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. | ||
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. | ||
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. | ||
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). | ||
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. | ||
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). | ||
Sequence rearrangements | RC-seq | Retrotransposon insertions | Retrotransposon Capture sequencing (RC-seq) enriches for mobile the 5′ and 3′ termini of mobile genetic elements. | |
TN-seq / INseq | Mariner transposon insertions | Transposon sequencing (TN-seq) and Insertion sequencing (INseq) study the Himar I Mariner transposon. | ||
TC-seq | DNA double strand break-mediated rearrangements | Translocation Capture sequencing (TC-seq) identifies AID-dependent chromosomal rearrangements. |