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 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.

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 RNA-CLAMP Enables Photo-activated Control of CRISPR-Cas9 Gene Editing by Site-specific Intramolecular Cross-linking of the sgRNA

2021 ◽  
Author(s):  
Dongyang Zhang ◽  
Shuaijiang Jin ◽  
Luping Liu ◽  
Ember Tota ◽  
Zijie Li ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Mammalian Cells ◽  
Gene Editing ◽  
Cross Linking ◽  
Stimuli Responsive ◽  
Spatiotemporal Resolution ◽  
Site Specific ◽  
Guide Rna ◽  
Dna Cleavage Activity ◽  
Versatile Tool

AbstractHere we introduce RNA-CLAMP, a technology which enables site-specific and enzymatic cross-linking (clamping) of two selected stem loops within an RNA of interest. Intramolecular clamping of the RNA can disrupt normal RNA function, whereas subsequent photo-cleavage of the crosslinker restores activity. We applied the RNA-CLAMP technique to the single guide RNA of the CRISPR-Cas9 gene editing system. By clamping two stem loops of the single-guide RNA (sgRNA) with a photo-cleavable cross-linker, gene editing was completely silenced. Visible light irradiation cleaved the crosslinker and restored gene editing with high spatiotemporal resolution. Furthermore, by designing two photo-cleavable linkers which are responsive to different wavelength of lights, we achieved multiplexed photo-activation of gene editing in mammalian cells. Notably, although the Cas9-sgRNA RNP is not capable of DNA cleavage activity upon clamping, it maintained the capability to bind to the target DNA. The RNA-CLAMP enabled photo-activated CRISPR-Cas9 gene editing platform offers clean background, free choice of activation wavelength and multiplexing capability. We believe that this technology to precisely and rapidly control gene editing will serve as a versatile tool in the future development of stimuli responsive gene editing technologies. Beyond gene editing, RNA-CLAMP provides a site-specific tool for manipulating the internal structure of functional RNAs.

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 Programmable CRISPR-responsive smart materials

Science ◽  
2019 ◽  
Vol 365 (6455) ◽  
pp. 780-785 ◽  
Author(s):  
Max A. English ◽  
Luis R. Soenksen ◽  
Raphael V. Gayet ◽  
Helena de Puig ◽  
Nicolaas M. Angenent-Mari ◽  
...  
Keyword(s):  
Smart Materials ◽  
Biological Information ◽  
Live Cells ◽  
Stimuli Responsive ◽  
Structural Element ◽  
Responsive Materials ◽  
Guide Rna ◽  
Poly Ethylene ◽  
Dna Hydrogels

Stimuli-responsive materials activated by biological signals play an increasingly important role in biotechnology applications. We exploit the programmability of CRISPR-associated nucleases to actuate hydrogels containing DNA as a structural element or as an anchor for pendant groups. After activation by guide RNA–defined inputs, Cas12a cleaves DNA in the gels, thereby converting biological information into changes in material properties. We report four applications: (i) branched poly(ethylene glycol) hydrogels releasing DNA-anchored compounds, (ii) degradable polyacrylamide-DNA hydrogels encapsulating nanoparticles and live cells, (iii) conductive carbon-black–DNA hydrogels acting as degradable electrical fuses, and (iv) a polyacrylamide-DNA hydrogel operating as a fluidic valve with an electrical readout for remote signaling. These materials allow for a range of in vitro applications in tissue engineering, bioelectronics, and diagnostics.

scholarly journals Using CRISPR-Cas9 for Therapeutic Protein Production (Review Article)

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Ashraf Ullah Khan ◽  
Zabi Ullah
Keyword(s):  
Nucleic Acids ◽  
Protein Production ◽  
Gene Editing ◽  
Ex Vivo ◽  
Somatic Cells ◽  
Review Article ◽  
Crispr Locus ◽  
Target Effects ◽  
Subsequent Integration

Existence of CRISPR/Cas9 systems in bacteria and archaea has been noted to be the reason for these organisms’ ability to disarm invading nucleic acids. Such immunity is noted to arise from the targeting of the invading nucleic acids by guiding RNAs (sgRNAs), their cleavage by Cas9 (an endonuclease), and their subsequent integration into CRISPR locus. Recent studies have shown that the CRISPR/Cas9 tool can be adopted for gene editing in eukaryotic cells and thus offering potential for its use to treat genetic conditions. In this review, CRISPR/Cas9 has been shown to be an effective genome-editing tool with studies showing efficacy in zygote editing, in-vivo editing of somatic cells and ex-vivo editing of somatic cells. Occurrence of off-target effects however make zygote editing in human cells ethically questionable due to possibility of introducing unwanted mutations that may be passed on to the progeny. Nevertheless, observations that such off-target effects arise mainly from the promiscuity of sgRNAs rather that errors in CRISPR/Cas9 system show promise for increased specificity by developing better sgRNAs.  Such increased specificity will facilitate the adoption of CRISPR/Cas9 for clinical use in treatment of conditions such as β-thalassemia, cystic fibrosis, Duchenne muscular dystrophy and HIV.

scholarly journals Peptide Nucleic Acids and Gene Editing: Perspectives on Structure and Repair

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 735 ◽  
Author(s):  
Nicholas G. Economos ◽  
Stanley Oyaghire ◽  
Elias Quijano ◽  
Adele S. Ricciardi ◽  
W. Mark Saltzman ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Gene Editing ◽  
Molecular Design ◽  
Peptide Nucleic Acids ◽  
Therapeutic Application ◽  
Monogenic Disorders ◽  
Structural Biochemistry ◽  
Nucleic Acid Structures ◽  
Target Effects

Unusual nucleic acid structures are salient triggers of endogenous repair and can occur in sequence-specific contexts. Peptide nucleic acids (PNAs) rely on these principles to achieve non-enzymatic gene editing. By forming high-affinity heterotriplex structures within the genome, PNAs have been used to correct multiple human disease-relevant mutations with low off-target effects. Advances in molecular design, chemical modification, and delivery have enabled systemic in vivo application of PNAs resulting in detectable editing in preclinical mouse models. In a model of β-thalassemia, treated animals demonstrated clinically relevant protein restoration and disease phenotype amelioration, suggesting a potential for curative therapeutic application of PNAs to monogenic disorders. This review discusses the rationale and advances of PNA technologies and their application to gene editing with an emphasis on structural biochemistry and repair.

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.

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