scholarly journals Target DNA mutagenesis-based fluorescence assessment of off-target activity of the CRISPR-Cas9 system

RSC Advances ◽  
2019 ◽  
Vol 9 (16) ◽  
pp. 9067-9074
Author(s):  
Dan Wang ◽  
Cuili Niu ◽  
Jingxin Han ◽  
Dejun Ma ◽  
Zhen Xi
Keyword(s):  
Living Cells ◽  
Luciferase Reporter ◽  
System P ◽  
Cas9 Nuclease ◽  
Target Dna ◽  
Target Activity ◽  
Dna Mutagenesis

Fluorescence assessment of off-target activity of Cas9 nuclease using an engineered dual-luciferase reporter in living cells.

A practical pH-compatible fluorescent sensor for hydrazine in soil, water and living cells

The Analyst ◽  
2020 ◽  
Vol 145 (22) ◽  
pp. 7380-7387 ◽  
Author(s):  
Huming Yan ◽  
Fangjun Huo ◽  
Yongkang Yue ◽  
Jianbin Chao ◽  
Caixia Yin
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Respiratory Tract ◽  
Human Body ◽  
Water Solubility ◽  
Fluorescent Sensor ◽  
Living Cells ◽  
Human Organs ◽  
System P ◽  
The Central Nervous System

The excellent water solubility of hydrazine (N2H4) allows it to easily invade the human body through the skin and respiratory tract, thereby damaging human organs and the central nervous system.

scholarly journals Cas12a is a dynamic and precise RNA-guided nuclease without off-target activity on λ-DNA

2021 ◽  
Author(s):  
Bijoya Paul ◽  
Loic Chaubet ◽  
Emma Verver ◽  
Guillermo Montoya
Keyword(s):  
Dna Binding ◽  
Genome Editing ◽  
Optical Tweezers ◽  
Target Site ◽  
Target Dna ◽  
Multiple Binding ◽  
Target Sites ◽  
Dna Binding And Cleavage ◽  
Target Activity ◽  
Insight Into

Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we combined optical tweezers with fluorescence to monitor Cas12a binding onto λ-DNA, providing insight into its DNA binding and cleavage mechanisms. At low forces Cas12a binds DNA specifically with two off-target sites, while at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. Despite the multiple binding events, cleavage is only observed on the target site at low forces, when the DNA is flexible. Activity assays show that the preferential off-target sites are not cleaved, and the λ-DNA is severed at the target site. This precision is also observed in Cas12a variants where the specific dsDNA and the unspecific ssDNA cleavage are dissociated or nick the target DNA. We propose that Cas12a and its variants are precise endonucleases that efficiently scan the DNA for its target but only cleave the selected site in the λ-DNA.

Development of a microdevice-based human mesenchymal stem cell-mediated drug delivery system

2019 ◽  
Vol 7 (6) ◽  
pp. 2348-2357 ◽  
Author(s):  
Junfei Xia ◽  
Ang-Chen Tsai ◽  
Wenhao Cheng ◽  
Xuegang Yuan ◽  
Teng Ma ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Drug Delivery System ◽  
Delivery System ◽  
Drug Delivery Systems ◽  
Human Mesenchymal Stem Cell ◽  
Living Cells ◽  
Controlled Delivery ◽  
System P

Cell-mediated drug delivery systems utilize living cells as vehicles to achieve controlled delivery of drugs.

scholarly journals A review: Possible optimization of Cas9-sgRNA nuclease delivery via ingested lipid nanoparticles bioencapsulated within plant cell-based enfolding

2019 ◽  
Author(s):  
Tatiana Hillman
Keyword(s):  
Dna Sequences ◽  
Genetic Disorders ◽  
Single Point ◽  
Lipid Nanoparticles ◽  
Single Point Mutation ◽  
Cas9 Nuclease ◽  
Target Dna ◽  
Effective Delivery ◽  
Sgrna Design

The possibility of gene editing to correct disorders is one of the most impactful therapeutic agents, currently. CRISPR Cas9-sgRNA nucleases can be used to cleave and to delete harmful or pathogenic DNA sequences, which cause genetic disorders. Cas9 nuclease includes palindromic repeats that cut and delete a single point mutation or multiple DNA target site sequences. The Cas9, attached to a sgRNA or a guiding RNA, finds and then cleaves the target DNA sequence. The Cas9-sgRNA method of cleavage has corrected DNA mutations that cause cataracts in the eyes, cystic fibrosis, and chronic granulomatous disease. However, there are issues with an effective delivery of Cas9-sgRA to target DNA sequences. Delivering Cas-9 nucleases are negatively affected by off-target DNA sites, sgRNA design, off-target cleavage, Cas9 activation, and the method of delivery. This review focuses on oral and ingested delivery methods to effectively guide the transport of Cas9-sgRNA nucleases in vivo. This review presents possible alternatives for nuclease delivery within optimized lipid-nanoparticles, plant, algae, and bacterial-based orally ingested edibles. This review attempts to provide evidence in support of the higher effectiveness of ingesting therapeutic bioencapsulated edibles because the edibles can directly contact immune cells within the gastrointestinal tract for blood or lymph circulation.

scholarly journals Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Minh Thuan Nguyen Tran ◽  
Mohd Khairul Nizam Mohd Khalid ◽  
Qi Wang ◽  
Jacqueline K. R. Walker ◽  
Grace E. Lidgerwood ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Editing Efficiency ◽  
Base Editing ◽  
Target Dna ◽  
Dna Editing ◽  
Target Activity ◽  
Adenine Base

Abstract Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.

scholarly journals Structural basis for Cas9 off-target activity

2021 ◽  
Author(s):  
Martin Pacesa ◽  
Chun-Han Lin ◽  
Antoine Clery ◽  
Katja Bargsten ◽  
Matthew J. Irby ◽  
...  
Keyword(s):  
Rational Design ◽  
Target Prediction ◽  
Structural Basis ◽  
Guide Rna ◽  
Guide Rnas ◽  
Target Dna ◽  
Bona Fide ◽  
Dna Specificity ◽  
Target Binding ◽  
Target Activity

The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of non- canonical base pairing interactions and preservation of base stacking within the guide-off-target heteroduplex. Off-target sites containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping rather than RNA bulge formation. Additionally, PAM-distal mismatches result in duplex unpairing and induce a conformational change of the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.

PyA-cluster system for the detection and imaging of miRNAs in living cells through double-three-way junction formation

2018 ◽  
Vol 54 (54) ◽  
pp. 7471-7474 ◽  
Author(s):  
Jong Jin Ro ◽  
Ha Jung Lee ◽  
Byeang Hyean Kim
Keyword(s):  
Fluorescence Probe ◽  
Living Cells ◽  
Cluster System ◽  
The Novel ◽  
Extended Version ◽  
System P ◽  
Junction Formation

Herein, we describe an extended version of a fluorescence probe for detecting miRNAs through the novel application of a PyA-cluster system.

A new fluorescence turn-on chemosensor for nanomolar detection of Al3+ constructed from a pyridine–pyrazole system

2018 ◽  
Vol 42 (4) ◽  
pp. 2933-2941 ◽  
Author(s):  
Barnali Naskar ◽  
Kinsuk Das ◽  
Ramij R. Mondal ◽  
Dilip K. Maiti ◽  
Alberto Requena ◽  
...  
Keyword(s):  
Living Cells ◽  
Selective Detection ◽  
Turn On ◽  
System P ◽  
Nanomolar Detection ◽  
Fluorescence Turn On

A pyridine–pyrazole based fluorescence turn-on chemosensor provides access to selective detection of Al3+ in solution as well as in HepG2 living cells.

scholarly journals Real-Time Monitoring of Calcineurin Activity in Living Cells: Evidence for Two Distinct Ca2+-dependent Pathways in Fission Yeast

2006 ◽  
Vol 17 (11) ◽  
pp. 4790-4800 ◽  
Author(s):  
Lu Deng ◽  
Reiko Sugiura ◽  
Mai Takeuchi ◽  
Masahiro Suzuki ◽  
Hidemine Ebina ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Fission Yeast ◽  
Tandem Repeats ◽  
Living Cells ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Concentration Dependent Manner ◽  
Intracellular Ca ◽  
Calcineurin Activity

In fission yeast, calcineurin dephosphorylates and activates the Prz1 transcription factor. Here, we identified the calcineurin-dependent response element (CDRE) in the promoter region of prz1+ gene and monitored the calcineurin activity in living cells using a destabilized luciferase reporter gene fused to three tandem repeats of CDRE. Elevated extracellular CaCl2 caused an increase in calcineurin activity with an initial peak and then approached a sustained constant level in a concentration-dependent manner. In CaCl2-sensitive mutants such as Δpmc1, the response was markedly enhanced, reflecting its high intracellular Ca2+. Agents expected to induce Ca2+ influx showed distinct patterns of the CDRE-reporter activity, suggesting different mechanisms of calcineurin activation. Knockout of yam8+ or cch1+ encoding putative subunits of a Ca2+ channel abolished the activation of calcineurin upon exposure to various stimuli, including high extracellular NaCl and cell wall–damaging agents. However, knockout of yam8+ or cch1+ did not affect the activation of calcineurin upon stimulation by elevated extracellular Ca2+. The Pck2 protein kinase C-Pmk1 mitogen-activate protein kinase pathway was required for the stimulation of calcineurin via Yam8/Cch1-mediated Ca2+ influx, but it was not required for the stimulation by elevated extracellular Ca2+, suggesting two distinct pathways for calcineurin activation.

scholarly journals Quantification of the affinities of CRISPR–Cas9 nucleases for cognate protospacer adjacent motif (PAM) sequences

2020 ◽  
Vol 295 (19) ◽  
pp. 6509-6517 ◽  
Author(s):  
Vladimir Mekler ◽  
Konstantin Kuznedelov ◽  
Konstantin Severinov
Keyword(s):  
Genome Editing ◽  
Target Location ◽  
Dna Probes ◽  
Target Sequence ◽  
Francisella Novicida ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Protospacer Adjacent Motif ◽  
Target Dna

The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9–gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9–gRNA, SaCas9–gRNA, and FnCas9–gRNA, respectively) and of three engineered SpCas9–gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a “beacon” assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9–gRNA binding to fluorescently labeled target DNA derivatives called “Cas9 beacons.” We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9–gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.

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