Biological Plasticity Rescues Target Activity in CRISPR Knockouts

AbstractGene knockouts (KOs) are efficiently engineered through CRISPR-Cas9-induced frameshift mutations. While DNA editing efficiency is readily verified by DNA sequencing, a systematic understanding of the efficiency of protein elimination has been lacking. Here, we devised an experimental strategy combining RNA-seq and triple-stage mass spectrometry (SPS-MS3) to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins, or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR-Cas9 generated KO lines is necessary for phenotype interpretation.

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Characterization of Protein Function and Differential Protein Expression in Soybean under Soaking Condition

Korean Journal of Crop Science ◽

10.7740/kjcs.2014.60.1.114 ◽

2015 ◽

Vol 60 (1) ◽

pp. 114-122

Author(s):

Seong-Woo Cho ◽

◽

Tae-Sun Kim ◽

Soo-Jeong Kwon ◽

Swapan Kumar Roy ◽

...

Keyword(s):

Protein Expression ◽

Protein Function ◽

Differential Protein Expression ◽

Differential Protein

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Preparation and Characterization of β-glucosidase Films for Stabilization and Handling in Dry Configurations

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666191202145351 ◽

2020 ◽

Vol 21 (8) ◽

pp. 741-747

Author(s):

Liguang Zhang ◽

Yanan Shen ◽

Wenjing Lu ◽

Lengqiu Guo ◽

Min Xiang ◽

...

Keyword(s):

Tensile Strength ◽

Protein Function ◽

Protein Stabilization ◽

Functional Protein ◽

Elongation At Break ◽

Gelatin Films ◽

The Stability ◽

And Function ◽

General Method

Background: Although the stability of proteins is of significance to maintain protein function for therapeutical applications, this remains a challenge. Herein, a general method of preserving protein stability and function was developed using gelatin films. Method: Enzymes immobilized onto films composed of gelatin and Ethylene Glycol (EG) were developed to study their ability to stabilize proteins. As a model functional protein, β-glucosidase was selected. The tensile properties, microstructure, and crystallization behavior of the gelatin films were assessed. Result: Our results indicated that film configurations can preserve the activity of β-glucosidase under rigorous conditions (75% relative humidity and 37°C for 47 days). In both control films and films containing 1.8 % β-glucosidase, tensile strength increased with increased EG content, whilst the elongation at break increased initially, then decreased over time. The presence of β-glucosidase had a negligible influence on tensile strength and elongation at break. Scanning electron-microscopy (SEM) revealed that with increasing EG content or decreasing enzyme concentrations, a denser microstructure was observed. Conclusion: In conclusion, the dry film is a promising candidate to maintain protein stabilization and handling. The configuration is convenient and cheap, and thus applicable to protein storage and transportation processes in the future.

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Molecular and Pharmacological Characterization of the Interaction between Human Geranylgeranyltransferase Type I and Ras-Related Protein Rap1B

International Journal of Molecular Sciences ◽

10.3390/ijms22052501 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2501

Author(s):

Sonja Hinz ◽

Dominik Jung ◽

Dorota Hauert ◽

Hagen S. Bachmann

Keyword(s):

Protein Function ◽

Tumor Development ◽

Cysteine Residue ◽

Type I ◽

Pharmacological Characterization ◽

Anti Cancer ◽

And Function ◽

Caax Motif ◽

Important Drug

Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor development and metastasis. This led to the development of GGTase-I inhibitors as anti-cancer drugs blocking the protein function and membrane association of e.g., Rap subfamilies that are involved in cell differentiation and cell growth. In the present study, we developed a new NanoBiT assay to monitor the interaction of human GGTase-I and its substrate Rap1B. Different Rap1B prenylation-deficient mutants (C181G, C181S, and ΔCQLL) were designed and investigated for their interaction with GGTase-I. While the Rap1B mutants C181G and C181S still exhibited interaction with human GGTase-I, mutant ΔCQLL, lacking the entire CAAX motif (defined by a cysteine residue, two aliphatic residues, and the C-terminal residue), showed reduced interaction. Moreover, a specific, peptidomimetic and competitive CAAX inhibitor was able to block the interaction of Rap1B with GGTase-I. Furthermore, activation of both Gαs-coupled human adenosine receptors, A2A (A2AAR) and A2B (A2BAR), increased the interaction between GGTase-I and Rap1B, probably representing a way to modulate prenylation and function of Rap1B. Thus, A2AAR and A2BAR antagonists might be promising candidates for therapeutic intervention for different types of cancer that overexpress Rap1B. Finally, the NanoBiT assay provides a tool to investigate the pharmacology of GGTase-I inhibitors.

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Characterization of synthetic riboswitch in cell-free protein expression systems

RNA Biology ◽

10.1080/15476286.2020.1868149 ◽

2021 ◽

pp. 1-12

Author(s):

Yaroslav Chushak ◽

Svetlana Harbaugh ◽

Kathryn Zimlich ◽

Bryan Alfred ◽

Jorge Chávez ◽

...

Keyword(s):

Protein Expression ◽

Expression Systems ◽

Free Protein

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Avoided motifs: short amino acid strings missing from protein datasets

Biological Chemistry ◽

10.1515/hsz-2020-0383 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Pablo Mier ◽

Miguel A. Andrade-Navarro

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Protein Function ◽

Large Protein ◽

New Approach ◽

Cellular Context ◽

Human Proteins ◽

Context Specific ◽

Protein Datasets

Abstract According to the amino acid composition of natural proteins, it could be expected that all possible sequences of three or four amino acids will occur at least once in large protein datasets purely by chance. However, in some species or cellular context, specific short amino acid motifs are missing due to unknown reasons. We describe these as Avoided Motifs, short amino acid combinations missing from biological sequences. Here we identify 209 human and 154 bacterial Avoided Motifs of length four amino acids, and discuss their possible functionality according to their presence in other species. Furthermore, we determine two Avoided Motifs of length three amino acids in human proteins specifically located in the cytoplasm, and two more in secreted proteins. Our results support the hypothesis that the characterization of Avoided Motifs in particular contexts can provide us with information about functional motifs, pointing to a new approach in the use of molecular sequences for the discovery of protein function.

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Characterization of Ocular Surface Microbial Profiles Revealed Discrepancies between Conjunctival and Corneal Microbiota

Pathogens ◽

10.3390/pathogens10040405 ◽

2021 ◽

Vol 10 (4) ◽

pp. 405

Author(s):

Anna Matysiak ◽

Michal Kabza ◽

Justyna A. Karolak ◽

Marcelina M. Jaworska ◽

Malgorzata Rydzanicz ◽

...

Keyword(s):

Microbial Communities ◽

Ocular Surface ◽

Molecular Techniques ◽

Corneal Tissue ◽

Rna Seq ◽

Microbiome Composition ◽

Viral Sequences ◽

Pcr Techniques ◽

Unmapped Reads

The ocular microbiome composition has only been partially characterized. Here, we used RNA-sequencing (RNA-Seq) data to assess microbial diversity in human corneal tissue. Additionally, conjunctival swab samples were examined to characterize ocular surface microbiota. Short RNA-Seq reads, obtained from a previous transcriptome study of 50 corneal tissues, were mapped to the human reference genome GRCh38 to remove sequences of human origin. The unmapped reads were then used for taxonomic classification by comparing them with known bacterial, archaeal, and viral sequences from public databases. The components of microbial communities were identified and characterized using both conventional microbiology and polymerase chain reaction (PCR) techniques in 36 conjunctival swabs. The majority of ocular samples examined by conventional and molecular techniques showed very similar microbial taxonomic profiles, with most of the microorganisms being classified into Proteobacteria, Firmicutes, and Actinobacteria phyla. Only 50% of conjunctival samples exhibited bacterial growth. The PCR detection provided a broader overview of positive results for conjunctival materials. The RNA-Seq assessment revealed significant variability of the corneal microbial communities, including fastidious bacteria and viruses. The use of the combined techniques allowed for a comprehensive characterization of the eye microbiome’s elements, especially in aspects of microbiota diversity.

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Characterization of full-length transcriptome in Saccharum officinarum and molecular insights into tiller development

BMC Plant Biology ◽

10.1186/s12870-021-02989-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Haifeng Yan ◽

Huiwen Zhou ◽

Hanmin Luo ◽

Yegeng Fan ◽

Zhongfeng Zhou ◽

...

Keyword(s):

Carbon Fixation ◽

Developmental Stages ◽

Genomic Data ◽

Saccharum Officinarum ◽

Full Length ◽

Rna Seq ◽

Pyruvate Phosphate Dikinase ◽

Sugarcane Yield ◽

Tiller Bud

Abstract Background Although extensive breeding efforts are ongoing in sugarcane (Saccharum officinarum L.), the average yield is far below the theoretical potential. Tillering is an important component of sugarcane yield, however, the molecular mechanism underlying tiller development is still elusive. The limited genomic data in sugarcane, particularly due to its complex and large genome, has hindered in-depth molecular studies. Results Herein, we generated full-length (FL) transcriptome from developing leaf and tiller bud samples based on PacBio Iso-Seq. In addition, we performed RNA-seq from tiller bud samples at three developmental stages (T0, T1 and T2) to uncover key genes and biological pathways involved in sugarcane tiller development. In total, 30,360 and 20,088 high-quality non-redundant isoforms were identified in leaf and tiller bud samples, respectively, representing 41,109 unique isoforms in sugarcane. Likewise, we identified 1063 and 1037 alternative splicing events identified in leaf and tiller bud samples, respectively. We predicted the presence of coding sequence for 40,343 isoforms, 98% of which was successfully annotated. Comparison with previous FL transcriptomes in sugarcane revealed 2963 unreported isoforms. In addition, we characterized 14,946 SSRs from 11,700 transcripts and 310 lncRNAs. By integrating RNA-seq with the FL transcriptome, 468 and 57 differentially expressed genes (DEG) were identified in T1vsT0 and T2vsT0, respectively. Strong up-regulation of several pyruvate phosphate dikinase and phosphoenolpyruvate carboxylase genes suggests enhanced carbon fixation and protein synthesis to facilitate tiller growth. Similarly, up-regulation of linoleate 9S-lipoxygenase and lipoxygenase genes in the linoleic acid metabolism pathway suggests high synthesis of key oxylipins involved in tiller growth and development. Conclusions Collectively, we have enriched the genomic data available in sugarcane and provided candidate genes for manipulating tiller formation and development, towards productivity enhancement in sugarcane.

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Genome-Wide Identification and Characterization of Hsf and Hsp Gene Families and Gene Expression Analysis under Heat Stress in Eggplant (Solanum melongema L.)

Horticulturae ◽

10.3390/horticulturae7060149 ◽

2021 ◽

Vol 7 (6) ◽

pp. 149

Author(s):

Chao Gong ◽

Qiangqiang Pang ◽

Zhiliang Li ◽

Zhenxing Li ◽

Riyuan Chen ◽

...

Keyword(s):

Heat Stress ◽

Gene Families ◽

High Temperature Stress ◽

Pcr Analysis ◽

Rna Seq ◽

Genome Wide ◽

Motif Composition ◽

Hsp Genes ◽

Identification And Characterization

Under high temperature stress, a large number of proteins in plant cells will be denatured and inactivated. Meanwhile Hsfs and Hsps will be quickly induced to remove denatured proteins, so as to avoid programmed cell death, thus enhancing the thermotolerance of plants. Here, a comprehensive identification and analysis of the Hsf and Hsp gene families in eggplant under heat stress was performed. A total of 24 Hsf-like genes and 117 Hsp-like genes were identified from the eggplant genome using the interolog from Arabidopsis. The gene structure and motif composition of Hsf and Hsp genes were relatively conserved in each subfamily in eggplant. RNA-seq data and qRT-PCR analysis showed that the expressions of most eggplant Hsf and Hsp genes were increased upon exposure to heat stress, especially in thermotolerant line. The comprehensive analysis indicated that different sets of SmHsps genes were involved downstream of particular SmHsfs genes. These results provided a basis for revealing the roles of SmHsps and SmHsp for thermotolerance in eggplant, which may potentially be useful for understanding the thermotolerance mechanism involving SmHsps and SmHsp in eggplant.

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Identification of Potential Key lncRNAs in the Context of Mouse Myeloid Differentiation by Systematic Transcriptomics Analysis

Genes ◽

10.3390/genes12050630 ◽

2021 ◽

Vol 12 (5) ◽

pp. 630

Author(s):

Yongqing Lan ◽

Meng Li ◽

Shuangli Mi

Keyword(s):

Transcription Factor ◽

Cell Model ◽

Myeloid Differentiation ◽

Rna Seq ◽

Hematopoietic Differentiation ◽

Granulocytic Differentiation ◽

Expression Of Genes ◽

Non Coding Rnas

Hematopoietic differentiation is a well-orchestrated process by many regulators such as transcription factor and long non-coding RNAs (lncRNAs). However, due to the large number of lncRNAs and the difficulty in determining their roles, the study of lncRNAs is a considerable challenge in hematopoietic differentiation. Here, through gene co-expression network analysis over RNA-seq data generated from representative types of mouse myeloid cells, we obtained a catalog of potential key lncRNAs in the context of mouse myeloid differentiation. Then, employing a widely used in vitro cell model, we screened a novel lncRNA, named Gdal1 (Granulocytic differentiation associated lncRNA 1), from this list and demonstrated that Gdal1 was required for granulocytic differentiation. Furthermore, knockdown of Cebpe, a principal transcription factor of granulocytic differentiation regulation, led to down-regulation of Gdal1, but not vice versa. In addition, expression of genes involved in myeloid differentiation and its regulation, such as Cebpa, were influenced in Gdal1 knockdown cells with differentiation blockage. We thus systematically identified myeloid differentiation associated lncRNAs and substantiated the identification by investigation of one of these lncRNAs on cellular phenotype and gene regulation levels. This study promotes our understanding of the regulation of myeloid differentiation and the characterization of roles of lncRNAs in hematopoietic system.

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Characterization of alternative splicing events and prognostic signatures in breast cancer

BMC Cancer ◽

10.1186/s12885-021-08305-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Pihua Han ◽

Jingjun Zhu ◽

Guang Feng ◽

Zizhang Wang ◽

Yanni Ding

Keyword(s):

Breast Cancer ◽

Alternative Splicing ◽

Proportional Hazards ◽

Pearson Correlation ◽

Original Data ◽

Cox Proportional Hazards ◽

Prognostic Indicators ◽

Splicing Factors ◽

Rna Seq

Abstract Background Breast cancer (BRCA) is one of the most common cancers worldwide. Abnormal alternative splicing (AS) frequently observed in cancers. This study aims to demonstrate AS events and signatures that might serve as prognostic indicators for BRCA. Methods Original data for all seven types of splice events were obtained from TCGA SpliceSeq database. RNA-seq and clinical data of BRCA cohorts were downloaded from TCGA database. Survival-associated AS events in BRCA were analyzed by univariate COX proportional hazards regression model. Prognostic signatures were constructed for prognosis prediction in patients with BRCA based on survival-associated AS events. Pearson correlation analysis was performed to measure the correlation between the expression of splicing factors (SFs) and the percent spliced in (PSI) values of AS events. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were conducted to demonstrate pathways in which survival-associated AS event is enriched. Results A total of 45,421 AS events in 21,232 genes were identified. Among them, 1121 AS events in 931 genes significantly correlated with survival for BRCA. The established AS prognostic signatures of seven types could accurately predict BRCA prognosis. The comprehensive AS signature could serve as independent prognostic factor for BRCA. A SF-AS regulatory network was therefore established based on the correlation between the expression levels of SFs and PSI values of AS events. Conclusions This study revealed survival-associated AS events and signatures that may help predict the survival outcomes of patients with BRCA. Additionally, the constructed SF-AS networks in BRCA can reveal the underlying regulatory mechanisms in BRCA.

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