Single-cell spatial mapping reveals alteration of cell type composition and tissue microenvironment during early colorectal cancer formation.

Colorectal cancer (CRC) is the third leading cause of cancer mortality in the United States. Familial adenomatous polyposis (FAP) is a hereditary syndrome that raises the risk of developing CRC, with total colectomy as the only effective prevention. Even though FAP is rare (0.

Multiomic analysis of familial adenomatous polyposis reveals molecular pathways associated with early tumorigenesis.

Familial adenomatous polyposis (FAP) is a genetic disease causing hundreds of premalignant polyps in affected persons and is an ideal model to study transitions of early precancer states to colorectal cancer (CRC). We performed deep multiomic profiling of 93 samples, including normal mucosa, benign polyps and dysplastic polyps, from six persons with FAP. Transcriptomic, proteomic, metabolomic and lipidomic analyses revealed a dynamic choreography of thousands of molecular and cellular events that occur during precancerous transitions toward cancer formation.

Simultaneous profiling of host expression and microbial abundance by spatial metatranscriptome sequencing.

We developed an analysis pipeline that can extract microbial sequences from spatial transcriptomic (ST) data and assign taxonomic labels, generating a spatial microbial abundance matrix in addition to the default host expression matrix, enabling simultaneous analysis of host expression and microbial distribution. We called the pipeline spatial metatranscriptome (SMT) and applied it on both human and murine intestinal sections and validated the spatial microbial abundance information with alternative assays. Biological insights were gained from these novel data that showed host-microbe interaction at various spatial scales.

Single-cell analyses define a continuum of cell state and composition changes in the malignant transformation of polyps to colorectal cancer.

To chart cell composition and cell state changes that occur during the transformation of healthy colon to precancerous adenomas to colorectal cancer (CRC), we generated single-cell chromatin accessibility profiles and single-cell transcriptomes from 1,000 to 10,000 cells per sample for 48 polyps, 27 normal tissues and 6 CRCs collected from patients with or without germline APC mutations. A large fraction of polyp and CRC cells exhibit a stem-like phenotype, and we define a continuum of epigenetic and transcriptional changes occurring in these stem-like cells as they progress from homeostasis to CRC. Advanced polyps contain increasing numbers of stem-like cells, regulatory T cells and a subtype of pre-cancer-associated fibroblasts.

Nucleofection of phiC31 Integrase Protein Mediates Sequence-Specific Genomic Integration in Human Cells.

The phage-derived phiC31 integrase is a useful tool for mediating sequence-specific genomic integration in mammalian cells, recombining donor plasmids bearing the attB recognition site with introduced genomic attP sites or endogeneous pseudo-attP sites having partial identity to attP. In most prior studies, phiC31 integrase has been introduced as plasmid DNA or mRNA. The current report examines whether phiC31 integrase functions efficiently in mammalian cells when co-nucleofected as a purified protein, along with attB-containing donor plasmids or PCR fragments.

Plasmid-Mediated Gene Therapy in Mouse Models of Limb Girdle Muscular Dystrophy.

We delivered plasmid DNA encoding therapeutic genes to the muscles of mouse models of limb girdle muscular dystrophy (LGMD) 2A, 2B, and 2D, deficient in calpain3, dysferlin, and alpha-sarcoglycan, respectively. We also delivered the human follistatin gene, which has the potential to increase therapeutic benefit. After intramuscular injection of DNA, electroporation was applied to enhance delivery to muscle fibers.

Applications of Alternative Nucleases in the Age of CRISPR/Cas9.

Breakthroughs in the development of programmable site-specific nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases (MNs), and most recently, the clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins (including Cas9) have greatly enabled and accelerated genome editing. By targeting double-strand breaks to user-defined locations, the rates of DNA repair events are greatly enhanced relative to un-catalyzed events at the same sites. However, the underlying biology of each genome-editing nuclease influences the targeting potential, the spectrum of off-target cleavages, the ease-of-use, and the types of recombination events at targeted double-strand breaks.

The intron landscape of the mtDNA cytb gene among the Ascomycota: introns and intron-encoded open reading frames.

Fungal mitochondrial genes are frequently noted for the presence of introns. These introns are self-splicing and can be assigned to either group I or II introns and they can encode open reading frames (ORFs). This study examines the introns present within the cytochrome b (cytb) gene of ascomycetes fungi.

Three new active members of the I-OnuI family of homing endonucleases.

In vitro characterization of 3 LAGLIDADG-type homing endonucleases (HEs) (I-CcaI, I-CcaII, and I-AstI) that belong to the I-OnuI family showed that they are functional HEs that cleave their respective cognate target sites. These endonucleases are encoded within group ID introns and appear to be orthologues that have inserted into 3 different mitochondrial genes: rns, rnl, and cox3. The endonuclease activity of I-CcaI was tested using various substrates, and its minimum DNA recognition sequence was estimated to be 26 nt.

Programmable Genome Editing Tools and their Regulation for Efficient Genome Engineering.

Targeted genome editing has become a powerful genetic tool for studying gene function or for modifying genomes by correcting defective genes or introducing genes. A variety of reagents have been developed in recent years that can generate targeted double-stranded DNA cuts which can be repaired by the error-prone, non-homologous end joining repair system or via the homologous recombination-based double-strand break repair pathway provided a suitable template is available. These genome editing reagents require components for recognizing a specific DNA target site and for DNA-cleavage that generates the double-stranded break.

Insertion of Group II Intron-Based Ribozyme Switches into Homing Endonuclease Genes.

Fungal mitochondrial genomes act as "reservoirs" for homing endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing, targeted mutagenesis and gene therapy applications. Herein, we present strategies where homing endonuclease open reading frames (HEases ORFs) are interrupted with group II intron sequences.

Using Group II Introns for Attenuating the In Vitro and In Vivo Expression of a Homing Endonuclease.

In Chaetomium thermophilum (DSM 1495) within the mitochondrial DNA (mtDNA) small ribosomal subunit (rns) gene a group IIA1 intron interrupts an open reading frame (ORF) encoded within a group I intron (mS1247). This arrangement offers the opportunity to examine if the nested group II intron could be utilized as a regulatory element for the expression of the homing endonuclease (HEase). Constructs were generated where the codon-optimized ORF was interrupted with either the native group IIA1 intron or a group IIB type intron.

PCR-based bioprospecting for homing endonucleases in fungal mitochondrial rRNA genes.

Fungal mitochondrial genomes act as "reservoirs" for homing endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing and gene therapy applications. Bioprospecting and characterization of naturally occurring homing endonucleases offers an alternative to synthesizing artificial endonucleases.

A homing endonuclease with a switch: characterization of a twintron encoded homing endonuclease.

The small ribosomal subunit gene residing in the mitochondrial DNA of the thermophilic fungus Chaetomium thermophilum var. thermophilum La Touche DSM 1495 is interrupted by a twintron at position mS1247. The mS1247 twintron represents the first mixed twintron found in fungal mtDNA, composed of an external group I intron encoding a LAGLIDADG open reading frame that is interrupted by an internal group II intron.