, Site-specific DNA recombination in mammalian cellsby the Cre recombinase of bacteriophage P1, Proc. Natl. Acad. Sci. U.S.A. 85(1988) 5166–5170.

[71] T. Aida, K. Chiyo, T. Usami, H. Ishikubo, R. Imahashi, Y. Wada, et al., Cloning-freeCRISPR/Cas system facilitates functional cassette knock-in in mice, GenomeBiol. 16 (2015) 87.

[72] H. Yang, H. Wang, C.S. Shivalila, A.W. Cheng, L. Shi, R. Jaenisch, One-stepgeneration of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering, Cell 154 (2013) 1370–1379.

[73] Y. Niu, B. Shen, Y. Cui, Y. Chen, J. Wang, L. Wang, et al., Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting inone-cell embryos, Cell 156 (2014) 836–843.

[74] Y. Shao, Y. Guan, L. Wang, Z. Qiu, M. Liu, Y. Chen, et al., CRISPR/Cas-mediatedgenome editing in the rat via direct injection of one-cell embryos, Nat. Protoc.9 (2014) 2493–2512.

[75] A.E. Friedland, Y.B. Tzur, K.M. Esvelt, M.P. Colaiacovo, G.M. Church, J.A. Calarco,Heritable genome editing in C. elegans via a CRISPR-Cas9 system, Nat. Methods10 (2013) 741–743.

[76] W.Y. Hwang, Y. Fu, D. Reyon, M.L. Maeder, S.Q. Tsai, J.D. Sander, et al., Efficientgenome editing in zebrafish using a CRISPR-Cas system, Nat. Biotechnol. 31(2013) 227–229.

[77] T. Hai, F. Teng, R. Guo, W. Li, Q. Zhou, One-step generation of knockout pigsby zygote injection of CRISPR/Cas system, Cell Res. 24 (2014) 372–375.

[78] R.J. Platt, S. Chen, Y. Zhou, M.J. Yim, L. Swiech, H.R. Kempton, et al., CRISPR-Cas9knockin mice for genome editing and cancer modeling, Cell 159 (2014)440–455.

[79] W. Xue, S. Chen, H. Yin, T. Tammela, T. Papagiannakopoulos, N.S. Joshi, et al.,CRISPR-mediated direct mutation of cancer genes in the mouse liver, Nature514 (2014) 380–384.

[80] F.J. Sanchez-Rivera, T. Papagiannakopoulos, R. Romero, T. Tammela, M.R. Bauer,A. Bhutkar, et al., Rapid modelling of cooperating genetic events in cancerthrough somatic genome editing, Nature 516 (2014) 428–431.

[81] P.K. Mazur, A. Herner, S.S. Mello, M. Wirth, S. Hausmann, F.J. Sanchez-Rivera,et al., Combined inhibition of BET family proteins and histone deacetylasesas a potential epigenetics-based therapy for pancreatic ductal adenocarcinoma,Nat. Med. 21 (2015) 1163–1171.

[82] S.H. Chiou, I.P. Winters, J. Wang, S. Naranjo, C. Dudgeon, F.B. Tamburini, et al.,Pancreatic cancer modeling using retrograde viral vector delivery and in vivoCRISPR/Cas9-mediated somatic genome editing, Genes Dev. 29 (2015)1576–1585.

[83] D. Maddalo, E. Manchado, C.P. Concepcion, C. Bonetti, J.A. Vidigal, Y.C. Han,et al., In vivo engineering of oncogenic chromosomal rearrangements withthe CRISPR/Cas9 system, Nature 516 (2014) 423–427.

[84] A.T. Shaw, D.W. Kim, K. Nakagawa, T. Seto, L. Crino, M.J. Ahn, et al., Crizotinibversus chemotherapy in advanced ALK-positive lung cancer, N. Engl. J. Med.368 (2013) 2385–2394.

[85] R.B. Blasco, E. Karaca, C. Ambrogio, T.C. Cheong, E. Karayol, V.G. Minero, et al.,Simple and rapid in vivo generation of chromosomal rearrangements usingCRISPR/Cas9 technology, Cell Rep. 9 (2014) 1219–1227.

[86] L.E. Dow, J. Fisher, K.P. O’Rourke, A. Muley, E.R. Kastenhuber, G. Livshits, et al.,Inducible in vivo genome editing with CRISPR-Cas9, Nat. Biotechnol. 33 (2015)390–394.

[87] M. Jinek, F. Jiang, D.W. Taylor, S.H. Sternberg, E. Kaya, E. Ma, et al., Structuresof Cas9 endonucleases reveal RNA-mediated conformational activation, Science343 (2014) 1247997.

[88] K.M. Esvelt, P. Mali, J.L. Braff, M. Moosburner, S.J. Yaung, G.M. Church,Orthogonal Cas9 proteins for RNA-guided gene regulation and editing, Nat.Methods 10 (2013) 1116–1121.

[89] M.E. Tanenbaum, L.A. Gilbert, L.S. Qi, J.S. Weissman, R.D. Vale, A protein-taggingsystem for signal amplification in gene expression and fluorescence imaging,Cell 159 (2014) 635–646.

[90] L.A. Gilbert, M.A. Horlbeck, B. Adamson, J.E. Villalta, Y. Chen, E.H. Whitehead,et al., Genome-scale CRISPR-mediated control of gene repression andactivation, Cell 159 (2014) 647–661.

[91] L.A. Gilbert, M.H. Larson, L. Morsut, Z. Liu, G.A. Brar, S.E. Torres, et al.,CRISPR-mediated modular RNA-guided regulation of transcription ineukaryotes, Cell 154 (2013) 442–451.

[92] P. Mali, J. Aach, P.B. Stranges, K.M. Esvelt, M. Moosburner, S. Kosuri, et al.,CAS9 transcriptional activators for target specificity screening and pairednickases for cooperative genome engineering, Nat. Biotechnol. 31 (2013)833–838.

[93] D. Bikard, W. Jiang, P. Samai, A. Hochschild, F. Zhang, L.A. Marraffini,Programmable repression and activation of bacterial gene expression usingan engineered CRISPR-Cas system, Nucleic Acids Res. 41 (2013) 7429–7437.

[94] P. Perez-Pinera, D.D. Kocak, C.M. Vockley, A.F. Adler, A.M. Kabadi, L.R. Polstein,et al., RNA-guided gene activation by CRISPR-Cas9-based transcription factors,Nat. Methods 10 (2013) 973–976.

[95] A.W. Cheng, H. Wang, H. Yang, L. Shi, Y. Katz, T.W. Theunissen, et al.,Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guidedtranscriptional activator system, Cell Res. 23 (2013) 1163–1171.

[96] S. Konermann, M.D. Brigham, A.E. Trevino, J. Joung, O.O. Abudayyeh, C. Barcena,et al., Genome-scale transcriptional activation by an engineered CRISPR-Cas9complex, Nature 517 (2015) 583–588.

[97] M.L. Maeder, S.J. Linder, V.M. Cascio, Y. Fu, Q.H. Ho, J.K. Joung, CRISPRRNA-guided activation of endogenous human genes, Nat. Methods 10 (2013)977–979.

[98] J.G. Zalatan, M.E. Lee, R. Almeida, L.A. Gilbert, E.H. Whitehead, M. La Russa,et al., Engineering complex synthetic transcriptional programs with CRISPRRNA scaffolds, Cell 160 (2015) 339–350.

[99] N. Dimitrova, J.R. Zamudio, R.M. Jong, D. Soukup, R. Resnick, K. Sarma, et al.,LincRNA-p21 activates p21 in cis to promote Polycomb target gene expressionand to enforce the G1/S checkpoint, Mol. Cell 54 (2014) 777–790.

ARTICLE IN PRESS
















































































































































































[100] R.J. Flockhart, D.E. Webster, K. Qu, N. Mascarenhas, J. Kovalski, M. Kretz, et al.,BRAFV600E remodels the melanocyte transcriptome and induces BANCR toregulate melanoma cell migration, Genome Res. 22 (2012) 1006–1014.

[101] T. Trimarchi, E. Bilal, P. Ntziachristos, G. Fabbri, R. Dalla-Favera, A. Tsirigos, et al.,Genome-wide mapping and characterization of Notch-regulated longnoncoding RNAs in acute leukemia, Cell 158 (2014) 593–606.

[102] N. Leveille, C.A. Melo, K. Rooijers, A. Diaz-Lagares, S.A. Melo, G. Korkmaz, et al.,Genome-wide profiling of p53-regulated enhancer RNAs uncovers a subsetof enhancers controlled by a lncRNA, Nat. Commun. 6 (2015) 6520.

[103] T. Kim, R. Cui, Y.J. Jeon, P. Fadda, H. Alder, C.M. Croce, MYC-repressed longnoncoding RNAs antagonize MYC-induced cell proliferation and cell cycleprogression, Oncotarget 6 (2015) 18780–18789.

[104] U. Braunschweig, S. Gueroussov, A.M. Plocik, B.R. Graveley, B.J. Blencowe,Dynamic integration of splicing within gene regulatory pathways, Cell 152(2013) 1252–1269.

[105] J.K. Choi, J.B. Bae, J. Lyu, T.Y. Kim, Y.J. Kim, Nucleosome deposition and DNAmethylation at coding region boundaries, Genome Biol. 10 (2009) R89.

[106] Y. Fu, D. Dominissini, G. Rechavi, C. He, Gene expression regulation mediatedthrough reversible m(6)A RNA methylation, Nat. Rev. Genet. 15 (2014)293–306.

[107] H.L. Zhou, G. Luo, J.A. Wise, H. Lou, Regulation of alternative splicing by localhistone modifications: potential roles for RNA-guided mechanisms, NucleicAcids Res. 42 (2014) 701–713.

[108] B. Chen, L.A. Gilbert, B.A. Cimini, J. Schnitzbauer, W. Zhang, G.W. Li, et al.,Dynamic imaging of genomic loci in living human cells by an optimizedCRISPR/Cas system, Cell 155 (2013) 1479–1491.

[109] T. Wang, J.J. Wei, D.M. Sabatini, E.S. Lander, Genetic screens in human cellsusing the CRISPR-Cas9 system, Science 343 (2014) 80–84.

[110] O. Shalem, N.E. Sanjana, E. Hartenian, X. Shi, D.A. Scott, T.S. Mikkelsen, et al.,Genome-scale CRISPR-Cas9 knockout screening in human cells, Science 343(2014) 84–87.

[111] J. Shi, E. Wang, J.P. Milazzo, Z. Wang, J.B. Kinney, C.R. Vakoc, Discovery of cancerdrug targets by CRISPR-Cas9 screening of protein domains, Nat. Biotechnol.33 (2015) 661–667.

[112] K. Birsoy, T. Wang, W.W. Chen, E. Freinkman, M. Abu-Remaileh, D.M. Sabatini,An essential role of the mitochondrial electron transport chainin cell proliferation is to enable aspartate synthesis, Cell 162 (2015) 540–551.

[113] T. Wang, K. Birsoy, N.W. Hughes, K.M. Krupczak, Y. Post, J.J. Wei, et al.,Identification and characterization of essential genes in the human genome,Science 350 (2015) 1096–1101.

[114] G.M. Findlay, E.A. Boyle, R.J. Hause, J.C. Klein, J. Shendure, Saturation editingof genomic regions by multiplex homology-directed repair, Nature 513 (2014)120–123.

[115] B. Zetsche, J.S. Gootenberg, O.O. Abudayyeh, I.M. Slaymaker, K.S. Makarova,P. Essletzbichler, et al., Cpf1 is a single RNA-guided endonuclease of a class2 CRISPR-Cas system, Cell 163 (2015) 759–771.

[116] S.Q. Tsai, Z. Zheng, N.T. Nguyen, M. Liebers, V.V. Topkar, V. Thapar, et al.,GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Casnucleases, Nat. Biotechnol. 33 (2015) 187–197.

[117] N. Crosetto, A. Mitra, M.J. Silva, M. Bienko, N. Dojer, Q. Wang, et al., Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing,Nat. Methods 10 (2013) 361–365.

[118] J.E. Dahlman, O.O. Abudayyeh, J. Joung, J.S. Gootenberg, F. Zhang, S. Konermann,Orthogonal gene knockout and activation with a catalytically active Cas9nuclease, Nat. Biotechnol. 33 (2015) 1159–1161.

[119] S. Kiani, A. Chavez, M. Tuttle, R.N. Hall, R. Chari, D. Ter-Ovanesyan, et al., Cas9gRNA engineering for genome editing, activation and repression, Nat. Methods12 (2015) 1051–1054.

[120] B.P. Kleinstiver, M.S. Prew, S.Q. Tsai, V.V. Topkar, N.T. Nguyen, Z. Zheng, et al.,Engineered CRISPR-Cas9 nucleases with altered PAM specificities, Nature 523(2015) 481–485.

[121] B.P. Kleinstiver, M.S. Prew, S.Q. Tsai, N.T. Nguyen, V.V. Topkar, Z. Zheng, et al.,Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 bymodifying PAM recognition, Nat. Biotechnol. 33 (2015) 1293–1298.

[122] F.A. Ran, L. Cong, W.X. Yan, D.A. Scott, J.S. Gootenberg, A.J. Kriz, et al., In vivogenome editing using Staphylococcus aureus Cas9, Nature 520 (2015) 186–191.

[123] E. Deltcheva, K. Chylinski, C.M. Sharma, K. Gonzales, Y. Chao, Z.A. Pirzada, et al.,CRISPR RNA maturation by trans-encoded small RNA and host factor RNaseIII, Nature 471 (2011) 602–607.

[124] L. Nissim, S.D. Perli, A. Fridkin, P. Perez-Pinera, T.K. Lu, Multiplexed andprogrammable regulation of gene networks with an integrated RNA andCRISPR/Cas toolkit in human cells, Mol. Cell 54 (2014) 698–710.

[125] S.Q. Tsai, N. Wyvekens, C. Khayter, J.A. Foden, V. Thapar, D. Reyon, et al., DimericCRISPR RNA-guided FokI nucleases for highly specific genome editing, Nat.Biotechnol. 32 (2014) 569–576.

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Health & Medicine

Crispr and cancer