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Table 2 A gambit of technological methods to interrogate the genome’s complexity in every possible way

From: Gene editing in the context of an increasingly complex genome

Broad area Technique Investigates Description Citation
RNA transcription, translation, and binding ChIRP-seq RNA-DNA binding Chromatin Isolation by RNA purification sequencing (ChIRP-seq) is used to determine regions of the genome that are bound by a specific RNA species. [54]
CLASH RNA-RNA binding Crosslinking, Ligation, And Sequencing of Hybrids (CLASH) is capable of determining RNA-RNA binding interactions. [56]
GRO-seq Active RNA transcription Global Run-On sequencing (GRO-seq) determines the sites in the genome at which active transcription is occurring by targeting transcriptionally-engaged RNA polymerases. [189]
NET-seq Active RNA transcription Native elongating transcript sequencing (NET-seq) determines, at nucleotide resolution, the sites in the genome at which active transcription is occurring by targeting the 3’ends of nascent transcripts associated with RNA polymerases. [190]
Ribo-seq Active RNA translation Ribosome sequencing (Ribo-seq) is capable of identifying ribosome-bound messenger RNAs (mRNAs), i.e., mRNAs that are under active translation. [191]
TRAP-seq Active RNA translation Translating Ribosome Affinity Purification sequencing (TRAP-seq) quantifies all mRNAs that are associated with 80s ribosome. [192]
RIP-seq RNA–protein binding RNA Immunoprecipitation sequencing (RIP-seq) is used to determine RNA species that are bound to a RNA binding protein (RBP) of interest. [57,58,59]
HITS-CLIP RNA-protein binding High Throughput Sequencing Crosslinking and Immunoprecipitation (HITS-CLIP) is used to determine RNA species that are bound to a RBP of interest.
HITS-CLIP is similar to RIP-seq with an added in vivo UV crosslinking step that improves specificity at the RNA-protein boundary.
[193]
PAR-CLIP RNA-protein binding Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) determines RNA species that are bound to a RBP of interest. PAR-CLIP improves on HITS-CLIP and RIP-seq through the inclusion photoreactive ribonucleoside analogs, which further improves specificity at the RNA-protein boundary during crosslinking. [194]
iCLIP RNA-protein binding Individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) determines RNA species that are bound to a RBP of interest, and provides base-level specificity at the RNA-protein boundary. [195]
PARE-seq miRNA target RNA Parallel Analysis of RNA Ends sequencing (PARE-seq) looks at the 5′ ends of polyadenylated products of miRNA-mediated mRNA decay to identify miRNA-target RNA pairs. [196, 197]
TIF-seq PEAT RNA transcript isoforms Transcript Isoform Sequencing (TIF-seq) allows for the identification of transcript isoforms by mapping their exact 5’ start and 3’end boundaries. [198, 199]
RNA form and structure SHAPE-seq RNA secondary and tertiary conformation Selective 2’-Hydroxyl Acylation analyzed by Primer Extension sequencing (SHAPE-seq) utilizes SHAPE chemistry followed by multiplexed paired-end deep sequencing of primer extension products and bioinformatic analysis using a maximum likelihood model to infer secondary and tertiary RNA structure. [200]
PARS RNA secondary structure Parallel analysis of RNA structure (PARS) determines RNA secondary structure simultaneously for thousands of RNA molecules via enzymatic footprinting with different RNAses. [201]
Frag-seq RNA secondary structure Fragmentation sequencing (Frag-seq) determines RNA secondary structure transcriptome-wide via P1 endonuclease, which cleaves single-stranded nucleic acids. [202]
ICE RNA inosines Inosine Chemical Erasing (ICE) identifies inosines on RNA species in the context of adenosine-to-inosine (A-to-I) conversion, a post-transcriptional modification that diversifies the transcriptome in various pathways. [203]
MeRIP-seq RNA methylation of the N6 position of adenosine (m6A) Methylated RNA Immunoprecipitation sequencing (MeRIP-Seq) identifies RNA species with methylation of the N6 position of adenosine (m6A), a post-transcriptional RNA modification. [204]
Cap-seq / CIP-TAP RNA 5′ capping Cap sequencing (Cap-seq) and Calf Intestinal alkaline Phosphatase Tobacco Acid Pyrophosphatase (CIP-TAP) both enrich for the 5′ ends of Pol II RNA species and differ based on the following: Cap-seq is selective for long-capped RNAs; CIP-TAP is selective for capped small RNAs (csRNAs). Both therefore define Pol II transcription start sites (TSSs). [205, 206]
DNA-protein interactions DNase-seq Global mapping of active regulatory chromatin, i.e., nucleosome-depleted DNase-seq identifies regulatory regions by targeting DNase I hypersensitive (HS) sites. [207]
FAIRE-seq Global mapping of active regulatory chromatin, i.e., nucleosome-depleted Formaldehyde-Assisted Isolation of Regulatory Elements sequencing (FAIRE-seq) identifies regions of active chromatin that coincide with DNase I HS sites and others. [208, 209]
MNase-seq (MAINE-seq) Global mapping of histone-bound DNA, i.e., nucleosome positioning MNase-Assisted Isolation of Nucleosomes Sequencing (MAINE-seq) identifies histone-bound DNA via digestion by micrococcal nuclease (MN). [210]
ATAC-seq Global mapping of both active regulatory chromatin and histone-bound DNA Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) identifies regions of DNA via hyperactive Tn5 transposase, which inserts adapters into accessible regions of chromatin. [211]
ChIA-PET Detects global chromatin interactions and infers 3-D structure Chromatin Interaction Analysis by Paired-End Tag sequencing (ChIA-PET) isolates chromatin interactions by formaldehyde cross-linking, sonication, and then chromatin immunoprecipitation (ChIP). Paired chromatin DNA fragments are then connected with linkers. [212]
3-C, 4-C, 5-C, Hi-C Captures interactions within and between chromosomes and infers 3-D structure Chromosome conformation capture (3C), chromosome conformation capture on chip (4C), 3C-carbon copy (5C), and high-throughput chromosome conformation capture are methods used to identify chromatin interactions at short ranges between 2 loci (3C) or long ranges via multiple loci (Hi-C). [213,214,215,216]
Sequence rearrangements RC-seq Retrotransposon insertions Retrotransposon Capture sequencing (RC-seq) enriches for mobile the 5′ and 3′ termini of mobile genetic elements. [217, 218]
TN-seq / INseq Mariner transposon insertions Transposon sequencing (TN-seq) and Insertion sequencing (INseq) study the Himar I Mariner transposon. [219, 220]
TC-seq DNA double strand break-mediated rearrangements Translocation Capture sequencing (TC-seq) identifies AID-dependent chromosomal rearrangements. [221, 222]