A Review: Computational approaches to design sgRNA of CRISPR-Cas9

2021 ◽  
Vol 16 ◽  
Author(s):  
Mohsin Ali Nasir ◽  
Samia Nawaz ◽  
Jia Huang
Keyword(s):  
In Silico ◽  
Gene Editing ◽  
Crucial Point ◽  
Computational Tools ◽  
Computational Approaches ◽  
Guide Rna ◽  
Fast Progression ◽  
Pros And Cons ◽  
Points Of View ◽  
Target Effects

: Clustered regularly interspaced short palindromic repeats along with CRISPR-associated protein mechanisms preserve the memory of previous experiences with DNA invaders, in particular spacers that are embedded in CRISPR arrays between coordinate repeats. There has been a fast progression in the comprehension of this immune system and its implementations; however, there are numerous points of view that anticipate explanations to make the field an energetic research zone. The efficiency of CRISPR-Cas depends on well considered single guide RNA. For this purpose, many bioinformatics methods and tools were created to support the design of greatly active and precise single guide RNA. In-silico single guide RNA architecture is a crucial point for effective gene editing by means of the CRISPR technique. Persistent attempts are prepared to improve in-silico single guide RNA formulation by great on-target effectiveness and decreased off-target effects. This review offers a summary of the CRISPR computational tools to help different researchers to pick a specific tool for their work according to their pros and cons, along with new thoughts to make new computational tools to overcome all existing limitations.

scholarly journals Chemical Synthesis of Stimuli-Responsive Guide RNA for Conditional Control of CRISPR-Cas9 Gene Editing

2021 ◽  
Author(s):  
Chunmei Gu ◽  
Lu Xiao ◽  
Jiachen Shang ◽  
Xiao Xu ◽  
Luo He ◽  
...  
Keyword(s):  
Chemical Synthesis ◽  
Nucleic Acids ◽  
Gene Editing ◽  
Live Cells ◽  
Stimuli Responsive ◽  
Guide Rna ◽  
Conditional Control ◽  
Target Effects

CRISPR-Cas9 promotes changes in identity or abundance of nucleic acids in live cells and is a programmable modality of broad biotechnological and therapeutic interest. To reduce off-target effects, tools for...

scholarly journals Differential efficacies of Cas nucleases on microsatellites involved in human disorders and associated off-target mutations

2019 ◽  
Author(s):  
Lucie Poggi ◽  
Lisa Emmenegger ◽  
Stéphane Descorps-Declère ◽  
Bruno Dumas ◽  
Guy-Franck Richard
Keyword(s):  
Gene Editing ◽  
Low Frequency ◽  
Strand Break ◽  
Double Strand Break ◽  
Yeast Genome ◽  
Guide Rna ◽  
Human Disorders ◽  
Dna End Resection ◽  
End Resection ◽  
Target Effects

AbstractMicrosatellite expansions are the cause of more than 20 neurological or developmental human disorders. Shortening expanded repeats using specific DNA endonucleases may be envisioned as a gene editing approach. Here, a new assay was developed to test several CRISPR-Cas nucleases on microsatellites involved in human diseases, by measuring at the same time double-strand break rates, DNA end resection and homologous recombination efficacy. Broad variations in nuclease performances were detected on all repeat tracts. Streptococcus pyogenes Cas9 was the most efficient of all. All repeat tracts did inhibit double-strand break resection. We demonstrate that secondary structure formation on the guide RNA was a major determinant of nuclease efficacy. Using deep sequencing, off-target mutations were assessed genomewide. Out of 221 CAG/CTG or GAA/TTC trinucleotide repeats of the yeast genome, three were identified as carrying statistically significant low frequency mutations, corresponding to off-target effects.

scholarly journals In silico experiments validate that twenty nucleotide spacer sequence provides precise targeting of bacterial genes in CRISPR-Cas9

2019 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Genome Editing ◽  
In Silico ◽  
Bacterial Genome ◽  
Specific Gene ◽  
Guide Rna ◽  
Theoretical Predictions ◽  
Modulation Of Gene Expression ◽  
Target Effects

As a genome editing tool useful for modulating the expression of different genes, CRISPR-Cas9 is known for its precision in targeting specific genes. To do this, CRISPR-Cas9 utilizes a guide RNA for guiding the Cas9 endonuclease to specific stretches of the DNA for genome editing or modulation of gene expression. Guide RNA comprises a spacer sequence and a protospacer adjacent motif (PAM) sequence. Both components work together to help target Cas9 to a specific stretch of DNA within a gene. In particular, spacer sequence provides a unique address for localizing Cas9 to specific stretch of DNA. But, possibility exists that there could be off-target effects for particular spacer sequence used in guide RNA. Specifically, spacer sequence might engage in complementary base pairing with other stretches of DNA in the bacterial genome, and result in additional genome editing or modulation of gene expression at genes that are not targeted. Results from an in silico experiment conducted with the rpoH gene of Pseudomonas aeruginosa PAO1 revealed that all spacer sequences derived from different stretches of the rpoH gene did not elicit off-target effects in the genome of the bacterium. This concurs with theoretical predictions that the probability of off-target effects from a 20 nucleotide long spacer region is vanishingly small. Hence, a 20 nucleotide spacer sequence in guide RNA should provide a unique DNA address for precise targeting of specific gene in the genome of a bacterium. Collectively, off-target effects of CRISPR-Cas9 is a valid concern for both genetic engineering and genome editing applications as targeting of additional genes from the desired one would result in unpredictable physiological and biochemical impacts on the cell. Using the rpoH gene of Pseudomonas aeruginosa PAO1 as example, results from an in silico experiment examining possible off-target effects of different 20 nucleotide spacer sequence able to target the sense and antisense strand of the gene revealed no off-target effects. Specifically, each spacer sequence used could only target the intended rpoH gene, which concurs with theoretical predictions of vanishingly small possibility of off-target effects on a bacterial genome from a 20 nucleotide spacer sequence. Overall, the results highlight that use of a 20 nucleotide spacer sequence in guide RNA could offer precise targeting of specific gene in a bacterium.

scholarly journals In silico experiments validate that twenty nucleotide spacer sequence provides precise targeting of bacterial genes in CRISPR-Cas9

2019 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Genome Editing ◽  
In Silico ◽  
Bacterial Genome ◽  
Specific Gene ◽  
Guide Rna ◽  
Theoretical Predictions ◽  
Modulation Of Gene Expression ◽  
Target Effects

As a genome editing tool useful for modulating the expression of different genes, CRISPR-Cas9 is known for its precision in targeting specific genes. To do this, CRISPR-Cas9 utilizes a guide RNA for guiding the Cas9 endonuclease to specific stretches of the DNA for genome editing or modulation of gene expression. Guide RNA comprises a spacer sequence and a protospacer adjacent motif (PAM) sequence. Both components work together to help target Cas9 to a specific stretch of DNA within a gene. In particular, spacer sequence provides a unique address for localizing Cas9 to specific stretch of DNA. But, possibility exists that there could be off-target effects for particular spacer sequence used in guide RNA. Specifically, spacer sequence might engage in complementary base pairing with other stretches of DNA in the bacterial genome, and result in additional genome editing or modulation of gene expression at genes that are not targeted. Results from an in silico experiment conducted with the rpoH gene of Pseudomonas aeruginosa PAO1 revealed that all spacer sequences derived from different stretches of the rpoH gene did not elicit off-target effects in the genome of the bacterium. This concurs with theoretical predictions that the probability of off-target effects from a 20 nucleotide long spacer region is vanishingly small. Hence, a 20 nucleotide spacer sequence in guide RNA should provide a unique DNA address for precise targeting of specific gene in the genome of a bacterium. Collectively, off-target effects of CRISPR-Cas9 is a valid concern for both genetic engineering and genome editing applications as targeting of additional genes from the desired one would result in unpredictable physiological and biochemical impacts on the cell. Using the rpoH gene of Pseudomonas aeruginosa PAO1 as example, results from an in silico experiment examining possible off-target effects of different 20 nucleotide spacer sequence able to target the sense and antisense strand of the gene revealed no off-target effects. Specifically, each spacer sequence used could only target the intended rpoH gene, which concurs with theoretical predictions of vanishingly small possibility of off-target effects on a bacterial genome from a 20 nucleotide spacer sequence. Overall, the results highlight that use of a 20 nucleotide spacer sequence in guide RNA could offer precise targeting of specific gene in a bacterium.

scholarly journals Conditional control of RNA-guided nucleic acid cleavage and gene editing

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Shao-Ru Wang ◽  
Ling-Yu Wu ◽  
Hai-Yan Huang ◽  
Wei Xiong ◽  
Jian Liu ◽  
...  
Keyword(s):  
Rna Structure ◽  
Gene Editing ◽  
Chemical Activation ◽  
Live Cells ◽  
Guide Rna ◽  
Dna And Rna ◽  
Conditional Control ◽  
Acid Cleavage ◽  
Versatile Tool ◽  
Target Effects

AbstractProkaryotes use repetitive genomic elements termed CRISPR (clustered regularly interspaced short palindromic repeats) to destroy invading genetic molecules. Although CRISPR systems have been widely used in DNA and RNA technology, certain adverse effects do occur. For example, constitutively active CRISPR systems may lead to a certain risk of off-target effects. Here, we introduce post-synthetic masking and chemical activation of guide RNA (gRNA) to controlling CRISPR systems. An RNA structure profiling probe (2-azidomethylnicotinic acid imidazolide) is used. Moreover, we accomplish conditional control of gene editing in live cells. This proof-of-concept study demonstrates promising potential of chemical activation of gRNAs as a versatile tool for chemical biology.

scholarly journals Application of CRISPR/Cas9 technology in wild apple (Malus sieverii) for paired sites gene editing

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yan Zhang ◽  
Ping Zhou ◽  
Tohir A. Bozorov ◽  
Daoyuan Zhang
Keyword(s):  
Abiotic Stress ◽  
Human Activities ◽  
Genetic Improvement ◽  
Gene Editing ◽  
New Technologies ◽  
Tianshan Mountains ◽  
Biotic And Abiotic Stress ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Albino Phenotype

Abstract Background Xinjiang wild apple is an important tree of the Tianshan Mountains, and in recent years, it has undergone destruction by many biotic and abiotic stress and human activities. It is necessary to use new technologies to research its genomic function and molecular improvement. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability varies depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Results In this study, we used 2 systems of vectors with paired sgRNAs targeting to MsPDS. As expected, we successfully induced the albino phenotype of calli and buds in both systems. Conclusions We conclude that CRISPR/Cas9 is a powerful system for editing the wild apple genome and expands the range of plants available for gene editing.

scholarly journals Non-viral delivery of CRISPR–Cas9 complexes for targeted gene editing via a polymer delivery system

Gene Therapy ◽  
2021 ◽  
Author(s):  
Jonathan O’Keeffe Ahern ◽  
Irene Lara-Sáez ◽  
Dezhong Zhou ◽  
Rodolfo Murillas ◽  
Jose Bonafont ◽  
...  
Keyword(s):  
Delivery System ◽  
Gene Editing ◽  
Transfection Efficiency ◽  
Attractive Alternative ◽  
Safe Delivery ◽  
Guide Rna ◽  
Recessive Dystrophic Epidermolysis Bullosa ◽  
Targeted Gene Editing ◽  
Genomic Editing ◽  
Polymeric Vectors

AbstractRecent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(β-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15–20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR–Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.

scholarly journals Spatiotemporal control of CRISPR/Cas9 gene editing

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Chenya Zhuo ◽  
Jiabin Zhang ◽  
Jung-Hwan Lee ◽  
Ju Jiao ◽  
Du Cheng ◽  
...  
Keyword(s):  
Genetic Engineering ◽  
Gene Editing ◽  
Genetic Regulation ◽  
Clinical Translation ◽  
Small Molecule Activation ◽  
Simple Design ◽  
Advantages And Disadvantages ◽  
Spatiotemporal Control ◽  
Critical Challenge ◽  
Target Effects

AbstractThe clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) gene editing technology, as a revolutionary breakthrough in genetic engineering, offers a promising platform to improve the treatment of various genetic and infectious diseases because of its simple design and powerful ability to edit different loci simultaneously. However, failure to conduct precise gene editing in specific tissues or cells within a certain time may result in undesirable consequences, such as serious off-target effects, representing a critical challenge for the clinical translation of the technology. Recently, some emerging strategies using genetic regulation, chemical and physical strategies to regulate the activity of CRISPR/Cas9 have shown promising results in the improvement of spatiotemporal controllability. Herein, in this review, we first summarize the latest progress of these advanced strategies involving cell-specific promoters, small-molecule activation and inhibition, bioresponsive delivery carriers, and optical/thermal/ultrasonic/magnetic activation. Next, we highlight the advantages and disadvantages of various strategies and discuss their obstacles and limitations in clinical translation. Finally, we propose viewpoints on directions that can be explored to further improve the spatiotemporal operability of CRISPR/Cas9.

Elucidating Protein-protein Interactions Through Computational Approaches and Designing Small Molecule Inhibitors Against them for Various Diseases

2018 ◽  
Vol 18 (20) ◽  
pp. 1719-1736 ◽  
Author(s):  
Sharanya Sarkar ◽  
Khushboo Gulati ◽  
Manikyaprabhu Kairamkonda ◽  
Amit Mishra ◽  
Krishna Mohan Poluri
Keyword(s):  
Small Molecule ◽  
Protein Interactions ◽  
Small Molecule Inhibitors ◽  
Computational Techniques ◽  
Protein Protein Interactions ◽  
Computational Tools ◽  
Computational Approaches ◽  
Protein Protein Interaction ◽  
Wide Range ◽  
Therapeutic Measures

Background: To carry out wide range of cellular functionalities, proteins often associate with one or more proteins in a phenomenon known as Protein-Protein Interaction (PPI). Experimental and computational approaches were applied on PPIs in order to determine the interacting partners, and also to understand how an abnormality in such interactions can become the principle cause of a disease. Objective: This review aims to elucidate the case studies where PPIs involved in various human diseases have been proven or validated with computational techniques, and also to elucidate how small molecule inhibitors of PPIs have been designed computationally to act as effective therapeutic measures against certain diseases. Results: Computational techniques to predict PPIs are emerging rapidly in the modern day. They not only help in predicting new PPIs, but also generate outputs that substantiate the experimentally determined results. Moreover, computation has aided in the designing of novel inhibitor molecules disrupting the PPIs. Some of them are already being tested in the clinical trials. Conclusion: This review delineated the classification of computational tools that are essential to investigate PPIs. Furthermore, the review shed light on how indispensable computational tools have become in the field of medicine to analyze the interaction networks and to design novel inhibitors efficiently against dreadful diseases in a shorter time span.

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