Mechanisms used for cDNA synthesis and site-specific integration of RNA into DNA genomes by a reverse transcriptase-Cas1 fusion protein.

Reverse transcriptase-Cas1 (RT-Cas1) fusion proteins found in some CRISPR systems enable spacer acquisition from both RNA and DNA, but the mechanism of RNA spacer acquisition has remained unclear. Here, we found that Marinomonas mediterranea RT-Cas1/Cas2 adds short 3'-DNA (dN) tails to RNA protospacers, enabling their direct integration into CRISPR arrays as 3'-dN-RNAs or 3'-dN-RNA/cDNA duplexes at rates comparable to similarly configured DNAs. Reverse transcription of RNA protospacers is initiated at 3' proximal sites by multiple mechanisms, including recently described de novo initiation, protein priming with any dNTP, and use of short exogenous or synthesized DNA oligomer primers, enabling synthesis of near full-length cDNAs of diverse RNAs without fixed sequence requirements. The integration of 3'-dN-RNAs or single-stranded DNAs (ssDNAs) is favored over duplexes at higher protospacer concentrations, potentially relevant to spacer acquisition from abundant pathogen RNAs or ssDNA fragments generated by phage defense nucleases. Our findings reveal mechanisms for site-specifically integrating RNA into DNA genomes with potential biotechnological applications.

Low-Cost Arduino Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) for Sensitive Nucleic Acid Detection.

This work presents a low-cost transcription loop-mediated isothermal amplification (RT-LAMP) instrument for nucleic acid detection, employing an Arduino Nano microcontroller. The cooling system includes customized printed circuit boards (PCBs) that serve as electrical resistors and incorporate fans. An aluminum block is designed to accommodate eight vials. The system also includes two PCB heaters-one for sample heating and the other for vial lid heating to prevent condensation. The color detection system comprises a TCS3200 color 8-sensor array coupled to one side of the aluminum heater body and a white 8-LED array coupled to the other side, controlled by two Multiplexer/Demultiplexer devices. LED light passes through the sample, reaching the color sensor and conveying color information crucial for detection. The top board is maintained at 110 ± 2 °C, while the bottom board is held at 65 ± 0.5 °C throughout the RT-LAMP assay. Validation tests successfully demonstrated the efficacy of the colorimetric RT-LAMP reactions using SARS-CoV-2 RNA amplification as a sample viability test, achieving 100% sensitivity and 97.3% specificity with 66 clinical samples. Our instrument offers a cost-effective (USD 100) solution with automated result interpretation and superior sensitivity compared to visual inspection. While the prototype was tested with SARS-CoV-2 RNA samples, its versatility extends to detecting other pathogens using alternative primers, showcasing its potential for broader applications in biosensing.

Insights into LINE-1 reverse transcription guide therapy development.

Subsets of long interspersed nuclear element 1 (LINE-1) retrotransposons can 'retrotranspose' throughout the human genome at a cost to host cell fitness, as observed in some cancers. Pharmacological inhibition of LINE-1 retrotransposition requires a comprehensive understanding of the LINE-1 ORF2p reverse transcriptase. Two recent publications, by Thawani et al. and Baldwin et al., report structures of LINE-1 ORF2p and address long-standing mechanistic gaps regarding LINE-1 retrotransposition. Both studies will be critical to design new specific inhibitors of the LINE-1 ORF2p reverse transcriptase.

Large Deletions, Cleavage of the Telomeric Repeat Sequence, and Reverse Transcriptase-Mediated DNA Damage Response Associated with Long Interspersed Element-1 ORF2p Enzymatic Activities.

L1 elements can cause DNA damage and genomic variation via retrotransposition and the generation of endonuclease-dependent DNA breaks. These processes require L1 ORF2p protein that contains an endonuclease domain, which cuts genomic DNA, and a reverse transcriptase domain, which synthesizes cDNA. The complete impact of L1 enzymatic activities on genome stability and cellular function remains understudied, and the spectrum of L1-induced mutations, other than L1 insertions, is mostly unknown. Using an inducible system, we demonstrate that an ORF2p containing functional reverse transcriptase is sufficient to elicit DNA damage response even in the absence of the functional endonuclease. Using a TK/Neo reporter system that captures misrepaired DNA breaks, we demonstrate that L1 expression results in large genomic deletions that lack any signatures of L1 involvement. Using an in vitro cleavage assay, we demonstrate that L1 endonuclease efficiently cuts telomeric repeat sequences. These findings support that L1 could be an unrecognized source of disease-promoting genomic deletions, telomere dysfunction, and an underappreciated source of chronic RT-mediated DNA damage response in mammalian cells. Our findings expand the spectrum of biological processes that can be triggered by functional and nonfunctional L1s, which have impactful evolutionary- and health-relevant consequences.

Major Drug Resistance Mutations on Reverse Transcriptase Gene in Human Immunodeficiency Virus Type-1 in Indonesia: A Systematic Review.

PURPOSE OF REVIEW: The prevalence of HIV-1 in Indonesia is on a concerning upward trajectory, with a concurrent rise in the development of drug-resistant strains, challenging the efficacy of antiretroviral therapy (ART). Many mutations have been found in the pol gene that makes HIV resistant to ART. We aim to review the major drug resistance mutations (DRMs) of reverse transcriptase (RT) of pol gene in HIV-1 cases in Indonesia. RECENT FINDINGS: A total of eleven articles reporting DRMs in HIV-1 subjects from various regions between 2015-2020 in Indonesia are included. The prevalence of major DRMs on the RT gene in studies included varies from 3.4% to 34%. The CRF01_AE subtype stands out as the predominant variant. Notably, the prevalence of major DRMs in ART-experienced individuals is 22.1%, while ART-naïve individuals show a lower rate of 4.4%. Among the RT gene mutations, M184I/V emerges as the most prevalent (10.5%) within the nucleos(t)ide reverse transcriptase inhibitors (NRTI) group, while K103N leads among the non-NRTI (NNRTI) group, with a frequency of 6.4%. Regionally, North Sulawesi records the highest prevalence of major DRMs in the RT gene at 21.1%, whereas Riau and Central Papua exhibit the lowest rates at 3.4%. Significant variations in drug resistance mutations within the RT gene across Indonesian regions highlight the importance of closely monitoring and evaluating the effectiveness of current antiretroviral therapy (ART) regimens. Considerably, more studies are needed to understand better and overcome the emergence of DRMs on HIV-1 patients in Indonesia.

Development of Moloney Murine Leukemia Virus Reverse Transcriptase Fused with Archaeal DNA-binding Protein Sis7a.

INTRODUCTION: Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) is a common enzyme used to convert RNA sequences into cDNA. However, it still has its shortcomings, especially in terms of processivity and thermostability. According to a previous patent, the fusion of polymerase enzyme to an archaeal DNA-binding protein has been proven to enhance its performance. Furthermore, recent studies have also stated that the fusion of a polymerase enzyme to an archaeal DNA-binding protein is predicted to improve its thermostability and processivity. AIM: As an early stage of enzyme development, this study aimed to design, express, and purify enzymatically active MMLV RT fused with archaeal DNA-binding protein. METHODS: RT fusion proteins were designed and evaluated using in silico methods. The RT fusion enzyme was then expressed in Escherichia coli BL21(DE3) and purified. Its reverse transcriptional activity was proved using reverse transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS: This study showed that MMLV RT fusion with Sis7a protein at its C-terminal end using commercial linker (GGVDMI) produced the best in silico evaluation results. The RT fusion was successfully expressed and purified. It was also known that the optimal condition for expression of the RT fusion was using 0.5 mM IPTG with post-induction incubation at room temperature (± 26°C) for 16 hours. In addition, the activity assay proved that the RT fusion has the reverse transcriptional activity. CONCLUSION: This study shows that the designed MMLV RT Sis7a fusion can be expressed and purified, is enzymatically active, and has the potential to be developed as an improved RT enzyme. Further study is still needed to prove its thermostability and processivity, and further characterize, and plan production scale-up of the MMLV RT Sis7a fusion for commercial use.

Keratocystoma: A Distinctive Salivary Gland Neoplasm Characterized by RUNX2 Rearrangements.

Keratocystoma is a rare salivary gland lesion that has been reported primarily in children and young adults. Because of a scarcity of reported cases, very little is known about it, including its molecular underpinnings, biological potential, and histologic spectrum. Purported to be a benign neoplasm, keratocystoma bears a striking histologic resemblance to benign lesions like metaplastic Warthin tumor on one end of the spectrum and squamous cell carcinoma on the other end. This overlap can cause diagnostic confusion, and it raises questions about the boundaries and definition of keratocystoma as an entity. This study seeks to utilize molecular tools to evaluate the pathogenesis of keratocystoma as well as its relationship with its histologic mimics. On the basis of targeted RNA sequencing (RNA-seq) results on a sentinel case, RUNX2 break-apart fluorescence in situ hybridization (FISH) was successfully performed on 4 cases diagnosed as keratocystoma, as well as 13 cases originally diagnosed as tumors that morphologically resemble keratocystoma: 6 primary squamous cell carcinomas, 3 metaplastic/dysplastic Warthin tumors, 2 atypical squamous cysts, 1 proliferating trichilemmal tumor, and 1 cystadenoma. RNA-seq and/or reverse transcriptase-PCR were attempted on all FISH-positive cases. Seven cases were positive for RUNX2 rearrangement, including 3 of 4 tumors originally called keratocystoma, 2 of 2 called atypical squamous cyst, 1 of 1 called proliferating trichilemmal tumor, and 1 of 6 called squamous cell carcinoma. RNA-seq and/or reverse transcriptase-PCR identified IRF2BP2::RUNX2 in 6 of 7 cases; for the remaining case, the partner remains unknown. The cases positive for RUNX2 rearrangement arose in the parotid glands of 4 females and 3 males, ranging from 8 to 63 years old (mean, 25.4 years; median, 15 years). The RUNX2 -rearranged cases had a consistent histologic appearance: variably sized cysts lined by keratinizing squamous epithelium, plus scattered irregular squamous nests, with essentially no cellular atypia or mitotic activity. The background was fibrotic, often with patchy chronic inflammation and/or giant cell reaction. One case originally called squamous cell carcinoma was virtually identical to the other cases, except for a single focus of small nerve invasion. The FISH-negative case that was originally called keratocystoma had focal cuboidal and mucinous epithelium, which was not found in any FISH-positive cases. The tumors with RUNX2 rearrangement were all treated with surgery only, and for the 5 patients with follow-up, there were no recurrences or metastases (1 to 120 months), even for the case with perineural invasion. Our findings solidify that keratocystoma is a cystic neoplastic entity, one which appears to consistently harbor RUNX2 rearrangements, particularly IRF2BP2::RUNX2 . Having a diagnostic genetic marker now allows for a complete understanding of this rare tumor. They arise in the parotid gland and affect a wide age range. Keratocystoma has a consistent morphologic appearance, which includes large squamous-lined cysts that mimic benign processes like metaplastic Warthin tumor and also small, irregular nests that mimic squamous cell carcinoma. Indeed, RUNX2 analysis has considerable promise for resolving these differential diagnoses. Given that one RUNX2 -rearranged tumor had focal perineural invasion, it is unclear whether that finding is within the spectrum of keratocystoma or whether it could represent malignant transformation. Most important, all RUNX2 -rearranged cases behaved in a benign manner.

Structures, functions and adaptations of the human LINE-1 ORF2 protein.

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

Utility of reverse transcriptase - Multiplex ligation-dependant probe amplification (RT-MLPA) in the molecular classification of Diffuse Large B cell lymphoma (DLBCL) by cell-of-origin (COO).

Classifying diffuse large B cell lymphomas, not otherwise specified (DLBCL, NOS), is based on their cell-of-origin (COO) which is included in the WHO classification (2016), is essential to characterize them better in context of prognostication. While gene expression profiling (GEP) considered the gold standard and more recently, the Nanostring-based approach, classify these tumors accurately, many laboratories with limited resources and instrumentation need an alternate approach that is reliable, inexpensive, and with a reasonable turnaround. The Reverse Transcriptase Multiplex Ligation Dependant Probe Amplification (RT-MLPA) to subtype DLBCL, NOS cases, as designed by CALYM group appears to provide a good alternative but needs to be validated in other centres. Therefore, this study evaluated DLBCL, NOS and compared the results of RT-MLPA to that obtained by immunohistochemistry using the Hans algorithm. Materials and Methods: Sixty-five DLBCL, NOS cases were included and the RT-MLPA was set up and standardized using probes that were designed by the CALYM study group. Briefly, RNA was extracted converted to cDNA and the 21-gene expression classifier that also included probes to detect MYD88 mutations and EBER mRNA was performed by MLPA. The results were analyzed by the open home grown software designed by the same group and compared to the results obtained by IHC. Results: Forty-four of the sixty-five cases provided concordant results (k = 0.35) and if the MYD88 results were to be used as a classifier the concordance would have improved from 67.7% to 82%. The 21 discordant cases were divided into five categories to provide a possible explanation for the discordance. Further 26% and 31% of the samples tested were positive for MYD88 mutations and EBER mRNA, respectively. The test had a turnaround of three days. Conclusion: The test provided moderate (67.7%) concordance when compared with IHC and perhaps would have provided higher concordance if compared with GEP. The test also has the advantage of providing information on the MYD88 and EBV infection status. It was found to be reliable, easy to perform and standardize, requiring only routine instruments available in most molecular laboratories. The RT-MLPA assay therefore provides an alternative for laboratories that would require subtyping of DLBCL, NOS cases in the absence of an access to GEP or other instrument intensive methods.

To Be Mobile or Not: The Variety of Reverse Transcriptases and Their Recruitment by Host Genomes.

Reverse transcriptases (RT), or RNA-dependent DNA polymerases, are unorthodox enzymes that originally added a new angle to the conventional view of the unidirectional flow of genetic information in the cell from DNA to RNA to protein. First discovered in vertebrate retroviruses, RTs were since re-discovered in most eukaryotes, bacteria, and archaea, spanning essentially all domains of life. For retroviruses, RTs provide the ability to copy the RNA genome into DNA for subsequent incorporation into the host genome, which is essential for their replication and survival. In cellular organisms, most RT sequences originate from retrotransposons, the type of self-replicating genetic elements that rely on reverse transcription to copy and paste their sequences into new genomic locations. Some retroelements, however, can undergo domestication, eventually becoming a valuable addition to the overall repertoire of cellular enzymes. They can be beneficial yet accessory, like the diversity-generating elements, or even essential, like the telomerase reverse transcriptases. Nowadays, ever-increasing numbers of domesticated RT-carrying genetic elements are being discovered. It may be argued that domesticated RTs and reverse transcription in general is more widespread in cellular organisms than previously thought, and that many important cellular functions, such as chromosome end maintenance, may evolve from an originally selfish process of converting RNA into DNA.

RT-based Sanger sequencing of RNAs containing complex RNA repetitive elements.

Although next-generation sequencing (NGS) technologies have revolutionized our ability to sequence DNA with high-throughput, the chain termination-based Sanger sequencing method remains a widely used approach for DNA sequence analysis due to its simplicity, low cost and high accuracy. In particular, high accuracy makes Sanger sequencing the "gold standard" for sequence validation in basic research and clinical applications. During the early days of Sanger sequencing development, reverse transcriptase (RT)-based RNA sequencing was also explored and showed great promise, but the approach did not acquire popularity over time due to the limited processivity and low template unwinding capability of Avian Myeloblastosis Virus (AMV) RT, and other RT enzymes available at the time. RNA molecules have complex features, often containing repetitive sequences and stable secondary or tertiary structures. While these features are required for RNA biological function, they represent strong obstacles for retroviral RTs. Repetitive sequences and stable structures cause reverse transcription errors and premature primer extension stops, making chain termination-based methods unfeasible. MarathonRT is an ultra-processive RT encoded group II intron that can copy RNA molecules of any sequence and structure in a single cycle, making it an ideal RT enzyme for Sanger RNA sequencing. In this chapter, we upgrade the Sanger RNA sequencing method by replacing AMV RT with MarathonRT, providing a simple, robust method for direct RNA sequence analysis. The guidance for troubleshooting and further optimization are also provided.

End-to-end RT-PCR of long RNA and highly structured RNA.

RNA molecules play important roles in numerous normal cellular processes and disease states, from protein coding to gene regulation. RT-PCR, applying the power of polymerase chain reaction (PCR) to RNA by coupling reverse transcription with PCR, is one of the most important techniques to characterize RNA transcripts and monitor gene expression. The ability to analyze full-length RNA transcripts and detect their expression is critical to decipher their biological functions. However, due to the low processivity of retroviral reverse transcriptases (RTs), we can only monitor a small fraction of long RNA transcripts, especially those containing stable secondary and tertiary structures. The full-length sequences can only be deduced by computational analysis, which is often misleading. Group II intron-encoded RTs are a new type of RT enzymes. They have evolved specialized structural elements that unwind template structures and maintain close contact with the RNA template. Therefore, group II intron-encoded RTs are more processive than the retroviral RTs. The discovery, optimization and deployment of processive group II intron RTs provide us the opportunity to analyze RNA transcripts with single molecule resolution. MarathonRT, the most processive group II intron RT, has been extensively optimized for processive reverse transcription. In this chapter, we use MarathonRT to deliver a general protocol for long amplicon generation by RT-PCR, and also provide guidance for troubleshooting and further optimization.

Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay.

This study focuses on three key aspects: (a) crude throat swab samples in a viral transport medium (VTM) as templates for RT-LAMP reactions; (b) a biotinylated DNA probe with enhanced specificity for LFA readouts; and (c) a digital semi-quantification of LFA readouts. Throat swab samples from SARS-CoV-2 positive and negative patients were used in their crude (no cleaning or pre-treatment) forms for the RT-LAMP reaction. The samples were heat-inactivated but not treated for any kind of nucleic acid extraction or purification. The RT-LAMP (20 min processing time) product was read out by an LFA approach using two labels: FITC and biotin. FITC was enzymatically incorporated into the RT-LAMP amplicon with the LF-LAMP primer, and biotin was introduced using biotinylated DNA probes, specifically for the amplicon region after RT-LAMP amplification. This assay setup with biotinylated DNA probe-based LFA readouts of the RT-LAMP amplicon was 98.11% sensitive and 96.15% specific. The LFA result was further analysed by a smartphone-based IVD device, wherein the T-line intensity was recorded. The LFA T-line intensity was then correlated with the qRT-PCR Ct value of the positive swab samples. A digital semi-quantification of RT-LAMP-LFA was reported with a correlation coefficient of R2 = 0.702. The overall RT-LAMP-LFA assay time was recorded to be 35 min with a LoD of three RNA copies/µL (Ct-33). With these three advancements, the nucleic acid testing-point of care technique (NAT-POCT) is exemplified as a versatile biosensor platform with great potential and applicability for the detection of pathogens without the need for sample storage, transportation, or pre-processing.

PRIME EDITING - a new method of editing genes / PRIME EDITING - nowa metoda edycji genów.

The Prime Editing method introduces the expected manipulations within a given genome with a Cas9-nicase and pegRNA structure and a reverse transcriptase, which is responsible for the synthesis of the segment, which is then incorporated into the edited strand. This technique is based on the previously discovered CRISPR/Cas9 method. It differs from CRISPR/Cas9 in the absence of double cracks within the DNA helix, which is due to its complex structure, including the presence of additional elements, i. e. the reverse transcriptase and the matrix within the pegRNA. PE is used to modify the DNA double helix. The work deals mainly with the creation and improvement as well as testing of the modern Prime Editing method. Information on the structure and functioning of the system is provided, as well as the research carried out so far with the use of PE, carried out within the genomes of cells derived from plant, animal, and human organisms, is described. The paper also contains information on the potential benefits and hopes related to the use of this innovative method.

Evolutionary conservation of an ancient retroviral gagpol gene in Artiodactyla.

The genomes of mammals contain fingerprints of past infections by ancient retroviruses that invaded the germline of their ancestors. Most of these endogenous retroviruses (ERVs) contain only remnants of the original retrovirus; however, on rare occasions, ERV genes can be co-opted for a beneficial host function. While most studies of co-opted ERVs have focused on envelope genes, including the syncytins that function in placentation, there are examples of co-opted gag genes including one we recently discovered in simian primates. Here, we searched for other intact gag genes in non-primate mammalian lineages. We began by examining the genomes of extant camel species, which represent a basal lineage in the order Artiodactyla. This identified a gagpol gene with a large open reading frame (ORF) (>3,500 bp) in the same orthologous location in Artiodactyla species but that is absent in other mammals. Thus, this ERV was fixed in the common ancestor of all Artiodactyla at least 64 million years ago. The amino acid sequence of this gene, termed ARTgagpol, contains recognizable matrix, capsid, nucleocapsid, and reverse transcriptase domains in ruminants, with an RNase H domain in camels and pigs. Phylogenetic analysis and structural prediction of its reverse transcriptase and RNase H domains groups ARTgagpol with gammaretroviruses. Transcriptomic analysis shows ARTgagpol expression in multiple tissues suggestive of a co-opted host function. These findings identify the oldest and largest ERV-derived gagpol gene with an intact ORF in mammals, an intriguing milestone in the co-evolution of mammals and retroviruses. IMPORTANCE Retroviruses are unique among viruses that infect animals as they integrate their reverse-transcribed double-stranded DNA into host chromosomes. When this happens in a germline cell, such as sperm, egg, or their precursors, the integrated retroviral copies can be passed on to the next generation as endogenous retroviruses (ERVs). On rare occasions, the genes of these ERVs can be domesticated by the host. In this study we used computational similarity searches to identify an ancient ERV with an intact viral gagpol gene in the genomes of camels that is also found in the same genomic location in other even-toed ungulates suggesting that it is at least 64 million years old. Broad tissue expression and predicted preservation of the reverse transcriptase fold of this protein suggest that it may be domesticated for a host function. This is the oldest known intact gagpol gene of an ancient retrovirus in mammals.

Genetic and antiretroviral drug resistance mutations analysis of reverse transcriptase and protease gene from Pakistani people living with HIV-1.

BACKGROUND: Antiretroviral therapy (ART) effectiveness is compromised by the emergence of HIV drug resistance mutations (DRM) and can lead to the failure of ART. Apart from intrinsic viral factors, non-compliance with drugs and/or the use of sub-optimum therapy can lead to the emergence of DRMs. In Pakistan HIV currently exists as a concentrated epidemic, however, ART coverage is very low, and drug adherence is poor. ART is selected assuming without baseline genotyping. Pakistan has recently seen a rise in treatment failures, but the country's actual burden of DRM is still unknown. In this study, we perform the genetic and drug resistance analysis of the pol gene from Pakistani HIV-positive ART-naïve and ART-experienced individuals. METHODS: In this study, HIV-1 pol was sequenced from 146 HIV-1 positive individuals, divided into ART-naïve (n = 37) and ART-experienced (n = 109). The sequences were also used to determine HIV-1 subtypes, the prevalence of DRM, and pol genetic variability. RESULTS: DRM analysis identified numerous DRMs against reverse transcriptase inhibitors in both ART-naïve and ART-experienced groups, including a few that are classified as rare. Additionally, the ART-experienced group showed mutations associated with resistance to protease inhibitors. Genetic analysis showed negative selection pressure in both groups, but a higher rate of evolution in the ART-naïve group. CONCLUSION: High prevalence of DRMs, especially against previous first-line treatment in ART- naïve and the accumulation of DRMs in ART-experienced groups is concerning and warrants that a more extensive DRM survey be carried out to inform first-line and second-line ART regimen recommendations.

Generation of an Avian Myeloblastosis Virus (AMV) Reverse Transcriptase Prime Editor.

Prime editing (PE) is a novel, double-strand break (DSB)-independent gene editing technology that represents an exciting avenue for the treatment of inherited retinal diseases (IRDs). Given the extensive and heterogenous nature of the 280 genes associated with IRDs, genome editing has presented countless complications. However, recent advances in genome editing technologies have identified PE to have tremendous potential, with the capability to ameliorate small deletions and insertions in addition to all twelve possible transition and transversion mutations. The current PE system is based on the fusion of the Streptococcus pyogenes Cas9 (SpCas9) nickase H840A mutant and an optimized Moloney murine leukemia virus (MMLV) reverse-transcriptase (RT) in conjunction with a PE guide RNA (pegRNA). In this study, we developed a prime editor based on the avian myeloblastosis virus (AMV)-RT and showed its applicability for the installation of the PRPH2 c.828+1G>A mutation in HEK293 cells.

FISH-negative BCR::ABL1-positive e19a2 chronic myeloid leukaemia: the most cryptic of insertions.

BACKGROUND: Chronic myeloid leukaemia (CML) is one of the most well characterised human malignancies. Most patients have a cytogenetically visible translocation between chromosomes 9 and 22 which generates the pathognomonic BCR::ABL1 fusion gene. The derivative chromosome 22 ('Philadelphia' or Ph chromosome) usually harbours the fusion gene encoding a constitutively active ABL1 kinase domain. A small subset of patients have no visible translocation. Historically, these 'Philadelphia chromosome negative' patients caused diagnostic confusion between CML and other myeloproliferative neoplasms; it is now well established that the BCR::ABL1 fusion gene can be generated via submicroscopic intrachromosomal insertion of ABL1 sequence into BCR, or, more rarely, of BCR into ABL1. The fusion genes arising from cryptic insertions are not detectable via G-banded chromosome analysis [karyotype] but can nevertheless always be detected using fluorescence in situ hybridisation (FISH) and/or qualitative reverse transcriptase PCR. CASE PRESENTATION: A 43-year-old female presented with suspected CML in 2007; however, contemporaneous gold standard laboratory investigations, G-banded chromosome analysis and FISH, were both negative. The reverse transcriptase quantitative PCR (RT-qPCR) assay available at the time, which was capable of detecting the common BCR::ABL1 transcripts (e13a2/e14a2), was also negative. Upon review in 2009, the newly recommended reverse transcriptase multiplex PCR (capable of detecting all BCR::ABL1 transcripts including the atypical ones) subsequently detected an e19a2 fusion. The patient then responded to tyrosine kinase inhibitor therapy. In contrast, FISH studies of both samples with three commercially available probes remained consistently negative. Retrospective whole genome sequencing, undertaken as part of the 100,000 Genomes Project, has now revealed that the patient's BCR::ABL1 fusion gene arose via a uniquely small insertion of 122 kb ABL1 sequences into BCR. CONCLUSIONS: We present a patient with suspected chronic myeloid leukaemia whose genetic investigations were originally negative at the time of diagnosis despite the use of contemporaneous gold standard methods. This is the first report of a FISH-negative, BCR::ABL1 positive CML which demonstrates that, even after sixty years of research into one of the most well understood human malignancies, whole genome sequencing can yield novel diagnostic findings in CML.

Retrotransposons hijack alt-EJ for DNA replication and eccDNA biogenesis.

Retrotransposons are highly enriched in the animal genome1-3. The activation of retrotransposons can rewrite host DNA information and fundamentally impact host biology1-3. Although developmental activation of retrotransposons can offer benefits for the host, such as against virus infection, uncontrolled activation promotes disease or potentially drives ageing1-5. After activation, retrotransposons use their mRNA as templates to synthesize double-stranded DNA for making new insertions in the host genome1-3,6. Although the reverse transcriptase that they encode can synthesize the first-strand DNA1-3,6, how the second-strand DNA is generated remains largely unclear. Here we report that retrotransposons hijack the alternative end-joining (alt-EJ) DNA repair process of the host for a circularization step to synthesize their second-strand DNA. We used Nanopore sequencing to examine the fates of replicated retrotransposon DNA, and found that 10% of them achieve new insertions, whereas 90% exist as extrachromosomal circular DNA (eccDNA). Using eccDNA production as a readout, further genetic screens identified factors from alt-EJ as essential for retrotransposon replication. alt-EJ drives the second-strand synthesis of the long terminal repeat retrotransposon DNA through a circularization process and is therefore necessary for eccDNA production and new insertions. Together, our study reveals that alt-EJ is essential in driving the propagation of parasitic genomic retroelements. Our study uncovers a conserved function of this understudied DNA repair process, and provides a new perspective to understand-and potentially control-the retrotransposon life cycle.

Orchestrating nucleic acid-protein interactions at chromosome ends: telomerase mechanisms come into focus.

Telomerase is a special reverse transcriptase ribonucleoprotein dedicated to the synthesis of telomere repeats that protect chromosome ends. Among reverse transcriptases, telomerase is unique in using a stably associated RNA with an embedded template to synthesize a specified sequence. Moreover, it is capable of iteratively copying the same template region (repeat addition processivity) through multiple rounds of RNA-DNA unpairing and reannealing, that is, the translocation reaction. Biochemical analyses of telomerase over the past 3 decades in protozoa, fungi and mammals have identified structural elements that underpin telomerase mechanisms and have led to models that account for the special attributes of telomerase. Notably, these findings and models can now be interpreted and adjudicated through recent cryo-EM structures of Tetrahymena and human telomerase holoenzyme complexes in association with substrates and regulatory proteins. Collectively, these structures reveal the intricate protein-nucleic acid interactions that potentiate telomerase's unique translocation reaction and clarify how this enzyme reconfigures the basic reverse transcriptase scaffold to craft a polymerase dedicated to the synthesis of telomere DNA. Among the many new insights is the resolution of the telomerase 'anchor site' proposed more than 3 decades ago. The structures also highlight the nearly universal conservation of a protein-protein interface between an oligonucleotide/oligosaccharide-binding (OB)-fold regulatory protein and the telomerase catalytic subunit, which enables spatial and temporal regulation of telomerase function in vivo. In this Review, we discuss key features of the structures in combination with relevant functional analyses. We also examine conserved and divergent aspects of telomerase mechanisms as gleaned from studies in different model organisms.