Method for in situ determination of nucleic acid proximity

Abstract

Disclosed is an in situ method for detecting spatial proximity relationships between nucleic acid sequences, such as DNA, in a cell. The method includes: providing a sample of one or more cells comprising nucleic acids; fragmenting the nucleic acids present in the cells that leaves 5′ overhanging ends; filling in the overhanging ends with at least one labeled nucleotide; joining the filled in end of the fragmented nucleic acids that are in close physical proximity to create one or more end joined nucleic acid fragments having a junction; isolating the one or more end joined nucleic acid fragments using the labeled nucleotide; and determining the sequence at the junction of the one or more end joined nucleic acid fragments.

1 - 117 . (canceled) 
     
     
         118 . A method for altering one or more chromatin loops anchored on a pair of loop anchors of one or more specific genomic regions of a chromosome in one or more cells of a cell type from a sample, wherein the sample is from an organ or a tissue or primary cells or cultured cells, said method comprising:
 introducing one or more CRISPR systems into the one or more cells of the cell type from the sample, wherein the one or more CRISPR systems target a region within or around a loop anchor on one or more specific genomic regions of a chromosome in the one or more cells of the cell type, wherein the one or more specific genomic regions of the chromosome in the one or more cells of the cell type comprise one or more chromatin loops, wherein the one or more chromatin loops are identified using a chromosome conformation capture technology, and wherein the one or more CRISPR systems are CRISPR RNA-guided DNA endonuclease systems,   wherein said introducing one or more CRISPR systems into the one or more cells of the cell type introduces a sequence into the loop anchor of the one or more specific genomic regions of the chromosome in the one or more cells of the cell type, or   wherein said introducing one or more CRISPR systems into the one or more cells of the cell type removes a sequence from the loop anchor of the one or more specific genomic regions of the chromosome in the one or more cells of the cell type,   whereby the one or more chromatin loops anchored on the pair of loop anchors of the one or more specific genomic regions of the chromosome in the one or more cells of the cell type are altered.   
     
     
         119 . The method of  claim 118 , wherein at least one base in the loop anchor is mutated. 
     
     
         120 . The method of  claim 118 , wherein the sequence of the loop anchor of the one or more specific genomic regions of the chromosome in the one or more cells of the cell type is inverted. 
     
     
         121 . The method of  claim 120 , wherein a new chromatin loop is generated from the inverted loop anchor. 
     
     
         122 . The method of  claim 118 , wherein the loop anchor is a CTCF motif. 
     
     
         123 . The method of  claim 118 , further comprising introducing two loop anchors into the one or more specific genomic regions of the chromosome in the one or more cells of the cell type using one or more CRISPR systems targeting the one or more specific genome regions of the chromosome in the one or more cells of the cell type such that a new chromatin loop is established. 
     
     
         124 . The method of  claim 118 , wherein the chromosome conformation capture technology is a Hi-C technique and generates a Hi-C heatmap, and pairs of loci that show significantly closer proximity with one another than with the loci lying between the pairs of loci in the Hi-C heatmap are peak loci and represent loop anchors. 
     
     
         125 . The method of  claim 124 , further comprising comparing the peak loci with gene expression data obtained from the one or more cells of the cell type, wherein the specific genomic regions are actively transcribed regions. 
     
     
         126 . The method of  claim 118 , wherein the one or more chromatin loops are involved in the regulation of the expression of a gene. 
     
     
         127 . The method of  claim 126 , wherein the one or more chromatin loops link a promoter and a regulatory element. 
     
     
         128 . The method of  claim 127 , wherein the regulatory element is an enhancer. 
     
     
         129 . The method of  claim 118 , wherein the one or more specific genomic regions is associated with a disease or condition. 
     
     
         130 . The method of  claim 129 , wherein the one or more cells of the cell type are associated with the disease or condition. 
     
     
         131 . The method of  claim 129 , wherein the one or more chromatin loops are associated with the regulation of a gene associated with the disease or condition. 
     
     
         132 . The method of  claim 118 , wherein said introducing the one or more CRISPR systems into the one or more cells of the cell type comprises delivering one or more vectors encoding the one or more CRISPR systems into the one or more cells of the cell type. 
     
     
         133 . The method of  claim 132 , wherein the vectors comprise a tissue-specific promoter. 
     
     
         134 . The method of  claim 118 , wherein said introducing the one or more CRISPR systems into the one or more cells of the cell type comprises delivering a cell-permeable reagent, a pyrrole-imidazole polyamide, into the one or more cells of the cell type. 
     
     
         135 . The method of  claim 118 , wherein the CRISPR systems comprise a nickase. 
     
     
         136 . The method of  claim 118 , wherein the CRISPR systems comprise one or more homology directed repair (HDR) templates. 
     
     
         137 . The method of  claim 118 , further comprising performing DNA-DNA proximity ligation and high throughput sequencing on said one or more cells of the cell type following said introducing the CRISPR systems into the one or more cells of the cell type such that a DNA-DNA proximity ligation library is generated, optionally performing a_HYbrid Capture technique after the library is generated. 
     
     
         138 . The method of  claim 137 , further comprising generating cell clones by culturing the one or more cells of the cell type after said introducing the one or more CRISPR systems into the one or more cells of the cell type and selecting cell clones having the altered chromatin loops. 
     
     
         139 . The method of  claim 138 , wherein the cell clones are screened for a specific phenotype. 
     
     
         140 . The method of  claim 118 , further comprising generating cell clones by culturing the one or more cells of the cell type after said introducing the CRISPR systems into the one or more cells of the cell type and selecting cell clones having a desired expression of one or more genes. 
     
     
         141 . The method of  claim 118 , wherein the one or more cells of the cell type are animal or plant cells. 
     
     
         142 . The method of  claim 118 , wherein the one or more chromatin loops are less than 2 Mb.