Terminal Inverted Repeat Sequences Research Articles

A Modified Monomeric Red Fluorescent Protein Reporter for Assessing CRISPR Activity

Gene editing in human embryonic stem cells (hESCs) has been significantly enhanced by the discovery and development of CRISPR Cas9, a programmable nuclease system that can introduce targeted double-stranded breaks. The system relies on the optimal selection of a sgRNA sequence with low off-targets and high efficiency. We designed an improved monomeric red fluorescent protein reporter, GEmCherry2, for assessing CRISPR Cas9 activity and for optimizing sgRNA. By incorporating an out-of-frame sequence to the N-terminal of the red fluorescent protein mCherry, we created a visual tool for assessing the indel frequency after cutting with CRISPR Cas9. When a sgRNA-Cas9 construct is co-transfected with a corresponding GEmCherry2 construct, single nucleotide indels can move the GEmCherry2 sequence back in-frame and allow quantification and comparison of the efficiency of different sgRNA target sites by measuring red fluorescence. With this GEmCherry2 assay, we compared four target sites in the safe harbor AAVS1 locus and found significant differences in target site activity. We verified the activity using TIDE, which ranked our target sites in a similar order as the GEmCherry2 system. We also identified an AAV short inverted terminal repeat sequence within the Cas9 construct that, upon removal significantly improved transient transfection and expression in hESCs. Moreover, using GEmCherry2, we designed a sgRNA to target SORCS2 in hESCs and successfully introduced indels into the coding sequence of SORCS2.

☆DNA assembly technique simplifies the construction of infectious clone of fowl adenovirus

Plasmid bearing adenovirus genome is generally constructed with the method of homologous recombination in E. coli BJ5183 strain. Here, we utilized Gibson gene assembly technique to generate infectious clone of fowl adenovirus 4 (FAdV-4). Primers flanked with partial inverted terminal repeat (ITR) sequence of FAdV-4 were synthesized to amplify a plasmid backbone containing kanamycin-resistant gene and pBR322 origin (KAN-ORI). DNA assembly was carried out by combining the KAN-ORI fragment, virus genomic DNA and DNA assembly master mix. E. coli competent cells were transformed with the assembled product, and plasmids (pKFAV4) were extracted and confirmed to contain viral genome by restriction analysis and sequencing. Virus was successfully rescued from linear pKFAV4-transfected chicken LMH cells. This approach was further verified in cloning of human adenovirus 5 genome. Our results indicated that DNA assembly technique simplified the construction of infectious clone of adenovirus, suggesting its possible application in virus traditional or reverse genetics.

Characterization of a functional insertion sequence ISSau2 from Staphylococcus aureus

BackgroundISSau2 has been suggested as a member of the IS150 f subgroup in the IS3 family. It encodes a fusion transposase OrfAB produced by programmed − 1 translational frameshifting with two overlapping reading frames orfA and orfB. To better characterize ISSau2, the binding and cleaving activities of the ISSau2 transposase and its transposition frequency were studied.ResultsThe purified ISSau2 transposase OrfAB was a functional protein in vitro since it bound specifically to ISSau2 terminal inverted repeat sequences (IRs) and cleaved the transposon ends at the artificial mini-transposon pUC19-IRL-gfp-IRR. In addition, the transposition frequency of ISSau2 in vivo was approximately 1.76 ± 0.13 × 10− 3, based on a GFP hop-on assay. Furthermore, OrfB cleaved IRs with the similar catalytic activity of OrfAB, while OrfA had no catalytic activity. Finally, either OrfA or OrfB significantly reduced the transposition of ISSau2 induced by OrfAB.ConclusionWe have confirmed that ISSau2 is a member of IS150/IS3 family. The ISSau2 transposase OrfAB could bind to and cleave the specific fragments containing the terminal inverted repeat sequences and induce the transposition, suggesting that ISSau2 is at least partially functional. Meanwhile, both OrfA and OrfB inhibited the transposition by ISSau2. Our results will help understand biological roles of ISSau2 in its host S. aureus.

Impact of Inverted Terminal Repeat Integrity on rAAV8 Production Using the Baculovirus/Sf9 Cells System.

Adeno-associated virus (AAV) inverted terminal repeats (ITRs) are key elements of AAV. These guanine-cytosine-rich structures are involved in the replication and encapsidation of the AAV genome, along with its integration in and excision from the host genome. These sequences are the only AAV-derived DNA sequences conserved in recombinant AAV (rAAV), as they allow its replication, encapsidation, and long-term maintenance and expression in target cells. Due to the original vector design, plasmids containing the gene of interest flanked by ITRs and used for rAAV production often present incomplete, truncated, or imperfect ITR sequences. For example, pSUB201 and its derivatives harbor a truncated (14 nt missing on the external part of the ITR), flop-orientated ITR plus 46 bp of non-ITR viral DNA at each end of the rAAV genome. It has been shown that rAAV genomes can be replicated, even with incomplete, truncated, or imperfect ITR sequences, leading to the production of rAAV vectors in transfection experiments. Nonetheless, it was hypothesized that unmodified wild-type (WT) ITR sequences could lead to a higher yield of rAAV, with less non-rAAV encapsidated DNA originating from the production cells and/or baculovirus shuttle vector genomes. This work studied the impact of imperfect ITRs on the level of encapsidated rAAV genomes and baculovirus-derived DNA sequences using the baculovirus/Sf9 cells production system. Replacement of truncated ITRs with WT and additional wtAAV2 sequences has an impact on the two major features of rAAV production: (1) a rise from 10% to 40% of full capsids obtained, and (2) up to a 10-fold reduction in non-rAAV encapsidated DNA. Furthermore, this study considered the impact on these major parameters of additional ITR elements and ITRs coupled with various regulatory elements of different origins. Implementation of the use of complete ITRs in the frame of the baculovirus-based rAAV expression system is one step that will be required to optimize the quality of rAAV-based gene therapy drugs.

Evaluating Efficiencies of Dual AAV Approaches for Retinal Targeting.

Retinal gene therapy has come a long way in the last few decades and the development and improvement of new gene delivery technologies has been exponential. The recent promising results from the first clinical trials for inherited retinal degeneration due to mutations in RPE65 have provided a major breakthrough in the field and have helped cement the use of recombinant adeno-associated viruses (AAV) as the major tool for retinal gene supplementation. One of the key problems of AAV however, is its limited capacity for packaging genomic information to a maximum of around 4.8 kb. Previous studies have demonstrated that homologous recombination and/or inverted terminal repeat (ITR) mediated concatemerization of two overlapping AAV vectors can partially overcome the size limitation and help deliver larger transgenes. The aim of this study was to investigate and compare the use of different AAV dual-vector approaches in the mouse retina using a systematic approach comparing efficiencies in vitro and in vivo using a unique oversized reporter construct. We show that the hybrid approach relying on vector genome concatemerization by highly recombinogenic sequences and ITRs sequence overlap offers the best levels of reconstitution both in vitro and in vivo compared to trans-splicing and overlap strategies. Our data also demonstrate that dose and vector serotype do not affect reconstitution efficiency but a discrepancy between mRNA and protein expression data suggests a bottleneck affecting translation.

Two new miniature inverted-repeat transposable elements in the genome of the clam Donax trunculus.

Repetitive sequences are important components of eukaryotic genomes that drive their evolution. Among them are different types of mobile elements that share the ability to spread throughout the genome and form interspersed repeats. To broaden the generally scarce knowledge on bivalves at the genome level, in the clam Donax trunculus we described two new non-autonomous DNA transposons, miniature inverted-repeat transposable elements (MITEs), named DTC M1 and DTC M2. Like other MITEs, they are characterized by their small size, their A + T richness, and the presence of terminal inverted repeats (TIRs). DTC M1 and DTC M2 are 261 and 286bp long, respectively, and in addition to TIRs, both of them contain a long imperfect palindrome sequence in their central parts. These elements are present in complete and truncated versions within the genome of the clam D. trunculus. The two new MITEs share only structural similarity, but lack any nucleotide sequence similarity to each other. In a search for related elements in databases, blast search revealed within the Crassostrea gigas genome a larger element sharing sequence similarity only to DTC M1 in its TIR sequences. The lack of sequence similarity with any previously published mobile elements indicates that DTC M1 and DTC M2 elements may be unique to D. trunculus.

Crystallization of and selenomethionine phasing strategy for a SETMAR-DNA complex.

Transposable elements have played a critical role in the creation of new genes in all higher eukaryotes, including humans. Although the chimeric fusion protein SETMAR is no longer active as a transposase, it contains both the DNA-binding domain (DBD) and catalytic domain of the Hsmar1 transposase. The amino-acid sequence of the DBD has been virtually unchanged in 50 million years and, as a consequence, SETMAR retains its sequence-specific binding to the ancestral Hsmar1 terminal inverted repeat (TIR) sequence. Thus, the DNA-binding activity of SETMAR is likely to have an important biological function. To determine the structural basis for the recognition of TIR DNA by SETMAR, the design of TIR-containing oligonucleotides and SETMAR DBD variants, crystallization of DBD-DNA complexes, phasing strategies and initial phasing experiments are reported here. An unexpected finding was that oligonucleotides containing two BrdUs in place of thymidines produced better quality crystals in complex with SETMAR than their natural counterparts.

319. Rationally Designed AAV Inverted Terminal Repeats Enhance Gene Targeting

Basic and clinical applications of mammalian genetic engineering rely on selective pressure and/or, ironically, DNA damage at or near the modification site to obtain relevant efficiencies. In regards to the later situation, continuously evolving endonuclease technologies remain focused on the generation of a site-specific DNA double strand break to stimulate homology directed repair (HDR). The stimulation of HDR is thus dictated by the ability of the endonuclease platform to specifically recognize its cognate site, with safety concerns dictated by its promiscuity influenced by its persistence. To eliminate the serious safety concerns associated with designer endonucleases while maintaining efficient HDR, it was hypothesized that rational modification of a viral DNA repair substrate would enhance gene editing without the requirement for induced chromosomal damage. Specifically, modifications of the adeno-associated virus (AAV) inverted terminal repeat sequence (ITR), in a viral vector context, were evaluated for stimulation of HDR in human cells. The results demonstrate an approximate 30-fold enhancement of AAV gene targeting using a particular rationally designed ITR sequence. Characterization of this event suggests that differences in the inherent ITR-initiated transcriptional activity and altered interactions with host DNA repair proteins contribute to the enhancement in HDR. The collective results demonstrate for the first time that alterations in the ITR sequence can enhance AAV gene targeting. Importantly, the refinement of such vectors may offer a safer alternative to site-specific endonuclease technologies as on- and off-target DNA cleavage concerns are eliminated.

87. Rationally Designed Inverted Terminal Repeats Enhance Transduction of Embryonic Stem Cells

Despite the prevalence of AAV vectors in gene therapy, treatments combining AAV vectors and cell therapy have not progressed into the clinic despite previous reports of successful transduction of stem cells by AAV vectors. One potential explanation is AAV vector toxicity in stem and stem-like cells, as demonstrated in previous studies. In particular, the AAV inverted terminal repeat (ITR) sequence was shown to induce apoptosis in human embryonic stem cells (hESCs) in a p53 dependent manner. A bioinformatic analysis of putative transcription factor binding sites in the AAV2 ITR sequence revealed six putative p53 binding sites. A panel of rationally designed AAV2 ITRs was generated which abrogated putative p53 binding sites and demonstrated reduced p53 binding in an in vitro binding assay. The synthetic ITRs were capable of all of the vector production requirements and were verified by sequencing directly from vector preparations. AAV vector transduction and the hESC DNA damage response was altered by the synthetic ITRs. Importantly, our ITRs demonstrated reduced toxicity in hESCs compared to an AAV vector having the AAV2 ITR sequence. The collective results allude to modulation of the host's DNA damage response using rationally designed ITRs, which may enhance the relevancy of AAV vectors in stem cell therapies.

PiggyBac transposon vectors: the tools of the human gene encoding.

A transposon is a DNA segment, which is able to change its relative position within the entire genome of a cell. The piggyBac (PB) transposon is a movable genetic element that efficiently transposes between vectors and chromosomes through a "cut-and-paste" mechanism. During transposition, the PB transposase recognizes transposon-specific inverted terminal repeats (ITRs) sequences located on both ends of the transposon vector and eight efficiently moves the contents from its original positions and efficiently integrates them into TTAA chromosomal sites. PB has drawn much attention because of its transposition efficiency, safety and stability. Due to its priorities, PB can be used as a new genetic vehicle, a new tool for oncogene screening and a new method for gene therapy. PB has created a new outlook for human gene encoding.

Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

Clinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new “Free-ITR” qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field.

Identification, characterization and diversification of non-autonomous hAT transposons and unknown insertions in Brassica

Transposons, the mobile genetics elements played an important role in genome evolution and duplication. Several DNA transposons superfamilies are described in plants and animal genomes; CACTA, hAT, Mariner Mutator and Harbinger being the most diverse among plants. The hAT transposons are proliferating in many plant and animal species. The dot plot comparison, computational and molecular approaches were used for the characterization and diversification of non-autonomous hAT families in Brassica and 494 and 30 full length elements were collected with estimated copies of ~5052 and ~4110 from whole Brassica rapa and Brassica oleracea genomes respectively. The analysis of terminal inverted repeats (TIRs) of these hAT sequences classified them into 13 families on the basis of TIRs sequence similarities. The WebLogo of TIRs of hATs confirmed that few family based TIRs are highly conserved, while other showed variable nucleotide sequences. Transposons based sequence specific amplification polymorphism markers revealed the insertion preference of hATs in diverse Brassica genomes and found many elements as polymorphic across Brassica accessions. Some elements were A or C-genome specific, while most of them are present in Brassica diploids and allopolyploids revealing their presence before separation of A, B and C-Brassica genomes around 8 MYA. Several other unknown mobile insertions were identified with or without TIRs and TSDs of varied lengths, not common to known transposon superfamilies. The detailed study of these insertions revealed their distribution and mobile nature, which although less in numbers and small in sizes are playing a role in genome evolution.

Productive life cycle of adeno-associated virus serotype 2 in the complete absence of a conventional polyadenylation signal.

We showed that WT adeno-associated virus serotype 2 (AAV2) genome devoid of a conventional polyadenylation [poly(A)] signal underwent complete genome replication, encapsidation and progeny virion production in the presence of adenovirus. The infectivity of the progeny virion was also retained. Using recombinant AAV2 vectors devoid of a human growth hormone poly(A) signal, we also demonstrated that a subset of mRNA transcripts contained the inverted terminal repeat (ITR) sequence at the 3' end, which we designated ITR in RNA (ITRR). Furthermore, AAV replication (Rep) proteins were able to interact with the ITRR. Taken together, our studies suggest a new function of the AAV2 ITR as an RNA element to mediate transgene expression from poly(A)-deleted mRNA.

557. Impact of the Integrity of the ITRs on the Production of rAAV Vectors

The Adeno-Associated Virus's (AAV) Inverted Terminal Repeats (ITRs) are key features of the AAV. ITRs are implied in the replication, encapsidation of the AAV genome, along with its integration in the host genome and its excision. These sequences are the only AAV DNA sequences conserved in the recombinant AAV as they allow its replication, encapsidation and long-term maintenance in the host non-dividing target cells. The ITRs are highly GC rich and structured due to the presence of repetitive palindromic sequences.During its replication, the AAV is able to reconstitute incomplete or imperfect ITR sequences. Thus it is often found that rAAV genomes cloned into the backbones of the DNA plasmids used for production, have imperfect ITRs sequences which are shortened or contain mutations.In this work, we study the impact of those imperfect ITRs and their impact on the levels of encapsidated rAAV genomes and cellular/baculovirus contaminating DNA sequences, in the baculovirus/Sf9 cells production system.

Construction and Gene Expression Analysis of a Single-Stranded DNA Minivector Based on an Inverted Terminal Repeat of Adeno-Associated Virus

The plasmid vectors currently used for nonviral gene transfer have the disadvantage of carrying a bacterial backbone and an antibiotic resistance gene, which may cause side effects. The adeno-associated virus (AAV) genome is a linear single-stranded DNA (ssDNA) molecule with palindromic inverted terminal repeat (ITR) sequences forming double-stranded DNA (dsDNA) hairpin (HP) structures at each end. Based on the AAV genome, we constructed an AAV-ITR ssDNA minivector that consists of a GFP expression cassette flanked by both ITR sequences of 125 nucleotides. The minivectors were produced by digestion of the parental plasmids followed by denaturation. The self-complementary inverted T-shaped HP structure of the minivector was automatically formed. The HEK 293T cells were transfected with the AAV-ITR ssDNA minivector, plasmid, and dsDNA expression cassette. The results showed that AAV-ITR ssDNA minivector had relatively low gene expression efficiency in vitro. However, we found that the GFP expression efficiency of the D sequence-deleted AAV-ITR ssDNA minivector was significantly increased and was similar to those obtained with the plasmid and dsDNA expression cassette. Our data suggest that the AAV-ITR ssDNA minivector may be a new type of gene expression vector for gene therapy besides the virus and plasmid.

Autonomous and non-autonomous Tn3-family transposons and their role in the evolution of mobile genetic elements.

The Tn3 family of transposons includes diverse elements that encode homologous transposases and contain conserved terminal inverted repeat sequences (IRs). The recent identification of non-autonomous elements, named TIMEs (Tn3-derived Inverted-repeat Miniature Elements), has shed new light on the diversity and evolution of this transposon family. A common feature of TIMEs and other members of this family is their ability to mobilize genomic DNA for transposition as part of composite transposons. These elements significantly influence the structure and properties of plasmids and other mobile genetic elements (MGEs). They may contain and move by transposition (i) plasmid replication systems, (ii) toxin-antitoxin systems and (iii) site-specific recombination modules that can resolve plasmid multimers. Some Tn3 family elements may also transfer large segments of chromosomal DNA into plasmids, which increases the pool of mobile DNA that can take part in horizontal gene transfer.

Establishment of a novel cell line for the enhanced production of recombinant adeno-associated virus vectors for gene therapy.

Adeno-associated viral (AAV) vectors show great promise because of their excellent safety profile; however, pre-existing immune responses have necessitated the administration of high titer AAV, posing a significant challenge to the advancement of gene therapy involving AAV vectors. Recombinant AAV vectors contain minimum viral proteins necessary for their assembly and gene delivery functions. During the process of AAV assembly and production, AAV vectors acquire, inherently and submissively, various cellular proteins, but the identity of these proteins is poorly characterized. We reason that by identifying host cell proteins inherently associated with AAV vectors we may better understand the contribution of cellular components to AAV vector assembly and, ultimately, may improve the production of AAV vectors for gene therapy. In this study, three serotypes of recombinant AAV, namely AAV2, AAV5, and AAV8, were investigated. We used liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods to identify protein composition in purified AAV vectors, confirmed protein identities using western blotting, and explored the potential function of selected proteins in AAV vector production using small hairpin (shRNA) methods. Using LC-MS/MS, we identified 44 AAV-associated cellular proteins including Y-box binding protein (YB1). We showed for the first time that the establishment of a novel producer cell line by introducing an shRNA sequence down-regulating YB1 resulted in up to 45- and 9-fold increase in physical vector genome titers of AAV2 and AAV8, respectively, and up to 7-fold increase in AAV2 transduction vector genome titers. Our results revealed that YB1 gene knockdown promoted AAV2 rep expression and vector DNA production and reduced the number of empty particles in AAV2 products, suggesting that YB1 plays an important role in AAV vector assembly by competition with adenovirus E2A and AAV capsid proteins for binding to the inverted terminal repeat (ITR) sequence. The significance and implications of our findings in future improvement of AAV production are discussed.

Insights on genome size evolution from a miniature inverted repeat transposon driving a satellite DNA

The genome size in eukaryotes does not correlate well with the number of genes they contain. We can observe this so-called C-value paradox in amphibian species. By analyzing an amphibian genome we asked how repetitive DNA can impact genome size and architecture. We describe here our discovery of a Tc1/mariner miniature inverted-repeat transposon family present in Xenopus frogs. These transposons named miDNA4 are unique since they contain a satellite DNA motif. We found that miDNA4 measured 331bp, contained 25bp long inverted terminal repeat sequences and a sequence motif of 119bp present as a unique copy or as an array of 2–47 copies. We characterized the structure, dynamics, impact and evolution of the miDNA4 family and its satellite DNA in Xenopus frog genomes. This led us to propose a model for the evolution of these two repeated sequences and how they can synergize to increase genome size.

Rational Design for Enhanced Gene Therapy with DNA Transposons

Several genetic diseases caused by deficiencies in enzymes required throughout the body can be treated by enzyme replacement therapy at costs of upward of hundreds of thousands of dollars per year per patient. In the long run, such costs are prohibitive. Gene therapy is therefore an appealing option in that its aim is to treat patients after only a single or very few interventions. Clinical-grade viral vectors can efficiently deliver therapeutic expression cassettes, but such viruses can still be expensive to prepare. By contrast, clinical-grade nonviral nucleic acids are relatively inexpensive to produce but difficult to deliver into cells. In this issue of Molecular Therapy, two reports from the same group describe improvements to two limiting factors of nonviral gene therapy using DNA transposons delivered to the liver.1,2 The authors substantially increased efficiency of delivery of transgenes to the target organ and the amount of therapeutic protein exported per cell following transgene delivery.

Mobility and generation of mosaic non-autonomous transposons by Tn3-derived inverted-repeat miniature elements (TIMEs).

Functional transposable elements (TEs) of several Pseudomonas spp. strains isolated from black shale ore of Lubin mine and from post-flotation tailings of Zelazny Most in Poland, were identified using a positive selection trap plasmid strategy. This approach led to the capture and characterization of (i) 13 insertion sequences from 5 IS families (IS3, IS5, ISL3, IS30 and IS1380), (ii) isoforms of two Tn3-family transposons – Tn5563a and Tn4662a (the latter contains a toxin-antitoxin system), as well as (iii) non-autonomous TEs of diverse structure, ranging in size from 262 to 3892 bp. The non-autonomous elements transposed into AT-rich DNA regions and generated 5- or 6-bp sequence duplications at the target site of transposition. Although these TEs lack a transposase gene, they contain homologous 38-bp-long terminal inverted repeat sequences (IRs), highly conserved in Tn5563a and many other Tn3-family transposons. The simplest elements of this type, designated TIMEs (Tn3 family-derived Inverted-repeat Miniature Elements) (262 bp), were identified within two natural plasmids (pZM1P1 and pLM8P2) of Pseudomonas spp. It was demonstrated that TIMEs are able to mobilize segments of plasmid DNA for transposition, which results in the generation of more complex non-autonomous elements, resembling IS-driven composite transposons in structure. Such transposon-like elements may contain different functional genetic modules in their core regions, including plasmid replication systems. Another non-autonomous element “captured” with a trap plasmid was a TIME derivative containing a predicted resolvase gene and a res site typical for many Tn3-family transposons. The identification of a portable site-specific recombination system is another intriguing example confirming the important role of non-autonomous TEs of the TIME family in shuffling genetic information in bacterial genomes. Transposition of such mosaic elements may have a significant impact on diversity and evolution, not only of transposons and plasmids, but also of other types of mobile genetic elements.