scholarly journals Linear-double-stranded DNA (ldsDNA) based AND logic computation in mammalian cells

2018 ◽  
Author(s):  
Weijun Su ◽  
Chunze Zhang ◽  
Shuai Li
Keyword(s):  
Synthetic Biology ◽  
Mammalian Cells ◽  
Rapid Development ◽  
Genetic Circuits ◽  
End Joining ◽  
Double Stranded Dna ◽  
Logic Computation ◽  
Key Enzyme ◽  
Non Homologous End Joining

AbstractSynthetic biology employs engineering principles to redesign biological system for clinical or industrial purposes. The development and application of novel genetic devices for genetic circuits construction will facilitate the rapid development of synthetic biology. Here we demonstrate that mammalian cells could perform two- and three-input linear-double-stranded DNA (ldsDNA) based Boolean AND logic computation. Through hydrodynamic ldsDNA delivery, two-input ldsDNA-base AND-gate computation could be achieved in vivo. Inhibition of DNA-PKcs expression, a key enzyme in non-homologous end joining (NHEJ), could significantly downregulate the intensity of output signals from ldsDNA-based AND-gate. We further reveal that in mammalian cells ldsDNAs could undergo end processing and then perform AND-gate calculation to generate in-frame output proteins. Moreover, we show that ldsDNAs or plasmids with identical overlapping sequences could also serve as inputs of AND-gate computation. Our work establishes novel genetic devices and principles for genetic circuits construction, thus may open a new gate for the development of new disease targeting strategies and new protein genesis methodologies.

scholarly journals Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joonas A. Jamsen ◽  
Akira Sassa ◽  
Lalith Perera ◽  
David D. Shock ◽  
William A. Beard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Time Lapse ◽  
Strand Break ◽  
Structural Basis ◽  
End Joining ◽  
Strand Breaks ◽  
Nucleotide Pools ◽  
Nucleotide Insertion ◽  
Non Homologous End Joining

AbstractReactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

scholarly journals In Vivo Blunt-End Cloning Through CRISPR/Cas9-Facilitated Non-Homologous End-Joining

2015 ◽  
Author(s):  
Jonathan M Geisinger ◽  
Sören Turan ◽  
Sophia Hernandez ◽  
Laura P Spector ◽  
Michele P Calos
Keyword(s):  
Mammalian Cells ◽  
Genome Engineering ◽  
Fluorescent Protein ◽  
Double Strand Breaks ◽  
Hek293 Cells ◽  
End Joining ◽  
Independent Manner ◽  
Strand Breaks ◽  
Non Homologous End Joining

The ability to precisely modify the genome in a site-specific manner is extremely useful. The CRISPR/Cas9 system facilitates precise modifications by generating RNA-guided double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting this excision and repair to insert exogenous sequences in a homology-independent manner without loss of additional nucleotides. We successfully utilize this method in a human immortalized cell line and induced pluripotent stem cells to insert fluorescent protein cassettes into various loci, with efficiencies up to 35.8% in HEK293 cells. We also present a version of Cas9 fused to the FKBP12-L106P destabilization domain for investigating repair dynamics of Cas9-induced double-strand breaks. Our in vivo blunt-end cloning method and destabilization-domain-fused Cas9 variant increase the repertoire of precision genome engineering approaches.

The Synergy between CRISPR and Chemical Engineering

2019 ◽  
Vol 19 (3) ◽  
pp. 147-171
Author(s):  
Cia-Hin Lau ◽  
Chung Tin
Keyword(s):  
Viral Vectors ◽  
Chemical Engineering ◽  
Target Genes ◽  
New Technologies ◽  
Rapid Development ◽  
Genetic Circuits ◽  
Viral Packaging ◽  
Conditional Control ◽  
Logic Operations

Gene therapy and transgenic research have advanced quickly in recent years due to the development of CRISPR technology. The rapid development of CRISPR technology has been largely benefited by chemical engineering. Firstly, chemical or synthetic substance enables spatiotemporal and conditional control of Cas9 or dCas9 activities. It prevents the leaky expression of CRISPR components, as well as minimizes toxicity and off-target effects. Multi-input logic operations and complex genetic circuits can also be implemented via multiplexed and orthogonal regulation of target genes. Secondly, rational chemical modifications to the sgRNA enhance gene editing efficiency and specificity by improving sgRNA stability and binding affinity to on-target genomic loci, and hence reducing off-target mismatches and systemic immunogenicity. Chemically-modified Cas9 mRNA is also more active and less immunogenic than the native mRNA. Thirdly, nonviral vehicles can circumvent the challenges associated with viral packaging and production through the delivery of Cas9-sgRNA ribonucleoprotein complex or large Cas9 expression plasmids. Multi-functional nanovectors enhance genome editing in vivo by overcoming multiple physiological barriers, enabling ligand-targeted cellular uptake, and blood-brain barrier crossing. Chemical engineering can also facilitate viral-based delivery by improving vector internalization, allowing tissue-specific transgene expression, and preventing inactivation of the viral vectors in vivo. This review aims to discuss how chemical engineering has helped improve existing CRISPR applications and enable new technologies for biomedical research. The usefulness, advantages, and molecular action for each chemical engineering approach are also highlighted.

scholarly journals Characterization, modelling and mitigation of gene expression burden in mammalian cells

2019 ◽  
Author(s):  
T Frei ◽  
F Cella ◽  
F Tedeschi ◽  
J Gutierrez ◽  
GB Stan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Host Cells ◽  
Genetic Circuits ◽  
Circuit Performance ◽  
Resource Requirements ◽  
Synthetic Circuit ◽  
Resource Aware

AbstractDespite recent advances in genome engineering, the design of genetic circuits in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here we demonstrate that competition for limited transcriptional and translational resources dynamically couples otherwise independent co-expressed exogenous genes, leading to diminished performance and contributing to the divergence between intended and actual function. We also show that the expression of endogenous genes is likewise impacted when genetic payloads are expressed in the host cells. Guided by a resource-aware mathematical model and our experimental finding that post-transcriptional regulators have a large capacity for resource redistribution, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the novel use of endogenous miRNAs as integral components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells.

scholarly journals Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in Vivo Genome Editing

2020 ◽  
Vol 21 (4) ◽  
pp. 1380 ◽  
Author(s):  
Giovanni Pasquini ◽  
Virginia Cora ◽  
Anka Swiersy ◽  
Kevin Achberger ◽  
Lena Antkowiak ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mammalian Cells ◽  
Time Course ◽  
Sequencing Analysis ◽  
End Joining ◽  
Dsb Repair ◽  
Repair Gene ◽  
Repair Pathways

Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases. Genome editing represents a promising emerging field in the treatment of monogenic disorders, as it aims to correct disease-causing mutations within the genome. Genome editing relies on highly specific endonucleases and the capacity of the cells to repair double-strand breaks (DSBs). As DSB pathways are cell-cycle dependent, their activity in postmitotic retinal neurons, with a focus on photoreceptors, needs to be assessed in order to develop therapeutic in vivo genome editing. Three DSB-repair pathways are found in mammalian cells: Non-homologous end joining (NHEJ); microhomology-mediated end joining (MMEJ); and homology-directed repair (HDR). While NHEJ can be used to knock out mutant alleles in dominant disorders, HDR and MMEJ are better suited for precise genome editing, or for replacing entire mutation hotspots in genomic regions. Here, we analyzed transcriptomic in vivo and in vitro data and revealed that HDR is indeed downregulated in postmitotic neurons, whereas MMEJ and NHEJ are active. Using single-cell RNA sequencing analysis, we characterized the dynamics of DSB repair pathways in the transition from dividing cells to postmitotic retinal cells. Time-course bulk RNA-seq data confirmed DSB repair gene expression in both in vivo and in vitro samples. Transcriptomic DSB repair pathway profiles are very similar in adult human, macaque, and mouse retinas, but not in ground squirrel retinas. Moreover, human-induced pluripotent stem-cell-derived neurons and retinal organoids can serve as well suited in vitro testbeds for developing genomic engineering approaches in photoreceptors. Our study provides additional support for designing precise in vivo genome-editing approaches via MMEJ, which is active in mature photoreceptors.

scholarly journals DNA repair of clustered lesions in mammalian cells: involvement of non-homologous end-joining

2008 ◽  
Vol 36 (15) ◽  
pp. 4872-4882 ◽  
Author(s):  
S. Malyarchuk ◽  
R. Castore ◽  
L. Harrison
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
End Joining ◽  
Non Homologous End Joining

scholarly journals Regulating synthetic gene networks in 3D materials

2012 ◽  
Vol 109 (38) ◽  
pp. 15217-15222 ◽  
Author(s):  
Tara L. Deans ◽  
Anirudha Singh ◽  
Matthew Gibson ◽  
Jennifer H. Elisseeff
Keyword(s):  
Synthetic Biology ◽  
Gene Networks ◽  
Materials Science ◽  
Genetic Circuits ◽  
Therapeutic Applications ◽  
Regulatory Processes ◽  
Cellular Events ◽  
The Matrix

Combining synthetic biology and materials science will enable more advanced studies of cellular regulatory processes, in addition to facilitating therapeutic applications of engineered gene networks. One approach is to couple genetic inducers into biomaterials, thereby generating 3D microenvironments that are capable of controlling intrinsic and extrinsic cellular events. Here, we have engineered biomaterials to present the genetic inducer, IPTG, with different modes of activating genetic circuits in vitro and in vivo. Gene circuits were activated in materials with IPTG embedded within the scaffold walls or chemically linked to the matrix. In addition, systemic applications of IPTG were used to induce genetic circuits in cells encapsulated into materials and implanted in vivo. The flexibility of modifying biomaterials with genetic inducers allows for patterned placement of these inducers that can be used to generate distinct patterns of gene expression. Together, these genetically interactive materials can be used to characterize genetic circuits in environments that more closely mimic cells’ natural 3D settings, to better explore complex cell–matrix and cell–cell interactions, and to facilitate therapeutic applications of synthetic biology.

scholarly journals Sequence Conversion by Single Strand Oligonucleotide Donors via Non-homologous End Joining in Mammalian Cells

2010 ◽  
Vol 285 (30) ◽  
pp. 23198-23207 ◽  
Author(s):  
Jia Liu ◽  
Alokes Majumdar ◽  
Jilan Liu ◽  
Lawrence H. Thompson ◽  
Michael M. Seidman
Keyword(s):  
Mammalian Cells ◽  
Single Strand ◽  
End Joining ◽  
Non Homologous End Joining

DNA double-strand break repair within heterochromatic regions

2012 ◽  
Vol 40 (1) ◽  
pp. 173-178 ◽  
Author(s):  
Johanne M. Murray ◽  
Tom Stiff ◽  
Penny A. Jeggo
Keyword(s):  
Chromatin Structure ◽  
Mammalian Cells ◽  
Strand Break ◽  
Higher Order ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Dna Dsbs ◽  
A Cell ◽  
Non Homologous End Joining

DNA DSBs (double-strand breaks) represent a critical lesion for a cell, with misrepair being potentially as harmful as lack of repair. In mammalian cells, DSBs are predominantly repaired by non-homologous end-joining or homologous recombination. The kinetics of repair of DSBs can differ widely, and recent studies have shown that the higher-order chromatin structure can dramatically affect the pathway utilized, the rate of repair and the genetic factors required for repair. Studies of the repair of DSBs arising within heterochromatic DNA regions have provided insight into the constraints that higher-order chromatin structure poses on repair and the processing that is uniquely required for the repair of such DSBs. In the present paper, we provide an overview of our current understanding of the process of heterochromatic DSB repair in mammalian cells and consider the evolutionary conservation of the processes.

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