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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 -
Nature Biotechnology May 2022The targeted deletion, replacement, integration or inversion of genomic sequences could be used to study or treat human genetic diseases, but existing methods typically...
The targeted deletion, replacement, integration or inversion of genomic sequences could be used to study or treat human genetic diseases, but existing methods typically require double-strand DNA breaks (DSBs) that lead to undesired consequences, including uncontrolled indel mixtures and chromosomal abnormalities. Here we describe twin prime editing (twinPE), a DSB-independent method that uses a prime editor protein and two prime editing guide RNAs (pegRNAs) for the programmable replacement or excision of DNA sequences at endogenous human genomic sites. The two pegRNAs template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, which replace the endogenous DNA sequence between the prime-editor-induced nick sites. When combined with a site-specific serine recombinase, twinPE enabled targeted integration of gene-sized DNA plasmids (>5,000 bp) and targeted sequence inversions of 40 kb in human cells. TwinPE expands the capabilities of precision gene editing and might synergize with other tools for the correction or complementation of large or complex human pathogenic alleles.
Topics: Base Sequence; CRISPR-Cas Systems; Chromosome Inversion; DNA; Gene Editing; Humans; RNA, Guide, CRISPR-Cas Systems
PubMed: 34887556
DOI: 10.1038/s41587-021-01133-w -
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 -
Proceedings of the National Academy of... May 2023CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies....
CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK (ong dsDNA with 3'-verhangs mediated RISPR nock-in), by utilizing specially designed 3'-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3'-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.
Topics: Animals; CRISPR-Cas Systems; Base Sequence; Gene Editing; DNA; Homologous Recombination; Mammals
PubMed: 37216515
DOI: 10.1073/pnas.2221127120 -
British Journal of Cancer Sep 2022In the current era of precision medicine, the identification of genomic alterations has revolutionised the management of patients with solid tumours. Recent advances in... (Review)
Review
In the current era of precision medicine, the identification of genomic alterations has revolutionised the management of patients with solid tumours. Recent advances in the detection and characterisation of circulating tumour DNA (ctDNA) have enabled the integration of liquid biopsy into clinical practice for molecular profiling. ctDNA has also emerged as a promising biomarker for prognostication, monitoring disease response, detection of minimal residual disease and early diagnosis. In this Review, we discuss current and future clinical applications of ctDNA primarily in non-small cell lung cancer in addition to other solid tumours.
Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell-Free Nucleic Acids; Circulating Tumor DNA; Humans; Liquid Biopsy; Lung Neoplasms
PubMed: 35347327
DOI: 10.1038/s41416-022-01776-9 -
Nature Biotechnology Apr 2021Existing technologies for site-specific integration of kilobase-sized DNA sequences in bacteria are limited by low efficiency, a reliance on recombination, the need for...
Existing technologies for site-specific integration of kilobase-sized DNA sequences in bacteria are limited by low efficiency, a reliance on recombination, the need for multiple vectors, and challenges in multiplexing. To address these shortcomings, we introduce a substantially improved version of our previously reported Tn7-like transposon from Vibrio cholerae, which uses a Type I-F CRISPR-Cas system for programmable, RNA-guided transposition. The optimized insertion of transposable elements by guide RNA-assisted targeting (INTEGRATE) system achieves highly accurate and marker-free DNA integration of up to 10 kilobases at ~100% efficiency in bacteria. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplexed insertions in three genomic loci and facile, multi-loci deletions by combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in biomedically and industrially relevant bacteria and achieved target- and species-specific integration in a complex bacterial community. This work establishes INTEGRATE as a versatile tool for multiplexed, kilobase-scale genome engineering.
Topics: CRISPR-Cas Systems; DNA Transposable Elements; Gene Editing; Genome, Bacterial; Plasmids; RNA, Guide, CRISPR-Cas Systems; Vibrio cholerae
PubMed: 33230293
DOI: 10.1038/s41587-020-00745-y -
Nature Communications Aug 2023Genome editing, specifically CRISPR/Cas9 technology, has revolutionized biomedical research and offers potential cures for genetic diseases. Despite rapid progress, low...
Genome editing, specifically CRISPR/Cas9 technology, has revolutionized biomedical research and offers potential cures for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of unintended mutations represent major limitations for genome editing applications caused by the interplay with DNA double-strand break repair pathways. To address this, we conduct a large-scale compound library screen to identify targets for enhancing targeted genome insertions. Our study reveals DNA-dependent protein kinase (DNA-PK) as the most effective target to improve CRISPR/Cas9-mediated insertions, confirming previous findings. We extensively characterize AZD7648, a selective DNA-PK inhibitor, and find it to significantly enhance precise gene editing. We further improve integration efficiency and precision by inhibiting DNA polymerase theta (Polϴ). The combined treatment, named 2iHDR, boosts templated insertions to 80% efficiency with minimal unintended insertions and deletions. Notably, 2iHDR also reduces off-target effects of Cas9, greatly enhancing the fidelity and performance of CRISPR/Cas9 gene editing.
Topics: Gene Editing; CRISPR-Cas Systems; Protein Kinases; DNA Repair; DNA
PubMed: 37580318
DOI: 10.1038/s41467-023-40344-4 -
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 -
Nature Biotechnology Jan 2021CRISPR-guided DNA cytosine and adenine base editors are widely used for many applications but primarily create DNA base transitions (that is, pyrimidine-to-pyrimidine or...
CRISPR-guided DNA cytosine and adenine base editors are widely used for many applications but primarily create DNA base transitions (that is, pyrimidine-to-pyrimidine or purine-to-purine). Here we describe the engineering of two base editor architectures that can efficiently induce targeted C-to-G base transversions, with reduced levels of unwanted C-to-W (W = A or T) and indel mutations. One of these C-to-G base editors (CGBE1), consists of an RNA-guided Cas9 nickase, an Escherichia coli-derived uracil DNA N-glycosylase (eUNG) and a rat APOBEC1 cytidine deaminase variant (R33A) previously shown to have reduced off-target RNA and DNA editing activities. We show that CGBE1 can efficiently induce C-to-G edits, particularly in AT-rich sequence contexts in human cells. We also removed the eUNG domain to yield miniCGBE1, which reduced indel frequencies but only modestly decreased editing efficiency. CGBE1 and miniCGBE1 enable C-to-G edits and will serve as a basis for optimizing C-to-G base editors for research and therapeutic applications.
Topics: CRISPR-Cas Systems; Cytidine Deaminase; Cytosine; DNA; Gene Editing; Guanine; HEK293 Cells; Humans
PubMed: 32690971
DOI: 10.1038/s41587-020-0609-x