scholarly journals Competitive precision genome editing (CGE) assay for functional analysis at single base resolution

2021 ◽  
Author(s):  
Paivi Pihlajamaa ◽  
Otto Kauko ◽  
Biswajyoti Sahu ◽  
Teemu Kivioja ◽  
Jussi Taipale
Keyword(s):  
Genome Editing ◽  
Statistical Power ◽  
Target Sequence ◽  
Wild Type ◽  
Single Base ◽  
Functional Consequence ◽  
E Box ◽  
Single Base Resolution ◽  
Original Feature

The two major limitations of applying CRISPR/Cas9-technology for analysis of the effect of genotype on phenotype are the difficulty of cutting DNA exactly at the intended site, and the decreased cell proliferation and other phenotypic effects caused by the DNA cuts themselves. Here we report a novel competitive genome editing assay that allows analysis of the functional consequence of precise mutations. The method is based on precision genome editing, where a target sequence close to a feature of interest is cut, and the DNA is then repaired using a template that either reconstitutes the original feature, or introduces an altered sequence. Introducing sequence labels to both types of repair templates generates a large number of replicate cultures, increasing statistical power. In addition, the labels identify edited cells, allowing direct comparison between cells that carry wild-type and mutant features. Here, we apply the assay for multiplexed analysis of the role of E-box sequences on MYC binding and cellular fitness.

Precise genome editing at single-base resolution by novel CRISPR-nickase system

2016 ◽  
Vol 33 ◽  
pp. S64-S65
Author(s):  
Atsushi Satomura ◽  
Kouichi Kuroda ◽  
Mitsuyoshi Ueda
Keyword(s):  
Genome Editing ◽  
Single Base ◽  
Single Base Resolution

scholarly journals Mirror bisulfite sequencing — a method for single-base resolution of hydroxymethylcytosine

2015 ◽  
Author(s):  
Xueguang Sun ◽  
Darany Tan ◽  
Tzu Hung Chung ◽  
Xi-Yu Jia
Keyword(s):  
Bisulfite Sequencing ◽  
Biological Role ◽  
Human Tissues ◽  
Bisulfite Conversion ◽  
Single Base ◽  
Cpg Site ◽  
Genome Wide ◽  
Single Base Resolution ◽  
Dna Strand

While the role of 5-methylcytosine has been well studied, the biological role of 5-hydroxymethylcytosine still remains unclear due to the limited methods available for single-base detection of 5-hydroxymethylcytosine (5hmC). Here, we present Mirror bisulfite sequencing detects 5-hydroxymethylcytosines at a single CpG site by synthesizing a DNA strand to mirror the parental strand. This semi-conservative duplex is sequentially treated with β-glucosyltransferase and M.SssI methylase. A glucosyl-5hmCpG in the parental strand inhibits methylation of the mirroring CpG site, and after bisulfite conversion, a thymine in the mirroring strand indicates a 5hmCpG site in the parental strand whereas a cytosine indicates a non-5hmC site. Using this method, the 5hmC levels of various human tissues and paired liver tissues were mapped genome-wide.

scholarly journals cdk1- and cdk2-Mediated Phosphorylation of MyoD Ser200 in Growing C2 Myoblasts: Role in Modulating MyoD Half-Life and Myogenic Activity

1999 ◽  
Vol 19 (4) ◽  
pp. 3167-3176 ◽  
Author(s):  
Magali Kitzmann ◽  
Marie Vandromme ◽  
Valerie Schaeffer ◽  
Gilles Carnac ◽  
Jean-Claude Labbé ◽  
...  
Keyword(s):  
Half Life ◽  
Site Directed Mutagenesis ◽  
Cyclin B ◽  
Wild Type ◽  
E Box ◽  
Integral Role ◽  
Phosphopeptide Mapping

ABSTRACT We have examined the role of protein phosphorylation in the modulation of the key muscle-specific transcription factor MyoD. We show that MyoD is highly phosphorylated in growing myoblasts and undergoes substantial dephosphorylation during differentiation. MyoD can be efficiently phosphorylated in vitro by either purified cdk1-cyclin B or cdk1 and cdk2 immunoprecipitated from proliferative myoblasts. Comparative two-dimensional tryptic phosphopeptide mapping combined with site-directed mutagenesis revealed that cdk1 and cdk2 phosphorylate MyoD on serine 200 in proliferative myoblasts. In addition, when the seven proline-directed sites in MyoD were individually mutated, only substitution of serine 200 to a nonphosphorylatable alanine (MyoD-Ala200) abolished the slower-migrating hyperphosphorylated form of MyoD, seen either in vitro after phosphorylation by cdk1-cyclin B or in vivo following overexpression in 10T1/2 cells. The MyoD-Ala200 mutant displayed activity threefold higher than that of wild-type MyoD in transactivation of an E-box-dependent reporter gene and promoted markedly enhanced myogenic conversion and fusion of 10T1/2 fibroblasts into muscle cells. In addition, the half-life of MyoD-Ala200 protein was longer than that of wild-type MyoD, substantiating a role of Ser200 phosphorylation in regulating MyoD turnover in proliferative myoblasts. Taken together, our data show that direct phosphorylation of MyoD Ser200 by cdk1 and cdk2 plays an integral role in compromising MyoD activity during myoblast proliferation.

scholarly journals High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants

2020 ◽  
Author(s):  
Karthik Murugan ◽  
Arun S. Seetharam ◽  
Andrew J. Severin ◽  
Dipali G. Sashital
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Double Strand Breaks ◽  
Target Sequence ◽  
Wild Type ◽  
Strand Breaks ◽  
Cleavage Assay ◽  
Target Sequences

AbstractCas9 is an RNA-guided endonuclease in the bacterial CRISPR-Cas immune system and a popular tool for genome editing. The most commonly used Cas9 variant, Streptococcus pyogenes Cas9 (SpCas9), is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific than wild-type (WT) SpCas9. However, systematic comparisons of the cleavage activities of these Cas9 variants have not been reported. In this study, we employed our high-throughput in vitro cleavage assay to compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9, and HiFi Cas9). We observed that all Cas9s tested were able to cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on the target sequence and Cas9 variant. For targets with multiple mismatches, SaCas9 and engineered SpCas9 variants are more prone to nicking, while WT SpCas9 creates double-strand breaks (DSB). These differences in cleavage rates and DSB formation may account for the varied specificities observed in genome editing studies. Our analysis reveals mismatch position-dependent, off-target nicking activity of Cas9 variants which have been underreported in previous in vivo studies.

scholarly journals Alteration of Salmonella enterica Virulence and Host Pathogenesis through Targeting sdiA by Using the CRISPR-Cas9 System

2021 ◽  
Vol 9 (12) ◽  
pp. 2564
Author(s):  
Momen Askoura ◽  
Ahmad J. Almalki ◽  
Amr S. Abu Lila ◽  
Khaled Almansour ◽  
Farhan Alshammari ◽  
...  
Keyword(s):  
Genome Editing ◽  
Salmonella Enterica ◽  
Bacterial Genome ◽  
Infection Model ◽  
Wild Type ◽  
Signal Molecule ◽  
Enteric Infections ◽  
The Impact

Salmonella enterica is a common cause of many enteric infections worldwide and is successfully engineered to deliver heterologous antigens to be used as vaccines. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) RNA-guided Cas9 endonuclease is a promising genome editing tool. In the current study, a CRISPR-Cas9 system was used to target S.enterica sdiA that encodes signal molecule receptor SdiA and responds to the quorum sensing (QS) signaling compounds N-acylhomoserine lactones (AHLs). For this purpose, sdiA was targeted in both S.enterica wild type (WT) and the ΔssaV mutant strain, where SsaV has been reported to be an essential component of SPI2-T3SS. The impact of sdiA mutation on S. enterica virulence was evaluated at both early invasion and later intracellular replication in both the presence and absence of AHL. Additionally, the influence of sdiA mutation on the pathogenesis S. enterica WT and mutants was investigated in vivo, using mice infection model. Finally, the minimum inhibitory concentrations (MICs) of various antibiotics against S. enterica strains were determined. Present findings show that mutation in sdiA significantly affects S.enterica biofilm formation, cell adhesion and invasion. However, sdiA mutation did not affect bacterial intracellular survival. Moreover, in vivo bacterial pathogenesis was markedly lowered in S.enterica ΔsdiA in comparison with the wild-type strain. Significantly, double-mutant sdiA and ssaV attenuated the S. enterica virulence and in vivo pathogenesis. Moreover, mutations in selected genes increased Salmonella susceptibility to tested antibiotics, as revealed by determining the MICs and MBICs of these antibiotics. Altogether, current results clearly highlight the importance of the CRISPR-Cas9 system as a bacterial genome editing tool and the valuable role of SdiA in S.enterica virulence. The present findings extend the understanding of virulence regulation and host pathogenesis of Salmonellaenterica.

scholarly journals Highly specific chimeric DNA-RNA guided genome editing with enhanced CRISPR-Cas12a system

2021 ◽  
Author(s):  
Hanseop Kim ◽  
Wi-jae Lee ◽  
Chan Hyoung Kim ◽  
Yeounsun Oh ◽  
Lee Wha Gwon ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Deoxyribonucleic Acid ◽  
Specific Gene ◽  
Target Sequence ◽  
Sequence Specificity ◽  
Wild Type ◽  
Dna Cleaving ◽  
Target Dna ◽  
Near Future

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system is composed of a Cas12a effector that acts as a deoxyribonucleic acid (DNA)-cleaving endonuclease and a crispr ribonucleic acid (crRNA) that guides the effector to the target DNA. It is considered a key molecule for inducing target-specific gene editing in various living systems. Here, we improved the efficiency and specificity of the CRISPR-Cas12a system through protein and crRNA engineering. In particular, to optimize the CRISPR-Cas12a system at the molecular level, we used a chimeric DNA-RNA guide chemically similar to crRNA to maximize target sequence specificity. Compared to the wild type (wt)-Cas12a system, when using enhanced Cas12a system (en-Cas12a), the efficiency and target specificity improved on average by 7.41 and 7.60 times respectively. In our study, when the chimeric DNA-RNA guided en-Cas12a effector was used, the gene editing efficiency and accuracy were simultaneously increased. These findings could contribute to highly accurate genome editing, such as human gene therapy, in the near future.

Differential Role of Components of the Fibrinolytic System in the Formation and Removal of Thrombus Induced by Endothelial Injury

1999 ◽  
Vol 81 (04) ◽  
pp. 601-604 ◽  
Author(s):  
Hiroyuki Matsuno ◽  
Osamu Kozawa ◽  
Masayuki Niwa ◽  
Shigeru Ueshima ◽  
Osamu Matsuo ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Thrombus Formation ◽  
Endothelial Injury ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Wild Type ◽  
Type Plasminogen Activator ◽  
Pai 1

SummaryThe role of fibrinolytic system components in thrombus formation and removal in vivo was investigated in groups of six mice deficient in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), or plasminogen activator inhibitor-1 (PAI-1) (u-PA-/-, t-PA-/- or PAI-1-/-, respectively) or of their wild type controls (u-PA+/+, t-PA+/+ or PAI-1+/+). Thrombus was induced in the murine carotid artery by endothelial injury using the photochemical reaction between rose bengal and green light (540 nm). Blood flow was continuously monitored for 90 min on day 0 and for 20 min on days 1, 2 and 3. The times to occlusion after the initiation of endothelial injury in u-PA+/+, t-PA+/+ or PAI-1+/+ mice were 9.4 ± 1.3, 9.8 ± 1.1 or 9.7 ± 1.6 min, respectively. u-PA-/- and t-PA-/- mice were indistinguishable from controls, whereas that of PAI-1-/- mice were significantly prolonged (18.4 ± 3.7 min). Occlusion persisted for the initial 90 min observation period in 10 of 18 wild type mice and was followed by cyclic reflow and reocclusion in the remaining 8 mice. At day 1, persistent occlusion was observed in 1 wild type mouse, 8 mice had cyclic reflow and reocclusion and 9 mice had persistent reflow. At day 2, all injured arteries had persistent reflow. Persistent occlusion for 90 min on day 0 was observed in 3 u-PA-/-, in all t-PA-/- mice at day 1 and in 2 of the t-PA-/-mice at day 2 (p <0.01 versus wild type mice). Persistent patency was observed in all PAI-1-/- mice at day 1 and in 5 of the 6 u-PA-/- mice at day 2 (both p <0.05 versus wild type mice). In conclusion, t-PA increases the rate of clot lysis after endothelial injury, PAI-1 reduces the time to occlusion and delays clot lysis, whereas u-PA has little effect on thrombus formation and spontaneous lysis.

Cell-specific regulation of IRS-1 gene expression: role of E box and C/EBP binding site in HepG2 cells and CHO cells

Diabetes ◽  
1997 ◽  
Vol 46 (3) ◽  
pp. 354-362 ◽  
Author(s):  
K. Matsuda ◽  
E. Araki ◽  
R. Yoshimura ◽  
K. Tsuruzoe ◽  
N. Furukawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Cho Cells ◽  
Hepg2 Cells ◽  
E Box ◽  
Specific Regulation

Exploring power in response inhibition tasks using the bootstrap: The impact of number of participants, number of trials, effect magnitude, and study design

2019 ◽  
Author(s):  
Curtis David Von Gunten ◽  
Bruce D Bartholow
Keyword(s):  
Study Design ◽  
Effect Size ◽  
Statistical Power ◽  
Bootstrap Sampling ◽  
Reliable Measure ◽  
Internal Reliability ◽  
The Impact ◽  
Power Analyses ◽  
Effect Magnitude

A primary psychometric concern with laboratory-based inhibition tasks has been their reliability. However, a reliable measure may not be necessary or sufficient for reliably detecting effects (statistical power). The current study used a bootstrap sampling approach to systematically examine how the number of participants, the number of trials, the magnitude of an effect, and study design (between- vs. within-subject) jointly contribute to power in five commonly used inhibition tasks. The results demonstrate the shortcomings of relying solely on measurement reliability when determining the number of trials to use in an inhibition task: high internal reliability can be accompanied with low power and low reliability can be accompanied with high power. For instance, adding additional trials once sufficient reliability has been reached can result in large gains in power. The dissociation between reliability and power was particularly apparent in between-subject designs where the number of participants contributed greatly to power but little to reliability, and where the number of trials contributed greatly to reliability but only modestly (depending on the task) to power. For between-subject designs, the probability of detecting small-to-medium-sized effects with 150 participants (total) was generally less than 55%. However, effect size was positively associated with number of trials. Thus, researchers have some control over effect size and this needs to be considered when conducting power analyses using analytic methods that take such effect sizes as an argument. Results are discussed in the context of recent claims regarding the role of inhibition tasks in experimental and individual difference designs.

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