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Briefings in Bioinformatics Nov 2019It is easy for today's students and researchers to believe that modern bioinformatics emerged recently to assist next-generation sequencing data analysis. However, the... (Review)
Review
It is easy for today's students and researchers to believe that modern bioinformatics emerged recently to assist next-generation sequencing data analysis. However, the very beginnings of bioinformatics occurred more than 50 years ago, when desktop computers were still a hypothesis and DNA could not yet be sequenced. The foundations of bioinformatics were laid in the early 1960s with the application of computational methods to protein sequence analysis (notably, de novo sequence assembly, biological sequence databases and substitution models). Later on, DNA analysis also emerged due to parallel advances in (i) molecular biology methods, which allowed easier manipulation of DNA, as well as its sequencing, and (ii) computer science, which saw the rise of increasingly miniaturized and more powerful computers, as well as novel software better suited to handle bioinformatics tasks. In the 1990s through the 2000s, major improvements in sequencing technology, along with reduced costs, gave rise to an exponential increase of data. The arrival of 'Big Data' has laid out new challenges in terms of data mining and management, calling for more expertise from computer science into the field. Coupled with an ever-increasing amount of bioinformatics tools, biological Big Data had (and continues to have) profound implications on the predictive power and reproducibility of bioinformatics results. To overcome this issue, universities are now fully integrating this discipline into the curriculum of biology students. Recent subdisciplines such as synthetic biology, systems biology and whole-cell modeling have emerged from the ever-increasing complementarity between computer science and biology.
Topics: Animals; Computational Biology; DNA; History, 20th Century; History, 21st Century; Humans; Proteins
PubMed: 30084940
DOI: 10.1093/bib/bby063 -
Cold Spring Harbor Protocols Oct 2022The most common method for isolating plasmid DNA is derived from an alkaline lysis procedure. The procedure exploits the differential partitioning of plasmid and...
The most common method for isolating plasmid DNA is derived from an alkaline lysis procedure. The procedure exploits the differential partitioning of plasmid and chromosomal DNA when denatured by alkali and subsequently renatured by neutralization of the medium. The circular covalently closed nature of plasmid DNA allows the denatured DNA strands to quickly find each other and reanneal during the renaturation step. This is not the case for chromosomal DNA, which, upon neutralization, aggregates with denatured proteins through hydrophobic interactions. As a result, plasmid DNA remains in solution and can be easily separated from most of the other macromolecules that coprecipitate. For the subsequent purification step, one can use the silica membrane technology integrated in many commercial kits. This technology exploits the ability of DNA to bind to silica in the presence of chaotropic salts. DNA is retained by a silica-based column, whereas most of the polysaccharides and proteins flow through. After wash steps to eliminate residual contaminants and salts, DNA is selectively eluted under low-salt conditions. A kit-free but relatively more cumbersome alternative to this procedure is the traditional phenol-chloroform extraction method followed by ethanol precipitation. Both methods are detailed here.
Topics: Alkalies; Bacteria; Chloroform; DNA; DNA, Bacterial; Ethanol; Phenols; Plasmids; Salts; Silicon Dioxide
PubMed: 35960622
DOI: 10.1101/pdb.prot107852 -
Cellular and Molecular Gastroenterology... 2023Hepatitis B virus (HBV) DNA integration is an incidental event in the virus replication cycle and occurs in less than 1% of infected hepatocytes during viral infection.... (Review)
Review
Hepatitis B virus (HBV) DNA integration is an incidental event in the virus replication cycle and occurs in less than 1% of infected hepatocytes during viral infection. However, HBV DNA is present in the genome of approximately 90% of HBV-related HCCs and is the most common somatic mutation. Whole genome sequencing of liver tissues from chronic hepatitis B patients showed integration occurring at random positions in human chromosomes; however, in the genomes of HBV-related HCC patients, there are integration hotspots. Both the enrichment of the HBV-integration proportion in HCC and the emergence of integration hotspots suggested a strong positive selection of HBV-integrated hepatocytes to progress to HCC. The activation of HBV integration hotspot genes, such as telomerase (TERT) or histone methyltransferase (MLL4/KMT2B), resembles insertional mutagenesis by oncogenic animal retroviruses. These candidate oncogenic genes might shed new light on HBV-related HCC biology and become targets for new cancer therapies. Finally, the HBV integrations in individual HCC contain unique sequences at the junctions, such as virus-host chimera DNA (vh-DNA) presumably being a signature molecule for individual HCC. HBV integration may thus provide a new cell-free tumor DNA biomarker to monitor residual HCC after curative therapies or to track the development of de novo HCC.
Topics: Humans; Hepatitis B virus; Carcinoma, Hepatocellular; Liver Neoplasms; Carcinogenesis; DNA, Viral
PubMed: 36690297
DOI: 10.1016/j.jcmgh.2023.01.001 -
Nature Jun 2023CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for...
CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1-Cas2 integrase is necessary but not sufficient. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation, but many CRISPR-Cas systems lack Cas4. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1-Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited exonucleases for faithful acquisition of new CRISPR immune sequences.
Topics: CRISPR-Associated Proteins; CRISPR-Cas Systems; Cryoelectron Microscopy; DNA; Exonucleases; Integrases; Biocatalysis; Genome, Bacterial
PubMed: 37316664
DOI: 10.1038/s41586-023-06178-2 -
Annual Review of Genetics 2015Horizontal gene transfer plays a major role in microbial evolution, allowing microbes to acquire new genes and phenotypes. Integrative and conjugative elements (ICEs,... (Review)
Review
Horizontal gene transfer plays a major role in microbial evolution, allowing microbes to acquire new genes and phenotypes. Integrative and conjugative elements (ICEs, a.k.a. conjugative transposons) are modular mobile genetic elements integrated into a host genome and are passively propagated during chromosomal replication and cell division. Induction of ICE gene expression leads to excision, production of the conserved conjugation machinery (a type IV secretion system), and the potential to transfer DNA to appropriate recipients. ICEs typically contain cargo genes that are not usually related to the ICE life cycle and that confer phenotypes to host cells. We summarize the life cycle and discovery of ICEs, some of the regulatory mechanisms, and how the types of cargo have influenced our view of ICEs. We discuss how ICEs can acquire new cargo genes and describe challenges to the field and various perspectives on ICE biology.
Topics: Conjugation, Genetic; DNA Replication; DNA Transposable Elements; DNA, Bacterial; Drug Resistance, Bacterial; Gene Expression Regulation; Gene Transfer, Horizontal; Plasmids; Recombination, Genetic
PubMed: 26473380
DOI: 10.1146/annurev-genet-112414-055018 -
The CRISPR Journal Oct 2022While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair...
While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. In this study, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified traffic light reporter (TLR) system (TLR-multi-Cas variant 1 [MCV1]) that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knockins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knockins in mammalian cell lines.
Topics: Humans; CRISPR-Cas Systems; DNA; DNA, Single-Stranded; Endonucleases; Gene Editing; HEK293 Cells; K562 Cells
PubMed: 36070530
DOI: 10.1089/crispr.2022.0058 -
Nature Methods Oct 2021How noncoding DNA determines gene expression in different cell types is a major unsolved problem, and critical downstream applications in human genetics depend on...
How noncoding DNA determines gene expression in different cell types is a major unsolved problem, and critical downstream applications in human genetics depend on improved solutions. Here, we report substantially improved gene expression prediction accuracy from DNA sequences through the use of a deep learning architecture, called Enformer, that is able to integrate information from long-range interactions (up to 100 kb away) in the genome. This improvement yielded more accurate variant effect predictions on gene expression for both natural genetic variants and saturation mutagenesis measured by massively parallel reporter assays. Furthermore, Enformer learned to predict enhancer-promoter interactions directly from the DNA sequence competitively with methods that take direct experimental data as input. We expect that these advances will enable more effective fine-mapping of human disease associations and provide a framework to interpret cis-regulatory evolution.
Topics: Animals; Cell Line; DNA; Databases, Genetic; Epigenesis, Genetic; Gene Expression Regulation; Genome; Genomics; Humans; Machine Learning; Mice; Nerve Net; Quantitative Trait Loci
PubMed: 34608324
DOI: 10.1038/s41592-021-01252-x -
Molecular Cancer Jun 2023Chimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic...
BACKGROUND
Chimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic landscape of solid tumors. However, access to CAR-T cells is not adequate to meet clinical needs, in part due to high cost and long lead times for manufacturing clinical grade virus. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 and double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) via homology-directed repair (HDR), however yields with this approach have been limiting for clinical application (dsDNA) or access to large yields sufficient to meet the manufacturing demands outside early phase clinical trials is limited (ssDNA).
METHODS
We applied homology-independent targeted insertion (HITI) or HDR using CRISPR/Cas9 and nanoplasmid DNA to insert an anti-GD2 CAR into the T cell receptor alpha constant (TRAC) locus and compared both targeted insertion strategies in our system. Next, we optimized post-HITI CRISPR EnrichMENT (CEMENT) to seamlessly integrate it into a 14-day process and compared our knock-in with viral transduced anti-GD2 CAR-T cells. Finally, we explored the off-target genomic toxicity of our genomic engineering approach.
RESULTS
Here, we show that site directed CAR integration utilizing nanoplasmid DNA delivered via HITI provides high cell yields and highly functional cells. CEMENT enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of 5.5 × 10-3.6 × 10 CAR + T cells. CRISPR knock-in CAR-T cells were functionally comparable with viral transduced anti-GD2 CAR-T cells and did not show any evidence of off-target genomic toxicity.
CONCLUSIONS
Our work provides a novel platform to perform guided CAR insertion into primary human T-cells using nanoplasmid DNA and holds the potential to increase access to CAR-T cell therapies.
Topics: Humans; T-Lymphocytes; DNA; Recombinational DNA Repair; Immunotherapy, Adoptive
PubMed: 37365642
DOI: 10.1186/s12943-023-01799-7 -
Gut Mar 2022Infection by HBV is the main risk factor for hepatocellular carcinoma (HCC) worldwide. HBV directly drives carcinogenesis through integrations in the human genome. This...
OBJECTIVE
Infection by HBV is the main risk factor for hepatocellular carcinoma (HCC) worldwide. HBV directly drives carcinogenesis through integrations in the human genome. This study aimed to precisely characterise HBV integrations, in relation with viral and host genomics and clinical features.
DESIGN
A novel pipeline was set up to perform viral capture on tumours and non-tumour liver tissues from a French cohort of 177 patients mainly of European and African origins. Clonality of each integration event was determined with the localisation, orientation and content of the integrated sequence. In three selected tumours, complex integrations were reconstructed using long-read sequencing or Bionano whole genome mapping.
RESULTS
Replicating HBV DNA was more frequently detected in non-tumour tissues and associated with a higher number of non-clonal integrations. In HCC, clonal selection of HBV integrations was related to two different mechanisms involved in carcinogenesis. First, integration of viral enhancer nearby a cancer-driver gene may lead to a strong overexpression of oncogenes. Second, we identified frequent chromosome rearrangements at HBV integration sites leading to cancer-driver genes () alterations at distance. Moreover, HBV integrations have direct clinical implications as HCC with a high number of insertions develop in young patients and have a poor prognosis.
CONCLUSION
Deep characterisation of HBV integrations in liver tissues highlights new HBV-associated driver mechanisms involved in hepatocarcinogenesis. HBV integrations have multiple direct oncogenic consequences that remain an important challenge for the follow-up of HBV-infected patients.
Topics: Carcinogenesis; Carcinoma, Hepatocellular; Case-Control Studies; Cohort Studies; DNA, Viral; Female; Hepatitis B virus; Humans; Liver Neoplasms; Male; Virus Integration
PubMed: 33563643
DOI: 10.1136/gutjnl-2020-323153 -
Frontiers in Cellular and Infection... 2022The incidence of cancer is high worldwide, and biological factors such as viruses and bacteria play an important role in the occurrence of cancer. , , and other... (Review)
Review
The incidence of cancer is high worldwide, and biological factors such as viruses and bacteria play an important role in the occurrence of cancer. , , and other organisms have been identified as carcinogens. Cancer is a disease driven by the accumulation of genome changes. Viruses can directly cause cancer by changing the genetic composition of the human body, such as cervical cancer caused by DNA integration and liver cancer caused by DNA integration. Recently, bacterial DNA has been found around cancers such as pancreatic cancer, breast cancer and colorectal cancer, and the idea that bacterial genes can also be integrated into the human genome has become a hot topic. In the present paper, we reviewed the latest phenomenon and specific integration mechanism of bacterial DNA into the human genome. Based on these findings, we also suggest three sources of bacterial DNA in cancers: bacterial DNA around human tissues, free bacterial DNA in bacteremia or sepsis, and endogenous bacterial DNA in the human genome. Clarifying the theory that bacterial DNA integrates into the human genome can provide a new perspective for cancer prevention and treatment.
Topics: Female; Humans; DNA, Bacterial; Virus Integration; Carcinogenesis; Uterine Cervical Neoplasms; Genome, Human; DNA, Viral
PubMed: 36310856
DOI: 10.3389/fcimb.2022.996778