scholarly journals Molecular recording of sequential cellular events into DNA

2021 ◽  
Author(s):  
Theresa B Loveless ◽  
Courtney K Carlson ◽  
Vincent J Hu ◽  
Catalina A Dentzel Helmy ◽  
Guohao Liang ◽  
...  
Keyword(s):  
Pulse Sequences ◽  
Cell Lineage ◽  
Complete Sequence ◽  
Guide Rnas ◽  
Cellular Events ◽  
Information Recording ◽  
Dna Strands ◽  
High Throughput Dna Sequencing ◽  
Insertion Mutations ◽  
High Information

Genetically encoded DNA recorders noninvasively convert transient biological events into durable mutations in a cell's genome, allowing for the later reconstruction of cellular experiences using high-throughput DNA sequencing. Existing DNA recorders have achieved high-information recording, durable recording, prolonged recording over multiple timescales, multiplexed recording of several user-selected signals, and temporally resolved signal recording, but not all at the same time. We present a DNA recorder called peCHYRON (prime editing Cell HistorY Recording by Ordered iNsertion) that does. In peCHYRON, prime editor guide RNAs (pegRNAs) insert a variable triplet DNA sequence alongside a constant propagation sequence that deactivates the previous and activates the next step of insertion. This process results in the sequential accumulation of regularly spaced insertion mutations at a synthetic locus. Accumulated insertions are permanent throughout editing because peCHYRON uses a prime editor that avoids cutting both DNA strands, which risks deletions. Editing continues indefinitely because each insertion adds the complete sequence needed to initiate the next step. Constitutively expressed pegRNAs generate insertion patterns that support straightforward reconstruction of cell lineage relationships. Pulsed expression of different pegRNAs enables the reconstruction of pulse sequences, which may be coupled to biological stimuli for temporally-resolved multiplexed event recording.

Conserved segmental expression of Krox-20 in the vertebrate hindbrain and its relationship to lineage restriction

Development ◽  
1991 ◽  
Vol 113 (Supplement_2) ◽  
pp. 59-62 ◽  
Author(s):  
M. Angela Nieto ◽  
Leila C. Bradley ◽  
David G. Wilkinson
Keyword(s):  
Expression Pattern ◽  
Zinc Finger ◽  
Cell Lineage ◽  
Zinc Finger Gene ◽  
Early Stages ◽  
Cellular Events ◽  
Early Expression ◽  
Segmental Expression

The zinc-finger gene Krox-20 is expressed in two alternating segments, rhombomeres (r) 3 and 5, in the developing mouse hindbrain. This expression pattern is established prior to rhombomere formation in the mouse, but it is not known how the timing of expression relates to cellular events of segmentation, such as lineage restriction. We have cloned Krox-20 sequences from Xenopus and the chick and shown that its alternating expression pattern is conserved in these systems, suggesting that its role in hindbrain development is conserved. Analysis of the early stages of Krox-20 expression in the chick show that both domains of expression precede the restriction of cell lineage to specific rhombomeres, consistent with a role of this gene in early events of hindbrain segmentation. The finding that expression is not coincident with lineage restriction indicates that early expression may not reflect an irreversible commitment of cells to r3 and r5 and/or may be mosaic.

Recent progress in NMR microscopy towards cellular imaging

1990 ◽  
Vol 333 (1632) ◽  
pp. 469-475 ◽  
Keyword(s):  
Molecular Diffusion ◽  
Signal To Noise Ratio ◽  
Pulse Sequences ◽  
Cell Lineage ◽  
Experimental System ◽  
Cellular Imaging ◽  
Physical Parameters ◽  
Nmr Microscopy ◽  
Resolution Limits ◽  
Finite Line

Recent advances in NMR microscopy based on fundamental physical parameters and experimental factors are discussed. We consider fundamental resolution limits due to molecular diffusion and the experimental system bandwidth, as well as practical resolution limits arising from poor signal-to-noise ratio due to small imaging voxel size and finite line broadening due to signal attenuation brought about by diffusion. Several microscopic imaging pulse sequences are presented and applied to elucidating cellular imaging problems such as the cell lineage patterns in Xenopus laevis embryos. Experimental results obtained with 7.0 T NMR microscopy system are presented.

scholarly journals Programming large target genomic deletion and concurrent insertion via a prime editing-based method: PEDAR

2021 ◽  
Author(s):  
Tingting Jiang ◽  
Xiao-Ou Zhang ◽  
Zhiping Weng ◽  
Wen Xue
Keyword(s):  
Transfection Efficiency ◽  
Exogenous Dna ◽  
Gene Therapies ◽  
Guide Rnas ◽  
Pathogenic Mutations ◽  
Cas9 Nuclease ◽  
Dna Strands ◽  
Dna Template ◽  
Genomic Insertions ◽  
Deletion Junction

Genomic insertions, duplications, and insertion/deletions (indels) account for ~14% of human pathogenic mutations. Current gene editing methods cannot accurately or efficiently correct these abnormal genomic rearrangements, especially larger alterations (>100 bp). Thus, developing a method to accurately delete insertions/duplications and repair the deletion junction could improve the scope of gene therapies. Here, we engineer a novel gene editor, PE-Cas9, by conjugating Cas9 nuclease to reverse transcriptase. Combined with two prime editing guide RNAs (pegRNAs) targeting complementary DNA strands, PE-Cas9 can direct the replacement of a genomic fragment, ranging from to ~1-kb to >10-kb, with a desired sequence at the target site without requiring an exogenous DNA template. In a reporter cell line, this PE-Cas9-based deletion and repair (PEDAR) method restored mCherry expression through in-frame deletion of a disrupted GFP sequence. We further show that PEDAR efficiency could be enhanced by using pegRNAs with high cleavage activity or increasing transfection efficiency. In tyrosinemia mice, PEDAR removed a 1.38-kb pathogenic insertion within the Fah gene and precisely repaired the deletion junction to restore FAH expression in liver. This study highlights PEDAR as a tool for correcting pathogenic mutations.

scholarly journals DNA damage response inhibition at dysfunctional telomeres by modulation of telomeric DNA damage response RNAs

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Francesca Rossiello ◽  
Julio Aguado ◽  
Sara Sepe ◽  
Fabio Iannelli ◽  
Quan Nguyen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cultured Cells ◽  
Telomeric Dna ◽  
Cellular Events ◽  
Mouse Tissues ◽  
Damage Response ◽  
Dna Strands ◽  
Non Coding Rnas

Abstract The DNA damage response (DDR) is a set of cellular events that follows the generation of DNA damage. Recently, site-specific small non-coding RNAs, also termed DNA damage response RNAs (DDRNAs), have been shown to play a role in DDR signalling and DNA repair. Dysfunctional telomeres activate DDR in ageing, cancer and an increasing number of identified pathological conditions. Here we show that, in mammals, telomere dysfunction induces the transcription of telomeric DDRNAs (tDDRNAs) and their longer precursors from both DNA strands. DDR activation and maintenance at telomeres depend on the biogenesis and functions of tDDRNAs. Their functional inhibition by sequence-specific antisense oligonucleotides allows the unprecedented telomere-specific DDR inactivation in cultured cells and in vivo in mouse tissues. In summary, these results demonstrate that tDDRNAs are induced at dysfunctional telomeres and are necessary for DDR activation and they validate the viability of locus-specific DDR inhibition by targeting DDRNAs.

scholarly journals Transient, flexible gene editing in zebrafish neutrophils and macrophages for determination of cell-autonomous functions

2021 ◽  
Vol 14 (7) ◽  
Author(s):  
Abdulsalam I. Isiaku ◽  
Zuobing Zhang ◽  
Vahid Pazhakh ◽  
Harriet R. Manley ◽  
Ella R. Thompson ◽  
...  
Keyword(s):  
Gene Editing ◽  
Cell Lineage ◽  
Gal4 Driver ◽  
Guide Rnas ◽  
Experimental Systems ◽  
Physiological Processes ◽  
Lamin B Receptor ◽  
Efficient Gene ◽  
Transgenic Lines ◽  
The Many

ABSTRACT Zebrafish are an important model for studying phagocyte function, but rigorous experimental systems to distinguish whether phagocyte-dependent effects are neutrophil or macrophage specific have been lacking. We have developed and validated transgenic lines that enable superior demonstration of cell-autonomous neutrophil and macrophage genetic requirements. We coupled well-characterized neutrophil- and macrophage-specific Gal4 driver lines with UAS:Cas9 transgenes for selective expression of Cas9 in either neutrophils or macrophages. Efficient gene editing, confirmed by both Sanger and next-generation sequencing, occurred in both lineages following microinjection of efficacious synthetic guide RNAs into zebrafish embryos. In proof-of-principle experiments, we demonstrated molecular and/or functional evidence of on-target gene editing for several genes (mCherry, lamin B receptor, trim33) in either neutrophils or macrophages as intended. These new UAS:Cas9 tools provide an improved resource for assessing individual contributions of neutrophil- and macrophage-expressed genes to the many physiological processes and diseases modelled in zebrafish. Furthermore, this gene-editing functionality can be exploited in any cell lineage for which a lineage-specific Gal4 driver is available. This article has an associated First Person interview with the first author of the paper.

scholarly journals Precise genomic deletions using paired prime editing

2021 ◽  
Author(s):  
Junhong Choi ◽  
Wei Chen ◽  
Chase C. Suiter ◽  
Choli Lee ◽  
Florence M. Chardon ◽  
...  
Keyword(s):  
Time Window ◽  
Strand Breaks ◽  
Genomic Deletions ◽  
Small Indels ◽  
Guide Rnas ◽  
Large Deletions ◽  
Protospacer Adjacent Motif ◽  
Dna Strands ◽  
Enhance Efficiency ◽  
Flexible Programming

AbstractTechnologies that precisely delete genomic sequences in a programmed fashion can be used to study function as well as potentially for gene therapy. The leading contemporary method for programmed deletion uses CRISPR/Cas9 and pairs of guide RNAs (gRNAs) to generate two nearby double-strand breaks, which is often followed by deletion of the intervening sequence during DNA repair. However, this approach can be inefficient and imprecise, with errors including small indels at the two target sites as well as unintended large deletions and more complex rearrangements. Here we describe a prime editing-based method that we term PRIME-Del, which induces a deletion using a pair of prime editing gRNAs (pegRNAs) that target opposite DNA strands, effectively programming not only the sites that are nicked but also the outcome of the repair. We demonstrate that PRIME-Del achieves markedly higher precision in programming deletions than CRISPR/Cas9 and gRNA pairs. We also show that PRIME-Del can be used to couple genomic deletions with short insertions, enabling deletions whose junctions do not fall at protospacer-adjacent motif (PAM) sites. Finally, we demonstrate that lengthening the time window of expression of prime editing components can substantially enhance efficiency without compromising precision. We anticipate that PRIME-Del will be broadly useful in enabling precise, flexible programming of genomic deletions, including in-frame deletions, as well as for epitope tagging and potentially for programming rearrangements.

scholarly journals A temporally resolved, multiplex molecular recorder based on sequential genome editing

2021 ◽  
Author(s):  
Junhong Choi ◽  
Wei Chen ◽  
Anna Minkina ◽  
Florence M Chardon ◽  
Chase C Suiter ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Lineage ◽  
General System ◽  
Text Messages ◽  
Tandem Array ◽  
Digital Medium ◽  
Short Text ◽  
Guide Rna ◽  
Guide Rnas

DNA is naturally well-suited to serve as a digital medium for in vivo molecular recording. However, DNA-based memory devices described to date are constrained in terms of the number of distinct signals that can be concurrently recorded and/or by a failure to capture the precise order of recorded events. Here we describe DNA Ticker Tape, a general system for in vivo molecular recording that largely overcomes these limitations. Blank DNA Ticker Tape consists of a tandem array of partial CRISPR-Cas9 target sites, with all but the first site truncated at their 5' ends, and therefore inactive. Signals of interest are coupled to the expression of specific prime editing guide RNAs. Editing events are insertional, and record the identity of the guide RNA mediating the insertion while also shifting the position of the "write head" by one unit along the tandem array, i.e. sequential genome editing. In this proof-of-concept of DNA Ticker Tape, we demonstrate the recording and decoding of complex event histories or short text messages; evaluate the performance of dozens of orthogonal tapes; and construct "long tape" potentially capable of recording the order of as many as 20 serial events. Finally, we demonstrate how DNA Ticker Tape simplifies the decoding of cell lineage histories.

scholarly journals Transcription initiation defines kinetoplast RNA boundaries

2018 ◽  
Author(s):  
François M. Sement ◽  
Takuma Suematsu ◽  
Liye Zhang ◽  
Tian Yu ◽  
Lan Huang ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Noncoding Rna ◽  
Trna Genes ◽  
Mrna Stabilization ◽  
Guide Rnas ◽  
Polycistronic Transcription ◽  
Genomic Location ◽  
Dna Strands ◽  
Duplex Formation

AbstractMitochondrial genomes are often transcribed into polycistronic primary RNAs punctuated by tRNAs whose excision defines mature RNA boundaries. Although kinetoplast DNA lacks tRNA genes, it is commonly held that monophosphorylated 5′-ends of functional molecules typify precursor partitioning by an unknown endonuclease. To the contrary, we demonstrate that in Trypanosoma brucei individual mRNAs and rRNAs are independently synthesized as 3′ extended precursors. The transcription-defined 5′ terminus is converted into monophosphorylated state by the 5′ pyrophosphohydrolase complex, termed PPsome, which is activated by RNA editing substrate binding complex (RESC). Most guide RNAs lack PPsome recognition sites and, therefore, remain triphosphorylated. We provide evidence that both 5′ pyrophosphate removal and 3′ adenylation are essential for mRNA stabilization. Furthermore, we uncover a mechanism by which antisense RNA-controlled 3′-5′ exonucleolytic trimming defines mRNA 3′-end. We conclude that mitochondrial mRNAs and rRNAs are transcribed and processed as insulated units irrespective of their genomic location.SignificanceIt is commonly held that in trypanosomes both mitochondrial DNA strands are transcribed into polycistronic precursors. These primary RNAs are presumably partitioned into individual pre-mRNAs by a “cryptic” endonuclease. We challenged the polycistronic transcription/ endonuclease model after revealing precursor processing by 3′-5′ degradation. This work demonstrates individual transcription of each gene and mRNA 5′-end definition by the first incorporated nucleotide triphosphate. We have uncovered the stabilizing role of 5′ triphosphate to monophosphate conversion and identified a protein complex responsible for this reaction. We have discovered antisense noncoding RNA originating near mRNA 3′ end and showed that a duplex formation modulates exonuclease activity to delimit the mature 3′ end. Collectively, our findings reveal mechanisms by which transcription defines both mRNA termini.

Properties of Isolated Mitochondria of Agaricus Bisporus: Their Relationship to Dormancy and the Germination of Spores

1973 ◽  
Vol 31 ◽  
pp. 458-459
Author(s):  
K. S. McCarty ◽  
R. F. Weave ◽  
L. Kemper ◽  
F. S. Vogel
Keyword(s):  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Microscopic Examination ◽  
Agaricus Bisporus ◽  
Osmotic Shock ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Isolated Mitochondria ◽  
Dna Strands ◽  
Cytoplasmic Ribosomes

During the prodromal stages of sporulation in the Basidiomycete, Agaricus bisporus, mitochondria accumulate in the basidial cells, zygotes, in the gill tissues prior to entry of these mitochondria, together with two haploid nuclei and cytoplasmic ribosomes, into the exospores. The mitochondria contain prominent loci of DNA [Fig. 1]. A modified Kleinschmidt spread technique1 has been used to evaluate the DNA strands from purified whole mitochondria released by osmotic shock, mitochondrial DNA purified on CsCl gradients [density = 1.698 gms/cc], and DNA purified on ethidium bromide CsCl gradients. The DNA appeared as linear strands up to 25 u in length and circular forms 2.2-5.2 u in circumference. In specimens prepared by osmotic shock, many strands of DNA are apparently attached to membrane fragments [Fig. 2]. When mitochondria were ruptured in hypotonic sucrose and then fixed in glutaraldehyde, the ribosomes were released for electron microscopic examination.

Parathyroid gland before and after cisplatin treatment

1987 ◽  
Vol 45 ◽  
pp. 860-861
Author(s):  
S.K. Aggarwal ◽  
J.M. Fadool
Keyword(s):  
Parathyroid Gland ◽  
Wistar Rats ◽  
Antitumor Agent ◽  
The Body ◽  
Cross Linking ◽  
Limiting Factor ◽  
Hormonal Changes ◽  
Primary Mechanism ◽  
Before And After ◽  
Dna Strands

Cisplatin (CDDP) a potent antitumor agent suffers from severe toxic side effects with nephrotoxicity being the major dose-limiting factor, The primary mechanism of its action has been proposed to be through its cross-linking DNA strands. It has also been shown to inactivate various transport enzymes and induce hypocalcemia and hypomagnesemia that may be the underlying cause for some of its toxicities. The present is an effort to study its influence on the parathyroid gland for any hormonal changes that control calcium levels in the body.Male Swiss Wistar rats (Crl: (WI) BR) weighing 200-300 g and of 60 days in age were injected (ip) with cisplatin (7mg/kg in normal saline). The controls received saline injections only. The animals were injected (iv) with calcium (0.5 ml of 10% calcium gluconate/day) and were killed by decapitation on day 1 through 5. Trunk blood was collected in heparinized tubes.

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