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Nature Biotechnology Apr 2023Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We...
Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.
Topics: Humans; CRISPR-Cas Systems; Integrases; DNA Cleavage; Gene Editing; DNA; DNA End-Joining Repair
PubMed: 36424489
DOI: 10.1038/s41587-022-01527-4 -
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 -
Nucleic Acids Research May 2023Human papillomavirus (HPV) integration is a critical step in cervical cancer development; however, the oncogenic mechanism at the genome-wide transcriptional level is...
Human papillomavirus (HPV) integration is a critical step in cervical cancer development; however, the oncogenic mechanism at the genome-wide transcriptional level is still poorly understood. In this study, we employed integrative analysis on multi-omics data of six HPV-positive and three HPV-negative cell lines. Through HPV integration detection, super-enhancer (SE) identification, SE-associated gene expression and extrachromosomal DNA (ecDNA) investigation, we aimed to explore the genome-wide transcriptional influence of HPV integration. We identified seven high-ranking cellular SEs generated by HPV integration in total (the HPV breakpoint-induced cellular SEs, BP-cSEs), leading to intra-chromosomal and inter-chromosomal regulation of chromosomal genes. The pathway analysis revealed that the dysregulated chromosomal genes were correlated to cancer-related pathways. Importantly, we demonstrated that BP-cSEs existed in the HPV-human hybrid ecDNAs, explaining the above transcriptional alterations. Our results suggest that HPV integration generates cellular SEs that function as ecDNA to regulate unconstrained transcription, expanding the tumorigenic mechanism of HPV integration and providing insights for developing new diagnostic and therapeutic strategies.
Topics: Female; Humans; Human Papillomavirus Viruses; Papillomavirus Infections; Uterine Cervical Neoplasms; Virus Integration; Enhancer Elements, Genetic; DNA; Transcription, Genetic; Genome, Human; Carcinogenesis; Chromosome Breakpoints; Chromosomes, Human
PubMed: 36864748
DOI: 10.1093/nar/gkad105 -
Small (Weinheim An Der Bergstrasse,... Jun 2019Dynamic DNA structures, a type of DNA construct built using programmable DNA self-assembly, have the capability to reconfigure their conformations in response to... (Review)
Review
Dynamic DNA structures, a type of DNA construct built using programmable DNA self-assembly, have the capability to reconfigure their conformations in response to environmental stimulation. A general strategy to design dynamic DNA structures is to integrate reconfigurable elements into conventional static DNA structures that may be assembled from a variety of methods including DNA origami and DNA tiles. Commonly used reconfigurable elements range from strand displacement reactions, special structural motifs, target-binding DNA aptamers, and base stacking components, to DNA conformational change domains, etc. Morphological changes of dynamic DNA structures may be visualized by imaging techniques or may be translated to other detectable readout signals (e.g., fluorescence). Owing to their programmable capability of recognizing environmental cues with high specificity, dynamic DNA structures embody the epitome of robust and versatile systems that hold great promise in sensing and imaging biological analytes, in delivering molecular cargos, and in building programmable systems that are able to conduct sophisticated tasks.
Topics: DNA; Nanostructures; Nanotechnology; Nucleic Acid Conformation
PubMed: 30970183
DOI: 10.1002/smll.201900228 -
Journal of Biomedical Science Jul 2023Dysregulating cellular metabolism is one of the emerging cancer hallmarks. Mitochondria are essential organelles responsible for numerous physiologic processes, such as... (Review)
Review
Dysregulating cellular metabolism is one of the emerging cancer hallmarks. Mitochondria are essential organelles responsible for numerous physiologic processes, such as energy production, cellular metabolism, apoptosis, and calcium and redox homeostasis. Although the "Warburg effect," in which cancer cells prefer aerobic glycolysis even under normal oxygen circumstances, was proposed a century ago, how mitochondrial dysfunction contributes to cancer progression is still unclear. This review discusses recent progress in the alterations of mitochondrial DNA (mtDNA) and mitochondrial dynamics in cancer malignant progression. Moreover, we integrate the possible regulatory mechanism of mitochondrial dysfunction-mediated mitochondrial retrograde signaling pathways, including mitochondrion-derived molecules (reactive oxygen species, calcium, oncometabolites, and mtDNA) and mitochondrial stress response pathways (mitochondrial unfolded protein response and integrated stress response) in cancer progression and provide the possible therapeutic targets. Furthermore, we discuss recent findings on the role of mitochondria in the immune regulatory function of immune cells and reveal the impact of the tumor microenvironment and metabolism remodeling on cancer immunity. Targeting the mitochondria and metabolism might improve cancer immunotherapy. These findings suggest that targeting mitochondrial retrograde signaling in cancer malignancy and modulating metabolism and mitochondria in cancer immunity might be promising treatment strategies for cancer patients and provide precise and personalized medicine against cancer.
Topics: Humans; Calcium; Neoplasms; Mitochondria; DNA, Mitochondrial; Reactive Oxygen Species; Tumor Microenvironment
PubMed: 37525297
DOI: 10.1186/s12929-023-00956-w -
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