scholarly journals Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention

2017 ◽  
Author(s):  
Qiang Zhang ◽  
Hui-Li Xing ◽  
Zhi-Ping Wang ◽  
Hai-Yan Zhang ◽  
Fang Yang ◽  
...  
Keyword(s):  
High Frequency ◽  
High Specificity ◽  
Cleavage Sites ◽  
Wild Type ◽  
Fusion Strategy ◽  
Editing Efficiency ◽  
Wild Type Counterpart ◽  
Reliable Isolation ◽  
Specificity Score ◽  
Target Effects

AbstractSpecificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We report unexpected observations of high frequency off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants although the sgRNA was predicted to have a high specificity score. We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. Optimization of the mCherry vectors and subsequent validation found that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that in plants, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful in improving genome editing efficiency and specificity in plants and other organisms.

scholarly journals Efficient Peptide-Mediated In Vitro Delivery of Cas9 RNP

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 878
Author(s):  
Oskar Gustafsson ◽  
Julia Rädler ◽  
Samantha Roudi ◽  
Tõnis Lehto ◽  
Mattias Hällbrink ◽  
...  
Keyword(s):  
Freeze Drying ◽  
Cell Penetrating Peptide ◽  
Wild Type ◽  
Efficient Manner ◽  
Editing Efficiency ◽  
Freeze Thaw ◽  
Enzyme Digest ◽  
Clinical Potential ◽  
Generation Sequencing

The toolbox for genetic engineering has quickly evolved from CRISPR/Cas9 to a myriad of different gene editors, each with promising properties and enormous clinical potential. However, a major challenge remains: delivering the CRISPR machinery to the nucleus of recipient cells in a nontoxic and efficient manner. In this article, we repurpose an RNA-delivering cell-penetrating peptide, PepFect14 (PF14), to deliver Cas9 ribonucleoprotein (RNP). The RNP-CPP complex achieved high editing rates, e.g., up to 80% in HEK293T cells, while being active at low nanomolar ranges without any apparent signs of toxicity. The editing efficiency was similar to or better compared to the commercially available reagents RNAiMAX and CRISPRMax. The efficiency was thoroughly evaluated in reporter cells and wild-type cells by restriction enzyme digest and next-generation sequencing. Furthermore, the CPP-Cas9-RNP complexes were demonstrated to withstand storage at different conditions, including freeze-thaw cycles and freeze-drying, without a loss in editing efficiency. This CPP-based delivery strategy complements existing technologies and further opens up new opportunities for Cas9 RNP delivery, which can likely be extended to other gene editors in the future.

Normal Synaptonemal Complex and Abnormal Recombination Nodules in Two Alleles of the Drosophila Meiotic Mutant mei-W68

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1337-1356 ◽  
Author(s):  
Adelaide T C Carpenter
Keyword(s):  
Electron Microscopy ◽  
Synaptonemal Complex ◽  
High Frequency ◽  
Serial Section ◽  
The Other ◽  
Wild Type ◽  
Recombination Nodules ◽  
Section Electron ◽  
Serial Section Electron Microscopy ◽  
Section Electron Microscopy

Abstract The meiotic phenotypes of two mutant alleles of the mei-W68 gene, 1 and L1, were studied by genetics and by serial-section electron microscopy. Despite no or reduced exchange, both mutant alleles have normal synaptonemal complex. However, neither has any early recombination nodules; instead, both exhibit high numbers of very long (up to 2 μm) structures here named “noodles.” These are hypothesized to be formed by the unchecked extension of identical but much shorter structures ephemerally seen in wild type, which may be precursors of early recombination nodules. Although the mei-W68L1 allele is identical to the mei-W681 allele in both the absence of early recombination nodules and a high frequency of noodles (i.e., it is amorphic for the noodle phene), it is hypomorphic in its effects on exchange and late recombination nodules. The differential effects of this allele on early and late recombination nodules are consistent with the hypothesis that Drosophila females have two separate recombination pathways—one for simple gene conversion, the other for exchange.

scholarly journals Comparison of pathogenicity of subtype H9 avian influenza wild-type viruses from a wide geographic origin expressing mono-, di-, or tri-basic hemagglutinin cleavage sites

2020 ◽  
Vol 51 (1) ◽  
Author(s):  
Rokshana Parvin ◽  
Jan Schinkoethe ◽  
Christian Grund ◽  
Reiner Ulrich ◽  
Franziska Bönte ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Geographic Origin ◽  
Cleavage Sites ◽  
Wild Type

scholarly journals Synthesis of Trypsin-Resistant Variants of the Listeria-Active Bacteriocin Salivaricin P

2010 ◽  
Vol 76 (16) ◽  
pp. 5356-5362 ◽  
Author(s):  
Eileen F. O'Shea ◽  
Paula M. O'Connor ◽  
Paul D. Cotter ◽  
R. Paul Ross ◽  
Colin Hill
Keyword(s):  
Antimicrobial Activity ◽  
Gastrointestinal Tract ◽  
Cleavage Sites ◽  
Wild Type ◽  
Enhanced Resistance ◽  
Fold Reduction ◽  
Two Component ◽  
Specific Protease ◽  
The Individual ◽  
Protease Action

ABSTRACT Two-component salivaricin P-like bacteriocins have demonstrated potential as antimicrobials capable of controlling infections in the gastrointestinal tract (GIT). The anti-Listeria activity of salivaricin P is optimal when the individual peptides Sln1 and Sln2 are added in succession at a 1:1 ratio. However, as degradation by digestive proteases may compromise the functionality of these peptides within the GIT, we investigated the potential to create salivaricin variants with enhanced resistance to the intestinal protease trypsin. A total of 11 variants of the salivaricin P components, in which conservative modifications at the trypsin-specific cleavage sites were explored in order to protect the peptides from trypsin degradation while maintaining their potent antimicrobial activity, were generated. Analysis of these variants revealed that eight were resistant to trypsin digestion while retaining antimicrobial activity. Combining the complementary trypsin-resistant variants Sln1-5 and Sln2-3 resulted in a MIC50 of 300 nM against Listeria monocytogenes, a 3.75-fold reduction in activity compared to the level for wild-type salivaricin P. This study demonstrates the potential of engineering bacteriocin variants which are resistant to specific protease action but which retain significant antimicrobial activity.

Mutation at autosomal loci of Chinese hamster ovary cells: involvement of a high-frequency event silencing two linked alleles

1983 ◽  
Vol 3 (7) ◽  
pp. 1172-1181
Author(s):  
W E Bradley
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
High Frequency ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Low Frequency ◽  
Class Ii ◽  
Class I ◽  
Wild Type ◽  
Ovary Cells

Two classes of cell lines heterozygous at the galactokinase (glk) locus have been isolated from Chinese hamster ovary cells. Class I, selected by plating nonmutagenized wild-type cells at low density in medium containing 2-deoxygalactose at a partially selective concentration, underwent subsequent mutation to the glk-/- genotype at a low frequency (approximately 10(-6) per cell), which was increased by mutagenesis. Class II heterozygotes, isolated by sib selection from mutagenized wild-type cells, had a higher spontaneous frequency of mutation to the homozygous state (approximately 10(-4) per cell), which was not affected by mutagenesis. About half of the glk-/- mutants derived from a class II heterozygote, but not the heterozygote itself, were functionally hemizygous at the syntenic thymidine kinase (tk) locus. Similarly, a tk+/- heterozygote with characteristics analogous to the class II glk+/- cell lines underwent high-frequency mutation to tk-/-, and most of these mutants, but not the tk+/- heterozygote, were functionally hemizygous at the glk locus. A model is proposed, similar to that for the mutational events at the adenine phosphoribosyl transferase locus (W. E. C. Bradley and D. Letovanec, Somatic Cell Genet. 8:51-66, 1982), of two different events, high and low frequency, being responsible for mutation at either of the linked loci tk and glk. The low-frequency event may be a point mutation, but the high-frequency event, in many instances, involves coordinated inactivation of a portion of a chromosome carrying the two linked alleles. Class II heterozygotes would be generated as a result of a low-frequency event at one allele, and class I heterozygotes would be generated by a high-frequency event. Supporting this model was the demonstration that all class I glk+/- lines examined were functionally hemizygous at tk.

scholarly journals Prohormone convertases 1/3 and 2 together orchestrate the site-specific cleavages of progastrin to release gastrin-34 and gastrin-17

2008 ◽  
Vol 415 (1) ◽  
pp. 35-43 ◽  
Author(s):  
Jens F. Rehfeld ◽  
Xiaorong Zhu ◽  
Christina Norrbom ◽  
Jens R. Bundgaard ◽  
Anders H. Johnsen ◽  
...  
Keyword(s):  
Structural Factors ◽  
Cleavage Sites ◽  
Wild Type ◽  
Prohormone Convertases ◽  
Site Specific ◽  
G Cells ◽  
Processing Site ◽  
Vitro Effect

Cellular synthesis of peptide hormones requires PCs (prohormone convertases) for the endoproteolysis of prohormones. Antral G-cells synthesize the most gastrin and express PC1/3, 2 and 5/6 in the rat and human. But the cleavage sites in progastrin for each PC have not been determined. Therefore, in the present study, we measured the concentrations of progastrin, processing intermediates and α-amidated gastrins in antral extracts from PC1/3-null mice and compared the results with those in mice lacking PC2 and wild-type controls. The expression of PCs was examined by immunocytochemistry and in situ hybridization of mouse G-cells. Finally, the in vitro effect of recombinant PC5/6 on progastrin and progastrin fragments containing the relevant dibasic cleavage sites was also examined. The results showed that mouse G-cells express PC1/3, 2 and 5/6. The concentration of progastrin in PC1/3-null mice was elevated 3-fold. Chromatography showed that cleavage of the Arg36Arg37 and Arg73Arg74 sites were grossly decreased. Accordingly, the concentrations of progastrin products were markedly reduced, α-amidated gastrins (-34 and -17) being 25% of normal. Lack of PC1/3 was without effect on the third dibasic site (Lys53Lys54), which is the only processing site for PC2. Recombinant PC5/6 did not cleave any of the dibasic processing sites in progastrin and fragments containing the relevant dibasic processing sites. The complementary cleavages of PC1/3 and 2, however, suffice to explain most of the normal endoproteolysis of progastrin. Moreover, the results show that PCs react differently to the same dibasic sequences, suggesting that additional structural factors modulate the substrate specificity.

scholarly journals CRISPR-mediated isogenic cell-SELEX approach for generating highly specific aptamers against native membrane proteins

2020 ◽  
Author(s):  
Jonah C. Rosch ◽  
Emma H. Neal ◽  
Daniel A. Balikov ◽  
Mohsin Rahim ◽  
Ethan S. Lippmann
Keyword(s):  
Membrane Proteins ◽  
Glucose Transporter ◽  
High Specificity ◽  
Epithelial Cell Line ◽  
Target Cells ◽  
Wild Type ◽  
Simultaneous Exposure ◽  
Affinity Reagents ◽  
A Cell

AbstractIntroductionThe generation of affinity reagents that bind native membrane proteins with high specificity remains challenging. Most in vitro selection paradigms utilize different cell types for positive and negative rounds of selection (where the positive selection is against a cell that expresses the desired membrane protein and the negative selection is against a cell that lacks the protein). However, this strategy can yield affinity reagents that bind unintended membrane proteins on the target cells. To address this issue, we developed a systematic evolution of ligands by exponential enrichment (SELEX) scheme that utilizes isogenic pairs of cells generated via CRISPR techniques.MethodsUsing a Caco-2 epithelial cell line with constitutive Cas9 expression, we knocked out the SLC2A1 gene (encoding the GLUT1 glucose transporter) via lipofection with synthetic gRNAs. Cell-SELEX rounds were carried out against wild-type and GLUT1-null cells using a single-strand DNA (ssDNA) library. Next-generation sequencing (NGS) was used to quantify enrichment of prospective binders to the wild-type cells.Results10 rounds of cell-SELEX were conducted via simultaneous exposure of ssDNA pools to wild-type and GLUT1-null Caco-2 cells under continuous perfusion. The top binders identified from NGS were validated by flow cytometry and immunostaining for their specificity to the GLUT1 receptor.ConclusionsOur data indicate that highly specific aptamers can be isolated with a SELEX strategy that utilizes isogenic cell lines. This approach should be broadly useful for generating affinity reagents that selectively bind to membrane proteins in their native conformations on the cell surface.

scholarly journals An estimate of heterosis in Drosophila melanogaster

1971 ◽  
Vol 18 (1) ◽  
pp. 97-105 ◽  
Author(s):  
J. A. Sved
Keyword(s):  
Drosophila Melanogaster ◽  
High Frequency ◽  
Marker Chromosome ◽  
Selective Advantage ◽  
Wild Type ◽  
Set Up

SUMMARYTwenty-five population cages of D. melanogaster were set up, each containing a different wild-type second chromosome and the marker chromosome Cy. In all but one case where contamination apparently occurred, the Cy chromosome persisted in the population at high frequency, showing a selective advantage of Cy/ + heterozygotes over wild-type homozygotes. Overall, the results indicate that homozygosity of the entire second chromosome causes a depression in fitness of the order of 85%.

scholarly journals Identification of the cleavage sites in the α6A integrin subunit: structural requirements for cleavage and functional analysis of the uncleaved α6Aβ1 integrin

1997 ◽  
Vol 324 (1) ◽  
pp. 263-272 ◽  
Author(s):  
Gepke O. DELWEL ◽  
Ingrid KUIKMAN ◽  
Roel C. van der SCHORS ◽  
Annemieke A. de MELKER ◽  
Arnoud SONNENBERG
Keyword(s):  
Cleavage Site ◽  
K562 Cells ◽  
Proteolytic Cleavage ◽  
Human Platelets ◽  
Light Chains ◽  
Integrin Subunit ◽  
Cleavage Sites ◽  
Wild Type ◽  
Proper Position ◽  
Arginine Residues

The α6A and α6B integrin subunits are proteolytically cleaved during biosynthesis into a heavy chain (120 kDa) that is disulphide-linked to one of two light chains (31 or 30 kDa). Analysis of the structure of the α6A subunit on the carcinoma cell line T24 and human platelets demonstrated that the two light chains of α6 are not differentially glycosylated products of one polypeptide. Rather they possess different polypeptide backbones, which presumably result from proteolytic cleavage at distinct sites in the α6 precursor. Mutations were introduced in the codons for the R876KKR879, E883K884, R890K891 and R898K899 sequences, the potential proteolytic cleavage sites, and wild-type and mutant α6A cDNAs were transfected into K562 cells. The mutant α6A integrin subunits were expressed in association with endogenous β1 at levels comparable to that of wild-type α6Aβ1. A single α6 polypeptide chain (150 kDa) was precipitated from transfectants expressing α6A with mutations or deletions in the RKKR sequence. Mutations in the EK sequence yielded α6A subunits that were cleaved once into a heavy and a light chain, whereas α6A subunits with mutations in one of the two RK sequences were, like wild-type α6A, cleaved into one heavy and two light chains. Thus a change in the RKKR sequence prevents the cleavage of α6. The EK site is the secondary cleavage site, which is used only when the primary site (RKKR) is intact. Microsequencing of the N-termini of the two α6A light chains from platelets demonstrated that cleavage occurs after Arg879 and Lys884. Because α6RKKG, α6GKKR and α6RGGR subunits were not cleaved it seems that both the arginine residues and the lysine residues are essential for cleavage of RKKR. α6A mutants with the RKKR sequence shifted to the EK site, in such a way that the position of the arginine residue after which cleavage occurs corresponds exactly to Lys884, were partly cleaved, whereas α6A mutants with the RKKR sequence shifted to other positions in the α6A subunit, including one in which it was shifted two residues farther than the EK cleavage site, were not cleaved. In addition, α6A mutants with an α5-like cleavage site, i.e. arginine, lysine and histidine residues at positions -1, -2 and -6, were not cleaved. Thus both an intact RKKR sequence and its proper position are essential. After activation by the anti-β1 stimulatory monoclonal antibody TS2/16, both cleaved and uncleaved α6Aβ1 integrins bound to laminin-1. The phorbol ester PMA, which activates cleaved wild-type and mutant α6Aβ1, did not activate uncleaved α6Aβ1. Thus uncleaved α6Aβ1 is capable of ligand binding, but not of inside-out signalling. Our results suggest that cleavage of α6 is required to generate a proper conformation that enables the affinity modulation of the α6Aβ1 receptor by PMA.

Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae

1982 ◽  
Vol 2 (4) ◽  
pp. 437-442
Author(s):  
G R Taylor ◽  
B J Barclay ◽  
R K Storms ◽  
J D Friesen ◽  
R H Haynes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Frequency ◽  
Wild Type Strain ◽  
Biologically Active ◽  
Thymidylate Synthetase ◽  
Temperature Sensitive ◽  
Synthetase Activity ◽  
Enzymatic Conversion ◽  
Wild Type ◽  
Sensitive Mutant

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.

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