Biological and Technological Implications of Meganucleases

Huifen Xu1,BinLiu1, and Jose Pardinas2 ∗

1College of Pharmaceutical Science, Soochow University, Suzhou 215123, China 2Janssen R&D, Spring House, PA, 19477, USA

Meganucleases are a class of rare endonucleases encoded by introns and inteins which can recognize long non- palindromic double-stranded DNA sequences ranging from 12 to 40 base pairs. Meganucleases can recognize and cleave an intron-free homing homolog allele and insert an intron via a double-stranded break-repair mechanism. Thus, meganuclease can change an intron-free allele to an allele containing an intron. Given their ability to introduce one or more double-strand breaks in complex genomes, meganucleases are useful tools for biotechnology applications such as construction of high-resolution physical maps, genome analysis, cloning, and genome engineering.

KEYWORDS: Meganuclease, Gene Editing.

INTRODUCTION IP: 192.168.39.211 On: Fri, but24 Sep may 2021 reduce 06:46:58 the cleaving rate to varying degrees. Copyright: American Scientific Publishers Since their discovery in 1977, introns have beenDelivered defined by AlthoughIngenta quite a few meganuclease targets have been as sequences of variable length found within genes that experimentally identified, the sequences of most recogni- are frequently excised at the RNA transcript level. Introns tion sites are presently not precisely known. do not generally survive to participate in gene expres- Meganucleases are generally named according to the sion at the protein level. The discovery of self-splicing by nomenclature used for other endonucleases. If the enzyme Thomas Cech and coworkers was a key milestone in intron is encoded by an intron, its name is prefixed with “I-,” and excision research. Self-splicing introns are found mostly if encoded by an intein, its name is prefixed with “PI-.”2 in plants and the mitochondria and chloroplasts of some lower eukaryotes such as Tetrahymena, Green Euglena, fungus, and yeast. Self-splicing introns can be divided into MEGANUCLEASE FAMILIES two groups: Group I introns require guanosine (acid) for LAGLIDADG Family self-splicing and do not form a lariat, or lasso-like struc- The LAGLIDADG family, also called the ‘LAGLIDADG,’ ture. Group II introns do not require guanosine for self- ‘DOD,’ ‘dodecapeptide,’ ‘dodecamer’ and ‘decapeptide’ splicing and form a lariat.1 family, is a large family with more than 200 members.1 3 4 Meganucleases are encoded by introns and inteins and The LAGLIDADG family is the most diverse of the belong to a class of enzymes characterized by large, hence meganuclease families. It is found in a large number very rare, recognition sites. For instance, a meganucle- of organisms: The chloroplasts of plants and seaweed; ase recognition sequence of 18 bp should appear only the mitochondria of fungi and protozoa; and in bacte- once by chance in a 7 × 1010 bp sequence, which corre- ria and archaea. Its ability to introduce ancient introns sponds roughly to the length of 20 mammalian genomes. and inteins into intron-less alleles makes it widespread in In sharp contrast to standard endonucleases, the meganu- genomes. clease recognition sequence is not highly specific. For A meganuclease is classified as a member of this fam- example, a single base change does not affect recognition ily by the presence of one or two LAGLIDADG motif

∗ sequences. I-CreI, I-CeuI and other LAGLIDADG family Author to whom correspondence should be addressed. members that contain a single copy of the LAGLIDADG E-mail: [email protected] Received: 10 January 2014 motif act as homodimers. I-DmoI, PI-SceI and other Accepted: 8 April 2014 LAGLIDADG family members that contain two motif

Gene Gene Edit. 2015, Vol. 1, No. 1 2376-3949/2015/1/041/006 doi:10.1166/gge.2015.1005 41 Biological and Technological Implications of Meganucleases Xu et al. sequences act as monomers. All LAGLIDADG family meganucleases can recognize DNA sites 13–40 bp long and cleave DNA to produce 3 ends with 4 bp overhangs. The LAGLIDADG family is encoded by group II introns. The exonuclease and homing activities of meganucleases are closely associated with their structural evolution.5 6

GIY-YIG Family Y have a characteristic GIY-(X10–11)-YIG motif.7 GIY- YIG meganuclease has been found in T4 Phage. It can be used independently as either an enzyme8 or as a movable Group I intron element.9 The GIY-YIG intron’s open reading frame has been reported in fungal mitochondria10–12 as well as in algae mitochondria13 14 and chloroplasts.15 16

His-Cys Box Family The proteins of this small family are encoded by group I introns.17 These movable introns are found in mold, fungus and the highly-conserved sequences of the amoeba’s ribosomal subunits. The family I-PpoI from Physarum is the most extensively studied.18 Characteristically, the His-Cys box contains more than 100 highly-conserved histidine and cysteine residues.17 19 A number of studies have established the significance of the His-Cys box motif and of its conserved residues, and have elucidated in detail its nucleolytic cleavage mechanism. IP: 192.168.39.211 On: Fri, 24 Sep 2021 06:46:58 H–N–H Family Copyright: American Scientific Publishers Delivered by Ingenta The H–N–H family is the least typical biochemically and structurally, and it is the least restricted to the group I intron family. It has been identified in the non- specific nucleases20 21 and in proteins encoded by Group II introns.22 23 The two conserved histidine residues in the HNH proteins are flanked by 30–33 conserved aspartic Figure 1. Group I intron (left), Group II intron (right). The mov- residues.24 25 ing open reading frame and its product are represented in Most HNH family members are encoded by group I red; exons and their products are green; other nucleic acid sequences are black. introns, including I-HmuI, I-HmuII and I-TevIII. I-HmuI and I-HmuII are found in the two closely related B. subtilis bacteriophages SPO1 and SP83.26–28 HNH family mem- (f) Donor/template DNA separates from target DNA. bers are also encoded by I-TevIII from the nrdB intron of (g) After DNA repair-synthesis, part of the intron’s exon the phage RB3 gene.29 30 wings are often copied to the target DNA. All of the above can be summarized in one sentence: MEGANUCLEASE CLEAVING MECHANISM The Double-Strand Break Repair (SDBR) pathway uses (A) Group I intron homing cleavage mechanism the donor intron DNA as template, replicates it and inserts The group I intron homing restriction mechanism is it into an intron on the target DNA. In addition to the well- known to include the following steps: established DSBR pathway, there are also the Syntheses- (a) The gene is transcribed to produce pre-mRNA. Dependent Strand Annealing (SDSA) pathway and other (b) Intron-encoded generation of meganuclease. pathways.1 31–35 (c) Double-stranded target site incision. (B) Group II intron homing cleavage mechanism (d) Partial digestion of the nucleic acid sequence at the (a) The gene is transcribed to produce pre-mRNA. gap in the two chains. (b) Pre-mRNA is translated to produce aI2 protein. (e) The donor intron DNA is used as template to As described below, aI2 protein has many functions. carry out repair-synthesis of the complementary acceptor (c) The lariat-like aI2 intron is spliced out from the pre- strand (Fig. 1). mRNA by the aI2 protein.

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are asymmetric: The exon sequence upstream of the intron transcript is a little longer than the downstream sequence.36–41

MEGANUCLEASE ASSEMBLY On the basis of a report published by Science in 2000, we present a brief introduction to meganuclease assembly and related experimental methods (Fig. 2).48 As shown in the figure above, a Donor plasmid car- ries intron LtrB which incorporates the open reading frame encoding the LtrA protein flanked by exons E1 and E2 which serve as target recognition and cleavage sites. Tran- scription is driven from the upstream phage T7 promoter. The donor plasmid confers Chloramphenicol resistance. The recipient plasmid incorporates E1 and E2 followed by a downstream tetracycline resistance gene. The Recipient plasmid confers ampicillin resistance. T1 and T2 are E. Figure 2. Meganuclease assembly and experiments. coli transcription termination sequences. T is a bacteriophage transcription termination sequence. (d) The excised aI2 intron and the aI2 protein combine The donor and recipient plasmids are co-transformed to form an RNP complex which participates in the intro- into E. coli. The LtrA protein is expressed and cuts duction of double-stranded cuts in a specific area of the both donor and recipient plasmids at the E1E2 sites. recipient DNA—the two-exon junction area. Thus, the LtrB intron plus the bacteriophage T7 promoter (e) Using the pre-mRNA intron as template, a catalytic are excised in one piece, and inserted into the recipi- cDNA is synthesized by the reverse transcriptase function ent plasmid to generate a recombinant plasmid. Since the Bacteriophage T7 promoter ends up upstream of the tetra- of the aI2 protein. The recipient DNA chain is also called cycline resistance gene, the newly-formed recombinant the starting chain, the lasso-shapedIP: 192.168.39.211 piece the aI2 intron On: is Fri, 24 Sep 2021 06:46:58 plasmid will exhibit tetracycline resistance, indicating that also called the non-starting chain. BothCopyright: the starting American chain Scientific Publishers the translocation has taken place. and the non-starting chain, take part in this step.Delivered by Ingenta (f) The non-starting chain removes the lariat-like aI2 intron, and uses the single-stranded cDNA in the starting- APPLIED RESEARCH IN GENE chain as template to carry out complementary-strand MANIPULATION AND RESTRUCTURING synthesis. Rare-cut endonucleases can introduce only a few DSBs in (g) When using the pre-mRNA as template to synthe- complex genomes. This feature makes meganucleases use- size cDNA, the intron’s flanking exon sequences often ful in gene mapping, cloning, targeting, and for research tend to be included. However, in contrast to group into the various biological systems involved in double- I intron homing, these carried-over flanking sequences strand break repair (Table I).

Table I. Examples Of meganuclease therapeutic applications.

Year Gene Enzyme Application Ref. (#)

1995 Neo gene I-SceI Used in mammalian cells for gene recombination and [49] disruption 1995 Env gene I-SceI Identify chromosomal homologous recombination in [50] mammalian cells and provide a basic method for gene rearrangements using meganuclease 1998 Hypoxanthine phosphoribosyl- I-SceI In vivo recombinantion to introduce an enzymatic [51] transferase (hprt) gene function 2000 Tetracycline resistance (tet R) gene Ll. LtrB Active detection system developed and Applied in [46] prokaryotic systems to knockout the CCR5 gene 2006 LagoZ I-SceI In toto restructuring of chromosomes [52] 2012 39 different genomic targets I-CreI Demonstrated that chromosome polymorphisms have [47] a great impact on cleavage during recombination 2013 Xeroderma pigmentosum group C XPCm Used to correct the XPC gene in cells exhibiting [53] (XP-C) resistance to ultraviolet 2014 Herpes simplex virus type 1 (HSV-1) Used to reduce human corneal transplant rejection [48]

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insertion. Since meganucleases are very rare cutters, they are good tools with which to generate bacterial gene maps; they are particularly suitable for the analysis of chromosome structure. Most reports in the literature that employ meganuclease for gene mapping rely on the generation of PFGE patterns. For example, a team of researchers used I-SceI to produce genomic maps of eukaryotic cells. These workers used either cassettes containing selectable markers to mediate homologous end restructuring, or random transposons to insert restriction sites into the genome. I-SceI has been used to assemble a Saccharomyces cerevisiae chromosome XI map and to generate a cosmid library.42 Other investigators have used SwaI, PacI, PmeI to map the genome of Corynebacterium glutamicum (ATCC 13032), the results are shown below (Fig. 3): I-CeuI only cuts certain multi-copy bacterial rRNA genes. The assem- bled I-CeuI fragment map can reveal the structure of the rDNA gene region. I-CeuI has been used to draw the genome maps of Escherichia coli K-12 and Salmonella typhimurium LT2. These studies have shown that chromosome rearrangements in independent isolates of wild-type Salmonella typhi are due to homologous recombination between the 7 rrn genes that encode ribosomal RNA.43

Gene Cloning Meganuclease recognition and cleavage sites have been IP: 192.168.39.211 On: Fri, 24 Sep 2021 06:46:58 Copyright: American Scientificused to Publishers make vectors to clone large fragments. For exam- Delivered by ple,Ingenta a plasmid vector containing a multiple cloning site as well as the PI-SceI, I-PpoI, I-CeuI, and PI-TliI cutting Figure 3. Using different meganucleases to map the genome site.44 Other investigators have made cos plasmids with of Corynebacterium glutamicum (ATCC 13032). I-SceI sites for large-fragment cloning. The latter have engineered their cos constructs to have a Kanamycin resis- Gene Mapping tance gene ﬂanked by I-SceI, as well as other restriction Genome mapping is carried out using chromosomal land- endonuclease sites, so that kanamycin resistance can be marks that are natural cleavage sites for cutting and used to screen when the fragment is inserted. Moreover,

Figure 4. Meganuclease research course.

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I-SceI can be subsequently used to cut and religate the newer and more powerful members: ZFNs, TALENs, and plasmid to remove the Kanamycin resistance gene.45 CRISPR/Cas.

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