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Advances in Nucleases Used for Genome Editing Central JSM Biochemistry & Molecular Biology Bringing Excellence in Open Access Mini Review *Corresponding author Berges Bradford K, Department of Microbiology and Advances in Nucleases Used for Molecular Biology, Brigham Young University, Provo, UT 84602, USA, Tel: 1-801-422-8112; Email: brad.berges@ gmail.com Genome Editing Submitted: 01 September 2016 Solis-Leal A and Berges BK* Accepted: 15 October 2016 Published: 17 October 2016 Department of Microbiology and Molecular Biology, Brigham Young University, USA ISSN: 2333-7109 Abstract Copyright © 2016 Berges et al. Genome editing is an exciting technology that allows for specific manipulation of complex genomes. While the original tools for genome manipulation had low OPEN ACCESS efficiencies, genome editing tools discovered in the past fifteen years have been widely studied and great efforts have been required to improve their efficiency. Keywords This article summarizes how Zinc Finger Nuclease (ZFN), Transcription Activator-Like • Talen Effector Nuclease (TALEN) and Clustered Regularly Interspaced Palindromic Repeat • CRISPR/Cas9 (CRISPR)/Cas9 nucleases work, as well as the most meaningful advances achieved in • Zinc finger nuclease the development of these technologies. • Gene editing • Gene therapy ABBREVIATIONS ZFN: Zinc Finger Nuclease; TALEN: Transcription Activator- Like Effector Nuclease; CRISPR: Clustered Regularly Interspaced (NHEJ) [7]. Further, these enzymes can be engineered to target Palindromic Repeat; DNA: Deoxyribonucleic Acid; DSBs: Double- DNAa single strand site infrom very9 the complex homologous genomes chromosome (e.g., the human as a haploidcorrect Strand Breaks; HDR: Homology-Directed Repair; NHEJ: Non- copygenome to undertakeis 3x10 bp). repair While of the the HDR DSB, mechanism the NHEJ system uses a re-ligatestemplate Homologous End Joining; InDels: Insertions and Deletions; ZF : the cleaved ends and randomly inserts and deletes nucleotides Zinc Finger; BP: Base Pair; AA: Amino Acid; RVDs: Repeat Variable Di-residues; 5MC: 5-Methylated Cytosines; RNA: Ribonucleic pathway produces a faithful copy of the original DNA while NHEJ Acid; CrRNA: CRISPR RNA; TrACrRNA: Trans-Activating CRISPR resulting in mutations referred to as indels. Thus, the HDR RNA; GRNA: Guide RNA; PAM: Protospacer-Associated Motif is anAs error-prone our understanding system [8,9]. of these DNA repair mechanisms has INTRODUCTION grown, so has our ability to develop more effective ways to edit Gene editing entails the engineering of DNA mutations, which coding region of a gene, and when indels are produced this can resultcomplex in genomes.a shift in Genethe reading knockouts frame are ofachieved a protein by cleavingresulting the in ofcan short entail DNA gene sequences deletions, hasgene existed insertions, for decades,or gene modifications.the ability to While the technology required to efficiently promote gene editing completely remove a gene by targeting both ends for DSBs, and ifgene the inactivationends are joined [10]. together Likewise, this this can technique result in may removal be used of the to engineer specific mutations in complex or large genomes has been challenging. For over 40 years the gene editing field has interveningHDR is also gene an [2]. effective tool to insert genes into a complex madedeveloped and new new editing strategies tools andto more strategies efficiently have been engineer discovered such mutations [1]. In such time, substantial advances have been presence of a DSB, the new DNA can serve as a template for genome. By introducing foreign linear DNA into a cell in the thisand technologyimproved. Nucleases has been usedsuch extensivelyas CRISPR/Cas9, to produce TALEN genetically and ZFN, engineeredmake targeted model modifications organisms of complexto study genomes gene functions possible, and viralHDR, vectors,thus inserting plasmids desirable or even single-strandedgenes into specific oligonucleotides sites in the genome [11].This exogenous DNA may be introduced through technology, these systems have a promising future for therapies other biological processes [2]. As we continue to develop the [12,13].Genome editing tools for a wide variety of diseases such as cancer [3], HIV-1 [4], cystic in complex DNA molecules, precise locations need to be targeted fibrosis [5], Duchenne muscular dystrophy [6], among many In order to activate these DNA repair pathways at specific sites otherGenome genetic modifications disorders. The above nucleases produce double-strand breaks (DSBs) induceso as not many to affect cuts inany a othercomplex sequence genome, in molecularthe genome. engineering Although in DNA that lead to the activation of one of two pathways: restriction enzymes typically find their target sequence and homology-directed repair (HDR) or non-homologous end joining is accomplishing a greater efficiency of on-target cleavage and decreasing off-target effects to related DNA sequences. Off- Cite this article: Solis-Leal A, Berges BK (2016) Advances in Nucleases Used for Genome Editing. JSM Biochem Mol Biol 3(2): 1017. Berges et al. (2016) Email: Central Bringing Excellence in Open Access target effects could introduce DNA mutations leading to cancer, mutations (Sharkey and ELD-KKR) have been shown to increase TALEN improvements are not restricted to the FokI nuclease; orand developed so the use in ofrecent highly years specific/targeted and will be discussed nucleases below: is critical. Zinc editing efficiency by working together [22]. other improvements made to TALENs are related to the TALE FingerThe following Nuclease targeted (ZFN), nucleasesTranscription have Activator-Likebeen discovered Effector and/ Nuclease (TALEN) and Clustered Regularly Interspaced Short inscaffold. the RVDs Both enhance the structure TALEN andactivity the lengthor even can allow be itmodified in certain to enhance activity, sensitivity and specificity [23-25]. Mutations PalindromicZinc Finger Repeat nucleases (CRISPR)/Cas9. may be enhanced by the mutation of 3 or 7 cationic amino acids ZFNs consist of two different domains, a His2-Cys2 scenarios, including a report showing that DNA binding specificity zinc finger However,to glutamine such [23]. nucleotides In addition, can still 5-methylated be targeted ifcytosines the amino (5Mc) acids (ZF) and a catalytic domain which contains a FokI nuclease. restrict TALEN activity if they are found in the target sequence. The ZF consists of a ββα structure which binds between 3 and responsible for binding cytosine, His-Asp, are changed to Asn- 4 DNA base pairs (bp) in the presence of a zinc atom using the Gly or even to an asparagine monomer, making this TALE repeat α -helix domain. Typically a ZFN contains between 3 and 6 ZF, thus recognizing longer (and hence more specific) DNA target 33 amino acids long. This modification allows the formation of a sequences [14]. special RVD loop that promotes 5mCbinding [24,25]. RVD, and the N- and C-terminal domains are respectively 287 pairThe of ZFNsis FokI nuclease engineered is a non-specificto bind to two DNA-cleaving opposite strands domain of DNA, that Original TALE monomer residues are similar except at the andupon such dimerization a way that is theable two to produceFokI domains DSB in overlap DNA. Therefore, a sequence a theseaa and second-generation 231 aa long. However, TALENs a variety are known of TALE as Goldy, constructs Sunny with and Each ZF binding domain in the targeted DNA region is separated differing lengths have shown higher DNA cleavage efficiency; frombetween the twoother DNA ZF bindingsequences domain that byare a boundspacer bysequence distinct of ZFNs. 5 to Platinum TALENs. Goldy TALENs have an N-terminus of 158 aa, followed by the repeat/binding domain, then with63 aa in the 7bp [15]. C-terminus [26]. Sunny TALENs have an N-terminal region of Targeting specific DNA sequences using ZF is essential 207aa and a C-terminal regional of 63 aa. They also show a single to be able to use this tool. Though designing ZFs with unique mutation, P11H, in the C-terminus [27]. Platinum TALENs either specificity was not completely successful [16], different selection have an N-terminal region of 136aa and a C-terminal region of methods are providing new ways to specifically select for ZF with 63 aa, or have an N-terminal regionth and of 32 153nd aa and a C-terminal higher specificity [17]. One of these selection methods help ZF region of 47 aa. They also show non-RVD variants (Ala-Asp, Asp- to recognize specific sequences and allow to combine multiple Ala,CRISPR/Cas9 Asp-Asp, or Glu-Ala) in the 4 aa positions [28]. ZF DNA binding domains together, thus creating multi-fingered enzymes that are able to recognize longer DNA sequences and hence to cut more specifically. This novel method has been it hasAlthough since beenthe CRISPR/Cas9manipulated typeby researchers II system wasas an discovered excellent nucleasesnamed OPEN mentioned [18], and in is thisa very article promising are highly way to promising, target unique the as a novel type of prokaryotic adaptive immuneEscherichia system [29], coli, introductionsequences in ofeukaryotic the genes genomes. encoding Although these nucleases ZFNs and to the all other cells where DNA sequences showed a pattern of repeats separated genome editing tool. This system was found in research determined that small pieces of foreign DNA (usually thatTALENs need to be genetically modified is still a major challenge. fromby a spacer plasmids sequence or bacteriophages) (also called protospacer) are integrated [30]. into Subsequent
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