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Cut and paste the genome: Genome editing for research and therapy

Abstract

Genome engineering, or alternatively called ‘genome editing,’ has been one of the rapidly growing fields of biotechnology for the last few decades. Scientists are now making targeted modifications of genome in any organism of choice with improved precision. In this mini review, we provide basic, fundamental theory and mechanisms of the well-known genome editing technologies such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system. We also discuss its role in the field of genetic research and highlight its therapeutic potential as an indispensable tool for the development of personalized medicine to come in near future.

1Introduction

For the last few decades, the biotechnology to decipher individual human genome such as whole-genome sequencing (WGS, [1]) and whole-exome sequencing (WES, [2]) advanced greatly, lowering barriers to decoding the information embedded in human genome. Such technological advances are anticipated to revolutionize our understanding of clinical genetics and to deliver personalized medicine in near future. Nevertheless, the present challenge lies in converting this tremendous amount of data (i.e., billions of nucleotides that comprise DNA of an individual) into clinically relevant information, such as how genotype affects phenotype. To transform the data into functionally relevant knowledge, technologies such as targeted gene inactivation by homologous recombination [3] or targeted gene knockdown by RNAi [4] have been utilized as means of providing empirical information to elucidate the function of genes of interest. However, the former has limitations such as extremely low efficiency of correct insertion into the target chromosome (1 in 106∼1 in 109) [5] and potential adverse mutagenesis [6], while the latter has limitations including incomplete and temporary knockdown of target gene and undesired off-target effects [7]. More recently, genome engineering technologies, commonly referred to as ‘genome editing,’ have been emerged, enabling scientists to make targeted modifications to the genome in practically any organism of choice with improved precision [7a]. This technology utilizes engineered nucleases which are complex of sequence-specific DNA binding domains and nonspecific DNA cleavage modules [8]. In this brief review, we aim to provide basic information on this genome editing technologies and discuss the applications and the therapeutic potential of these technologies, as well as future prospects.

2Principle mechanisms of genome editing technologies

Genome editing technology involves customized programmable DNA-binding nucleases such as zinc-finger nucleases (ZFNs) [8], transcription activator-like effector nucleases (TALENs) [9], and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 (CRISPR-associated 9) nucleases [10]. Although each nuclease has unique mode of action, in principle, these nucleases recognize, bind to, and cleave chromosomal DNA to create site-specific DNA double strand breaks (DSBs). Subsequently, these DSBs trigger endogenous DNA repair systems, such as homology directed repair (HDR) or error-prone non-homologous end joining (NHEJ), resulting in targeted genome modification (Fig. 1) [11].

3Programmable DNA-binding nucleases

3.1Zinc-finger nucleases (ZFNs)

First discovered in transcription factor from Xenopus laevis [12], Cys2-His2 zinc-fingers are the most common sequence-specific DNA-binding motifs found in all eukaryotic organisms [13]. Each zinc-finger motif consists of about 30 amino acids in a ββα configuration, and two cysteines and two histidines coordinate a single zinc atom in individual zinc-finger [14]. Amino acids in positions −1, 3, and 6 on the α-helix contact 3 base pairs in the major groove of DNA [15]. Thus, theoretically, a combination of 6 separate zinc-fingers that each recognizes a 3 base pair DNA sequence can consequently recognize a specific DNA sequence of 18 base pairs, which is long enough sequence to specify a unique site in the human genome. The construction of such unnatural, synthetic zinc-fingers that recognized 18 base pairs of DNA sequence made application of zinc-finger proteins for recognizing specific DNA sequence possible [16]. However, it was the combination of zinc-finger proteins with cleavage domain of endonuclease that made possible to realize the concept of targeted genome engineering. Isolated from Flavobacterium okeanokoites, the type II restriction endonuclease Fok I recognizes the DNA sequence of 5’-GGATG-3’ and cleaves 9th and 13th base pairs from the recognition site, regardless of the sequence at the cleaved site [17]. When the cleavage domain of Fok I endonuclease was combined with zinc-finger protein to generate zinc-finger-Fok I fusion proteins, these hybrid fusion proteins (ZFNs) were able to cut DNA at the predetermined sequences [18].

Since Fok I cleavage domain must dimerize to catalyze DNA cleavage, during ZFN-mediated site specific DNA cleavage, ZFN target sites consisted of two zinc-finger binding sites separated by 5∼7 base pairs of spacer sequence are occupied by two ZFNs on each strand and the cleavage domain of each ZFN dimerize on the spacer sequence and cleaves it (Fig. 2). Currently there are 3 large sets of constructed ZF proteins (ZFPs) from independent groups, namely The Scripps Research Institute [19–21], Sangamo Biosciences [22], and ToolGen [23], are available. Recently published articles on the use of ZFNs are listed in Table 1. The contents of Table 1 are not based on scientific significance by any means. It is just examples of recent research trend involving use of ZFNs. As demonstrated in the Table below, researches on ZFNs are still active, and most of the cases the ZFNs are utilized as a tool for studying function of certain genes by inducing gene disruptions. However, there are also studies to correct mutations in human cells, implicating the therapeutic potential of genome editing using ZFNs.

3.2Transcription activator-like effector nucleases (TALENs)

TAL (transcription activator-like) proteins were first identified as secreted proteins from bacterial plant pathogen Xanthomonas, and the named based on observation that they activated transcription of endogenous pathogenic genes in plants [35, 36]. Once they enter the nucleus of host cells, they bind to effector-specific sequences on the promoters of host genes to initiate transcription [37]. TALENs are composed of DNA-binding domain (DBD), N-terminal, C-terminal, and FokI endonuclease domain. The DBD of TALEN is composed of ‘repeats’ that itself composed of 33∼35 amino acids [38].

Among these amino acids, the amino acid residues at position 12 and 13 of each repeat decide which nucleotide of DNA they bind to (i.e., Asparagine-Isoleucine (NI) ⟶ Adenine (A), Asparagine-Glycine (NG) ⟶ Thymine (T), Aspargine-Asparagine (NN) ⟶ Guanine (G), Histidine-Aspartic Acid (HD) ⟶ Cytosine (C)) so they are named as ‘repeat-variable di-residue (RVD) [39]. It is this one RVD to one nucleotide match between the RVD of TALENs and chromosomal DNA that underpins target sequence specificity of TALENs (Fig. 3A). Furthermore, this sequence specific interaction and simplicity of its coding system made generation of customized TALE domains that target DNA sequence of interest possible [40, 41].

The TALE domains can be assembled in predetermined combinations and linked to the Fok I nuclease to drive sequence-specific DSB of chromosomal DNA [42]. TALENs, in pairs, bind to opposing target sequences so that the linked Fok I nucleases come in contact with the spacer from opposite sides. The Fok I nucleases from each TALEN forms Fok I dimer and it cleaves double-stranded DNA (Fig. 3B). As indicated in the Table 2, TALEN-mediated genome editing is also widely used to facilitate gene disruption, addition, and correction.

3.3Clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9

The existence of CRISPR was first discovered by Nakata et al. in studying iap gene of E.coli [62]. What they found was 29 nucleotide-long repeats interspaced by 5 intervening 32 nucleotide-long, non-repetitive sequences. The actual word CRISPR for describing such repeat sequences was first coned by Jansen in 2002 [63]. The CRISPR loci are composed of a set of CRISPR-associated (Cas) genes and a series of repeats (direct repeats, approximately 20–50 base pairs) interspaced by unique, non-repetitive sequences (spacers).

These non-repetitive sequences correspond to the sequences of foreign genetic elements (protospacers) [64]. the protospacers are flanked by a short protospacer adjacent motif (PAM) that is either located on the 3’ (type II CRISPR) or 5’ (type I CRISPR) of foreign DNA [65]. Additionally, leader sequence, rich in A+T sequences, is known to serve as a promoter element for the CRISPR loci [66] (Fig. 4). CRISPR is an essential components of RNA-based adaptive immune systems of bacteria and archaea [67]. In response to foreign genetic element challenges, bacteria and archaea incorporate short fragments of foreign genetic elements into host genome as shown in Fig. 4. When CRISPR locus is transcribed, the long primary transcript is processed to produce a library of short CRISPR-derived RNAs (crRNAs) [68, 69]. Each crRNA has a complementary sequence to a previously encountered foreign genetic elements, and it mediates detection and subsequent destruction of foreign nucleic acids [70]. This unique immune system can be divided into 3 distinct CRISPR types (type I-III) based on gene conservation and locus organization [71]. Especially in the type II CRISPR/Cas system, crRNAs hybridize with trans-activating crRNAs (tracrRNAs) to facilitate RNA-guided sequence-specific DNA cleavage by Cas9 proteins. Cas9 utilizes RNA-DNA pairing to target foreign DNA, and Cas9-RNA complex-mediated DNA cleavage requires recognition of PAM where DNA strand separation and RNA-DNA hybrid formation occurs(Fig. 5) [72].

DNA sequence. In fact, reprogrammed RNA-guided nucleases have been demonstrated to be competent in facilitating gene disruption, addition, and correction in human cells and other model organisms (Table 3).

Compared to ZFNs and TALENs, CRISPR/Cas9 system has higher efficiency and shortest target length, and it can also facilitate multiplex targeting [79]. Furthermore, Cas9 can be used for applications other than usual category of gene modifications (disruption, addition, and correction), such as, but not limited to, transcriptional control or DNA labeling [80, 81].

4Perspectives: The role of genome editing in achieving personalized medicine

Although it is in its infancy, over the last few decades, genome editing has come of age and tremendous interest has been placed on its utility as a tool for basic/clinical genetics. Accumulating data also indicate its potential as a dexterous and powerful means of personalized medicine which is expected to provide individual patient with ‘tailored’ therapeutics near future. Promising results using these site-specific nucleases in therapeutic approaches against severe combined immune deficiency (SCID) [82], sickle cell disease [83], and hemophilia B [83], make the hopes of personalized medicine up ever more. For ‘personalized medicine’ to be realized, close cooperation of related biotechnologies and disciplines is required (Fig. 6).

For example, an ideal starting point of personalized medicine would be genomic information analysis of an individual patient. Biotechnologies such as WGS and WES would be a valuable tool for this task. From the genomic analysis by sequencing can identify specific genetic defects of patients (i.e., single nucleotide polymorphism, SNP), and the relationship between specific SNP and phenotypic manifestation can be systemically examined both in vitro and in vivo. At this stage comes the genome editing technology. With the genome editing technology (i.e., gene knock in/out, point mutation, gene correction, and etc.) the functional consequences of a certain SNP can be determined in details (research-oriented utilization of genomic editing). Furthermore, genomic editing can serve as a part of therapeutic process that requires genomic modifications (i.e., gene correction of induced pluripotent stem cells (iPS) from specific patient). Thus, the genome editing technology can be both research-oriented and therapy-oriented applications. Although there are still some unsolved issues regarding detailed mechanisms of individual genome editing such as how foreign sequences are selected and incorporated into the CRISPR loci of host, it is undoubtful that these technology eventually revolutionizes the field of genetic research as well as our understanding of diseases and opens a new paradigm in fighting currently incurable diseases.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgments

This study was supported by a Korea Science and Engineering Foundation grant funded by the Korean government (MEST) (2014030459) and a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (A120478).

References

1 

Ng PC, Kirkness EF2010Whole genome sequencingMethods in Molecular Biology628215226

2 

Rabbani B, Tekin M, Mahdieh N2014The promise of whole-exome sequencing in medical geneticsJournal of Human Genetics59515

3 

Capecchi MR2005Gene targeting in mice: Functional analysis of the mammalian genome for the twenty-first centuryNature Reviews Genetics6507512

4 

Mohr SE, Smith JA, Shamu CE, Neumuller RA, Perrimon N2014RNAi screening comes of age: Improved techniques and complementary approachesNature Reviews Molecular Cell Biology15591600

5 

Capecchi MR1989Altering the genome by homologous recombinationScience24412881292

6 

Vasquez KM, Marburger K, Intody Z, Wilson JH2001Manipulating the mammalian genome by homologous recombinationProceedings of the National Academy of Sciences of the United States of America9884038410

7 

Moore CB, Guthrie EH, Huang MT, Taxman DJ2010Short hairpin RNA (shRNA): Design, delivery, and assessment of gene knockdownMethods in Molecular Biology629141158

{ label needed for ref[@id='r7a'] } 

Wang W, Xu X, Li Z, Lendlein A, Ma N2014Genetic engineering of mesenchymal stem cells by non-viral gene deliveryClin Hemorheol Microcirc581948

8 

Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD2010Genome editing with engineered zinc finger nucleasesNature Reviews Genetics11636646

9 

Joung JK, Sander JD2013TALENs: A widely applicable technology for targeted genome editingNature Reviews Molecular Cell Biology144955

10 

Maggio I, Holkers M, Liu J, Janssen JM, Chen X, Goncalves MA2014Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cellsScientific Reports45105

11 

Kim H, Kim JS2014A guide to genome engineering with programmable nucleasesNature Reviews Genetics15321334

12 

Miller J, McLachlan AD, Klug A1985Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytesThe EMBO Journal416091614

13 

Pabo CO, Peisach E, Grant RA2001Design and selection of novel Cys2His2 zinc finger proteinsAnnual Review of Biochemistry70313340

14 

Beerli RR, Barbas CF3rd2002Engineering polydactyl zinc-finger transcription factorsNature Biotechnology20135141

15 

Pavletich NP, Pabo CO1991Zinc finger-DNA recognition: Crystal structure of a Zif268-DNA complex at 2.1 AScience252809817

16 

Liu Q, Segal DJ, Ghiara JB, Barbas CF3rd1997Design of polydactyl zinc-finger proteins for unique addressing within complex genomesProceedings of the National Academy of Sciences of the United States of America9455255530

17 

Sugisaki H, Kanazawa S1981New restriction endonucleases from Flavobacterium okeanokoites (FokI) and Micrococcus luteus (MluI)Gene167378

18 

Kim YG, Cha J, Chandrasegaran S1996Hybrid restriction enzymes: Zinc finger fusions to Fok I cleavage domainProceedings of the National Academy of Sciences of the United States of America9311561160

19 

Dreier B2005Development of zinc finger domains for recognition of the 5’-CNN-3’ family DNA sequences and their use in the construction of artificial transcription factorsThe Journal of Biological Chemistry2803558835597

20 

Dreier B, Beerli RR, Segal DJ, Flippin JD, Barbas CF3rd2001Development of zinc finger domains for recognition of the 5’-ANN-3’ family of DNA sequences and their use in the construction of artificial transcription factorsThe Journal of Biological Chemistry2762946629478

21 

Segal DJ, Dreier B, Beerli RR, Barbas CF3rd1999Toward controlling gene expression at will: Selection and design of zinc finger domains recognizing each of the 5’-GNN-3’ DNA target sequencesProceedings of the National Academy of Sciences of the United States of America9627582763

22 

Liu Q, Xia Z, Zhong X, Case CC2002Validated zinc finger protein designs for all 16 GNN DNA triplet targetsThe Journal of Biological Chemistry27738503856

23 

Bae KH2003Human zinc fingers as building blocks in the construction of artificial transcription factorsNature Biotechnology21275280

24 

Park A, Liegel RP, Ronchetti A, Ebert AD, Geurts A, Sidjanin DJ2014Targeted disruption of Tbc1d20 with zinc-fingernucleases causes cataracts and testicular abnormalities in miceBMC Genetics15135

25 

Salabi F, Nazari M, Chen Q, Nimal J, Tong J, Cao WG2014Myostatin knockout using zinc-finger nucleases promotesproliferation of ovine primary satellite cells in vitro Journal of Biotechnology192PA268280

26 

Sampson KE, Brinker A, Pratt J, Venkatraman N, Xiao Y, Blasberg J, Steiner T, Bourner M, Thompson DC2015Zinc finger nuclease-mediated gene knockout results in loss of transport activity for P-glycoprotein, BCRP, andMRP2 in Caco-2 cellsDrug metabolism and disposition: the biological fate of chemicals43199207

27 

Ding W, Hu Z, Zhu D, Jiang X, Yu L, Wang X, Zhang C, Wang L, Ji T, Li K, He D, Xia X, Liu D, Zhou J, Ma D, Wang H2014Zinc finger nucleases targeting the human papillomavirus E7 oncogene induce E7 disruptionand a transformed phenotype in HPV16/18-positive cervical cancer cellsClinical cancer research: an officialjournal of the American Association for Cancer Research2064956503

28 

Yi G2014CCR5 gene editing of resting CD4(+) T cells by transient ZFN expression from HIV envelope pseudotyped nonintegrating lentivirus confers HIV-1 resistance in humanized miceMolecular Therapy Nucleic Acids3e198

29 

Liu X2014Generation of mastitis resistance in cows by targeting human lysozyme gene to beta-casein locus using zinc-finger nucleasesProceedings Biological Sciences / The Royal Society28120133368

30 

Lombardo A2011Site-specific integration and tailoring of cassette design for sustainable gene transferNature Methods8861869

31 

Merling RK, Sweeney CL, Chu J, Bodansky A, Choi U, Priel DL, Kuhns DB, Wang H, Vasilevsky S, De Ravin SS, Winkler T, Dunbar CE, Zou J, Zarember KA, Gallin JI, Holland SM, Malech HL2015An AAVS1-targeted minigene platformfor correction of iPSCs from all five types of chronic granulomatous diseaseMolecular therapy: the journal ofthe American Society of Gene Therapy23147157

32 

Zhang W2014Targeted genome correction by a single adenoviral vector simultaneously carrying an inducible zinc finger nuclease and a donor templateJournal of Biotechnology 188C16

33 

Zou J, Mali P, Huang X, Dowey SN, Cheng L2011Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell diseaseBlood11845994608

34 

Sebastiano V2011In situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleasesStem Cells2917171726

35 

Bogdanove AJ, Voytas DF2011TAL effectors: Customizable proteins for DNA targetingScience33318431846

36 

Kay S, Hahn S, Marois E, Hause G, Bonas U2007A bacterial effector acts as a plant transcription factor and induces a cell size regulatorScience318648651

37 

Bogdanove AJ, Schornack S, Lahaye T2010TAL effectors: Finding plant genes for disease and defenseCurrent Opinion in Plant Biology13394401

38 

Cermak T2011Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targetingNucleic Acids Research39e82

39 

Moscou MJ, Bogdanove AJ2009A simple cipher governs DNA recognition by TAL effectorsScience3261501

40 

Miller JC2011A TALE nuclease architecture for efficient genome editingNature Biotechnology29143148

41 

Zhang F, Cong L, Lodato S, Kosuri S, Church GM, Arlotta P2011Efficient construction of sequence-specific TAL effectors for modulating mammalian transcriptionNature Biotechnology29149153

42 

Li T2011TAL nucleases (TALNs): Hybrid proteins composed of TAL effectors and FokI DNA-cleavage domainNucleic Acids Research39359372

43 

Zhang Z, Wu E, Qian Z, Wu WS2014A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targetsScientific Reports47338

44 

Elbaz I, Lerer-Goldshtein T, Okamoto H, Appelbaum L2014Reduced synaptic density and deficient locomotor response inneuronal activity-regulated pentraxin 2a mutant zebrafishFASEB Journal: Official Publication of the Federationof American Societies for Experimental Biology

45 

Choi J, Suzuki KI, Sakuma T, Shewade L, Yamamoto T, Buchholz DR2014Unliganded thyroid hormone receptor alpha regulates developmental timing via gene repression as revealed by gene disruption in Xenopus tropicalisEndocrinologyen20141554

46 

Kabir S, Hockemeyer D, de Lange T2014TALEN gene knockouts reveal no requirement for the conserved human shelterin protein Rap1 in telomere protection and length regulationCell Reports912731280

47 

Lugassy J2014Modulation of TCR responsiveness by the Grb2-family adaptor, GadsCellular Signalling27125134

48 

Tatsumi Y, Takeda M, Matsuda M, Suzuki T, Yokoi H2014TALEN-mediated mutagenesis in zebrafish reveals a role forr-spondin 2 in fin ray and vertebral developmentFEBS Letters58845434550

49 

Liu C, Xiao L, Li F, Zhang H, Li Q, Liu H, Fu S, Li C, Zhang X, Wang J, Staunstrup NH, Li Y, Yang H2014Generation of outbred Ace2 knockout mice by RNA transfection of TALENs displaying colitis reminiscentpathophysiology and inflammationTransgenic research

50 

Zhang Z, Zhu B, Ge W2015Genetic analysis of zebrafish gonadotropin (FSH and LH) functions by TALEN-mediated genedisruptionMolecular Endocrinology297698

51 

Wen L, Fu L, Guo X, Chen Y, Shi YB2015Histone methyltransferase Dot1L plays a role in postembryonic development inXenopus tropicalisFASEB Journal: Official Publication of the Federation of American Societies for ExperimentalBiology29385393

52 

Chen B2015Disruption of microRNA-21 by TALEN leads to diminished cell transformation and increased expression of cell-environment interaction genesCancer Letters356506516

53 

Krentz NA, Nian C, Lynn FC2014TALEN/CRISPR-mediated eGFP knock-in add-on at the OCT4 locus does not impact differentiation of human embryonic stem Cells towards endodermPloS One9e114275

54 

Li HL, Fujimoto N, Sasakawa N, Shirai S, Ohkame T, Sakuma T, Tanaka M, Amano N, Watanabe A, Sakurai H, Yamamoto T, Yamanaka S, Hotta A2015Precise correction of the dystrophin gene in duchenne muscular dystrophypatient induced pluripotent stem cells by TALEN and CRISPR-Cas9Stem cell reports4143154

55 

Ousterout DG2013Reading frame correction by targeted genome editing restores dystrophin expression in cells from Duchenne muscular dystrophy patientsMolecular Therapy: The Journal of the American Society of Gene Therapy2117181726

56 

Ramalingam S, Annaluru N, Kandavelou K, Chandrasegaran S2014TALEN-Mediated generation and genetic correction of disease-specific human induced pluripotent stem cellsCurrent Gene Therapy14461472

57 

Suzuki K2014Targeted gene correction minimally impacts whole-genome mutational load in human-disease-specific induced pluripotent stem cell clonesCell Stem Cell153136

58 

Ma N2013Transcription activator-like effector nuclease (TALEN)-mediated gene correction in integration-free beta-thalassemia induced pluripotent stem cellsThe Journal of Biological Chemistry2883467134679

59 

Sun N, Zhao H2014Seamless correction of the sickle cell disease mutation of the HBB gene in human induced pluripotent stem cells using TALENsBiotechnology and Bioengineering11110481053

60 

Low BE, Krebs MP, Joung JK, Tsai SQ, Nishina PM, Wiles MV2014Correction of the Crb1rd8 allele and retinal phenotype in C57BL/6N mice via TALEN-mediated homology-directed repairInvestigative Ophthalmology & Visual Science55387395

61 

Dupuy A2013Targeted gene therapy of xeroderma pigmentosum cells using meganuclease and TALENPloS One8e78678

62 

Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A1987Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene productJournal of Bacteriology16954295433

63 

Jansen R, Embden JD, Gaastra W, Schouls LM2002Identification of genes that are associated with DNA repeats in prokaryotesMolecular Microbiology4315651575

64 

Shah SA, Erdmann S, Mojica FJ, Garrett RA2013Protospacer recognition motifs: Mixed identities and functional diversityRNA Biology10891899

65 

Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, Almendros C2009Short motif sequences determine the targets of the prokaryotic CRISPR defence systemMicrobiology155733740

66 

Pougach K2010Transcription, processing and function of CRISPR cassettes in Escherichia coliMolecular Microbiology7713671379

67 

Wiedenheft B, Sternberg SH, Doudna JA2012RNA-guided genetic silencing systems in bacteria and archaeaNature482331338

68 

Deltcheva E2011CRISPR RNA maturation by trans-encoded small RNA and host factor RNase IIINature471602607

69 

Gesner EM, Schellenberg MJ, Garside EL, George MM, Macmillan AM2011Recognition and maturation of effector RNAs in a CRISPR interference pathwayNature Structural & Molecular Biology18688692

70 

Garneau JE2010The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNANature4686771

71 

Makarova KS2011Evolution and classification of the CRISPR-Cas systemsNature Reviews Microbiology9467477

72 

Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA2014DNA interrogation by the CRISPR RNA-guided endonuclease Cas9Nature5076267

73 

Mehrabian M2014CRISPR-Cas9-based knockout of the prion protein and its effect on the proteomePloS One9e114594

74 

Cho SW, Kim S, Kim JM, Kim JS2013Targeted genome engineering in human cells with the Cas9 RNA-guided endonucleaseNature Biotechnology31230232

75 

Cong L2013Multiplex genome engineering using CRISPR/Cas systemsScience339819823

76 

Mali P2013RNA-guided human genome engineering via Cas9Science339823826

77 

Wu Y, Zhou H, Fan X, Zhang Y, Zhang M, Wang Y, Xie Z, Bai M, Yin Q, Liang D, Tang W, Liao J, Zhou C, Liu W, Zhu P, Guo H, Pan H, Wu C, Shi H, Wu L, Tang F, Li J2015Correction of a genetic disease byCRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cellsCell research256779

78 

Long C, McAnally JR, Shelton JM, Mireault AA, Bassel-Duby R, Olson EN2014Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNAScience34511841188

79 

Chen L2014Advances in genome editing technology and its promising application in evolutionary and ecological studiesGiga Science324

80 

Hsu PD, Lander ES, Zhang F2014Development and applications of CRISPR-Cas9 for genome engineeringCell15712621278

81 

Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F2015Genome-scale transcriptional activation by an engineeredCRISPR-Cas9 complexNature517583588

82 

Urnov FD2005Highly efficient endogenous human gene correction using designed zinc-finger nucleasesNature435646651

83 

Li H2011 In vivo genome editing restores haemostasis in a mouse model of haemophiliaNature475217221

Figures and Tables

Fig.1

Schematics of genome editing process.

Schematics of genome editing process.
Fig.2

ZFN dimerize on the spacer sequence via sequence-specific interaction between ZFP and DNA. Amino acids inpositions −1, 3, and 6 on the α-helix recognizes and contact 3 base pairs in the major groove of DNA. FokI cleavage domains from each ZFN dimerize over the spacer sequence to facilitate DBS.

ZFN dimerize on the spacer sequence via sequence-specific interaction between ZFP and DNA. Amino acids inpositions −1, 3, and 6 on the α-helix recognizes and contact 3 base pairs in the major groove of DNA. FokI cleavage domains from each ZFN dimerize over the spacer sequence to facilitate DBS.
Fig.3

Structures of TALE and TALEN. A. Schematic presentation of TALE (TAL effector). Each TALE repeat contains 33–35 amino acids, and each RVD (amino acids at positions 12 and 13) recognizes a corresponding single base pair. RVD within a consensus repeat sequence is indicated in red. NLS: nuclear localization signal, AD: transcriptional activation domain. B. TALEN pair binds to specific DNA sequence recognized by TALEs. FokI cleavage domains from each TALE dimerize over the spacer sequence. In most cases, naturally occurring TALEs are preceded by T at the 5’-end [39] as indicated by red box.

Structures of TALE and TALEN. A. Schematic presentation of TALE (TAL effector). Each TALE repeat contains 33–35 amino acids, and each RVD (amino acids at positions 12 and 13) recognizes a corresponding single base pair. RVD within a consensus repeat sequence is indicated in red. NLS: nuclear localization signal, AD: transcriptional activation domain. B. TALEN pair binds to specific DNA sequence recognized by TALEs. FokI cleavage domains from each TALE dimerize over the spacer sequence. In most cases, naturally occurring TALEs are preceded by T at the 5’-end [39] as indicated by red box.
Fig.4

Schmatics of a generalized CRISPR locus. Upon introduction of foreign genetic elements from bacteriophages or plasmids, Cas proteins obtain spacers from the exogenous protospacer sequences and they are incorporated into the CRISPR locus of host genome.

Schmatics of a generalized CRISPR locus. Upon introduction of foreign genetic elements from bacteriophages or plasmids, Cas proteins obtain spacers from the exogenous protospacer sequences and they are incorporated into the CRISPR locus of host genome.
Fig.5

DNA cleavage by type II CRISPR nuclease system. In type II CRISPR, crRNA-tracrRNA hybrids complex with Cas9 to facilitate sequence-specific DNA cleavage. Filled triangle indicates cleavage by Cas ribonucleases. Small arrows indicate DNA cleavage by Cas9. sgRNA: single guide RNA.

DNA cleavage by type II CRISPR nuclease system. In type II CRISPR, crRNA-tracrRNA hybrids complex with Cas9 to facilitate sequence-specific DNA cleavage. Filled triangle indicates cleavage by Cas ribonucleases. Small arrows indicate DNA cleavage by Cas9. sgRNA: single guide RNA.
Fig.6

Example of genome editing utilized in personalized medicine.

Example of genome editing utilized in personalized medicine.
Table 1

Selected recent examples of ZFN-mediated genome editing in various organisms including human cells

Gene modificationOrganismGenesRefs
DisruptionMousetbc1d20, rab3gap1, rab3gap2, rab18[24]
Sheepmstn[25]
HumanMDR1, MRP2, BCRP[26]
HumanHPV E7[27]
Humanized mouseCCR5[28]
AdditionCowhLYZ[29]
HumanCCR5[30]
CorrectionHumanAAVS1[31, 32]
HumanHBB[33, 34]

Few selected, recent articles searched through Pubmed. The literature search was to overview the research trend regarding ZFN-mediated genome editing.

Table 2

Selected recent examples of TALEN-mediated genome editing in various organisms including human cells

Gene modificationOrganismGenesRefs
DisruptionMousec-kit, PU.1[43]
Zebrafishnptx2a[44]
X. tropicalis TRα [45]
HumanRap1[46]
HumanGads[47]
Zebrafishrspo2[48]
Mouseace2[49]
Zebrafishfshb, lhb[50]
X. tropicalis dot1l[51]
HumanmiR-21[52]
AdditionHumaneGFP[53]
CorrectionHumanDMD[54, 55]
HumanCCR5[56]
HumanHBB[57–59]
MouseCrb1[60]
HumanXPC[61]

Few selected, recent articles searched through Pubmed. The literature search was to overview the research trend regarding TALEN-mediated genome editing.

Table 3

Selected recent examples of CRISPR/Cas-mediated genome editing in various organisms including human cells

Gene modificationOrganismGenesRefs
DisruptionMousePrP[73]
HumanCCR5[74]
humanPVALB, EMX1[75]
AdditionHumanAAVS1[76]
CorrectionMouseCrygc[77]
HumanDMD[54]
MouseDMD[78]

Few selected, recent articles searched through Pubmed. The literature search was to overview the research trend regarding CRISPR/Cas-mediated genome editing.