scholarly journals Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light-Activated Guide RNA

2019 ◽  
Author(s):  
Wenyuan Zhou ◽  
Wes Brown ◽  
Anirban Bardhan ◽  
Michael Delaney ◽  
Amber S. Ilk ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Zebrafish Embryos ◽  
Complete Suppression ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Conditional Control ◽  
Rapid Restoration ◽  
Spatiotemporal Control ◽  
In Cells

AbstractWe developed a new method for conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos via photochemically activated, caged guide RNAs. Caged gRNAs are generated by substituting four nucleobases evenly distributed throughout the 5’-protospacer region with caged nucleobases during synthesis. Caging confers complete suppression of gRNA:target dsDNA hybridization and rapid restoration of CRISPR/Cas9 function upon optical activation. This tool offers simplicity and complete programmability in design, high spatiotemporal specificity in cells and zebrafish embryos, excellent off to on switching, and stability by preserving the ability to form Cas9:gRNA ribonucleoprotein complexes. caged gRNAs are novel tools for conditional control of gene editing thereby enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation.

ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

2020 ◽  
Author(s):  
Y.V. Mikhaylova ◽  
◽  
M.A. Tyumentseva ◽  
A.A. Shelenkov ◽  
Y.G. Yanushevich ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Correct Choice ◽  
Target Sequence ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Chemokine Receptor Ccr5 ◽  
Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

scholarly journals Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light‐Activated Guide RNA

2020 ◽  
Vol 132 (23) ◽  
pp. 9083-9088 ◽  
Author(s):  
Wenyuan Zhou ◽  
Wes Brown ◽  
Anirban Bardhan ◽  
Michael Delaney ◽  
Amber S. Ilk ◽  
...  
Keyword(s):  
Guide Rna ◽  
Spatiotemporal Control ◽  
In Cells

scholarly journals Eukaryotic Box C/D methylation machinery has two non-symmetric protein assembly sites

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Simone Höfler ◽  
Peer Lukat ◽  
Wulf Blankenfeldt ◽  
Teresa Carlomagno
Keyword(s):  
Three Dimensional ◽  
Protein Assembly ◽  
Motif Pair ◽  
Mutant Analysis ◽  
X Ray ◽  
Guide Rna ◽  
X Ray Crystallography ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes

AbstractBox C/D ribonucleoprotein complexes are RNA-guided methyltransferases that methylate the ribose 2’-OH of RNA. The central ‘guide RNA’ has box C and D motifs at its ends, which are crucial for activity. Archaeal guide RNAs have a second box C’/D’ motif pair that is also essential for function. This second motif is poorly conserved in eukaryotes and its function is uncertain. Conflicting literature data report that eukaryotic box C’/D’ motifs do or do not bind proteins specialized to recognize box C/D-motifs and are or are not important for function. Despite this uncertainty, the architecture of eukaryotic 2’-O-methylation enzymes is thought to be similar to that of their archaeal counterpart. Here, we use biochemistry, X-ray crystallography and mutant analysis to demonstrate the absence of functional box C’/D’ motifs in more than 80% of yeast guide RNAs. We conclude that eukaryotic Box C/D RNPs have two non-symmetric protein assembly sites and that their three-dimensional architecture differs from that of archaeal 2’-O-methylation enzymes.

Assembly of mitochondrial ribonucleoprotein complexes involves specific guide RNA (gRNA)-binding proteins and gRNA domains but does not require preedited mRNA

1994 ◽  
Vol 14 (4) ◽  
pp. 2629-2639
Author(s):  
L K Read ◽  
H U Göringer ◽  
K Stuart
Keyword(s):  
Complex Formation ◽  
Rna Editing ◽  
Binding Proteins ◽  
Macromolecular Complex ◽  
Differential Distribution ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Recognition Elements

RNA editing in kinetoplastids probably employs a macromolecular complex, the editosome, that is likely to include the guide RNAs (gRNAs) which specify the edited sequence. Specific ribonucleoprotein (RNP) complexes which form in vitro with gRNAs (H. U. Göringer, D. J. Koslowsky, T. H. Morales, and K. D. Stuart, Proc. Natl. Acad. Sci. USA, in press) are potential editosomes or their precursors. We find that several factors are important for in vitro formation of these RNP complexes and identify specific gRNA-binding proteins present in the complexes. Preedited mRNA promotes the in vitro formation of the four major gRNA-containing RNP complexes under some conditions but is required for the formation of only a subcomponent of one complex. The 5' gRNA sequence encompassing the RYAYA and anchor regions and the 3' gRNA oligo(U) tail are both important in complex formation, since their deletion results in a dramatic decrease of some complexes and the absence of others. UV cross-linking experiments identify several proteins which are in contact with gRNA and preedited mRNA in mitochondrial extracts. Proteins of 25 and 90 kDa are highly specific for gRNAs, and the 90-kDa protein binds specifically to gRNA oligo(U) tails. The gRNA-binding proteins exhibit a differential distribution between the four in vitro-formed complexes. These experiments reveal several proteins potentially involved in RNA editing and indicate that multiple recognition elements in gRNAs are used for complex formation.

scholarly journals Trypanosoma brucei Guide RNA Poly(U) Tail Formation Is Stabilized by Cognate mRNA

2000 ◽  
Vol 20 (3) ◽  
pp. 883-891 ◽  
Author(s):  
Michael T. McManus ◽  
Brian K. Adler ◽  
Victoria W. Pollard ◽  
Stephen L. Hajduk
Keyword(s):  
Trypanosoma Brucei ◽  
Small Rnas ◽  
Complex I ◽  
Pairing Interaction ◽  
Mrna Editing ◽  
Rich Region ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Base Pairing Interaction

ABSTRACT Guide RNAs (gRNAs) are small RNAs that provide specificity for uridine addition and deletion during mRNA editing in trypanosomes. Terminal uridylyl transferase (TUTase) adds uridines to pre-mRNAs during RNA editing and adds a poly(U) tail to the 3′ end of gRNAs. The poly(U) tail may stabilize the association of gRNAs with cognate mRNA during editing. Both TUTase and gRNAs associate with two ribonucleoprotein complexes, I (19S) and II (35S to 40S). Complex II is believed to be the fully assembled active editing complex, since it contains pre-edited mRNA and enzymes thought necessary for editing. Purification of TUTase from mitochondrial extracts resulted in the identification of two chromatographically distinct TUTase activities. Stable single-uridine addition to different substrate RNAs is performed by the 19S complex, despite the presence of a uridine-specific 3′ exonuclease within this complex. Multiple uridines are added to substrate RNAs by a 10S particle that may be an unstable subunit of complex I lacking the uridine-specific 3′ exonuclease. Multiple uridines could be stably added onto gRNAs by complex I when the cognate mRNA is present. We propose a model in which the purine-rich region of the cognate mRNA protects the uridine tail from a uridine exonuclease activity that is present within the complex. To test this model, we have mutated the purine-rich region of the pre-mRNA to abolish base-pairing interaction with the poly(U) tail of the gRNA. This RNA fails to protect the uridine tail of the gRNA from exoribonucleolytic trimming and is consistent with a role for the purine-rich region of the mRNA in gRNA maturation.

scholarly journals Controlling gene expression in mammalian cells using multiplexed conditional guide RNAs for Cas12a

2021 ◽  
Author(s):  
Lukas Oesinghaus ◽  
Friedrich C. Simmel
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Basic Research ◽  
Dependent Manner ◽  
Multiple Control ◽  
Guide Rnas ◽  
Single Transcript ◽  
Spatiotemporal Control ◽  
Expression In Mammalian Cells ◽  
Activate Gene

AbstractSpatiotemporal control of the activity of Cas proteins is of considerable interest for both basic research and therapeutics. Only few mechanisms have been demonstrated for regulating the activity of guide RNAs (gRNAs) for Cas12a in mammalian cells, however, and combining and compactly integrating multiple control instances on single transcripts has not been possible so far. Here, we show that conditional processing of the 3’ tail is a viable general approach towards switchable Pol II-transcribed Cas12a gRNAs that can activate gene expression in mammalian cells in an input-dependent manner. Processing of the 3’ tail can be achieved using microRNA and short hairpin RNA as inputs, via a guanine-responsive ribozyme, and also using an RNA strand displacement mechanism. We further show that Cas12a along with several independently switchable gRNAs can be integrated on a single transcript using stabilizing RNA triplexes, providing a route towards compact Cas12a-based gene regulation constructs with multi-input switching capabilities.

scholarly journals Orthogonal CRISPR-Cas genome editing and efficient inhibition with anti-CRISPRs in zebrafish embryos

2020 ◽  
Author(s):  
Paige R. Takasugi ◽  
Evan P. Drage ◽  
Sahar N. Kanishka ◽  
Marissa A. Higbee ◽  
James A. Gagnon
Keyword(s):  
Genome Editing ◽  
Streptococcus Pyogenes ◽  
High Efficiency ◽  
Bacterial Species ◽  
Zebrafish Embryos ◽  
Type Ii ◽  
Guide Rnas ◽  
Highly Effective ◽  
Orthogonal Systems ◽  
Spatiotemporal Control

AbstractThe CRISPR-Cas universe continues to expand. The type II CRISPR-Cas system from Streptococcus pyogenes (SpCas9) is most widely used for genome editing due to its high efficiency in cells and organisms. However, concentrating on a single CRISPR-Cas system limits options for multiplexed editing. We hypothesized that CRISPR-Cas systems originating from different bacterial species could operate simultaneously and independently due to their distinct single-guide RNAs (sgRNAs) or CRISPR-RNAs (crRNAs), and protospacer adjacent motifs (PAMs). Additionally, we hypothesized that CRISPR-Cas activity in zebrafish could be regulated through the expression of inhibitory anti-CRISPR (Acr) proteins. Here, we use a simple mutagenesis approach to demonstrate that CRISPR-Cas systems from Streptococcus pyogenes (SpCas9), Streptococcus aureus (SaCas9), and Lachnospiraceae bacterium (LbCas12a, previously known as LbCpf1) are highly effective, orthogonal systems capable of operating simultaneously in zebrafish. We also demonstrate that type II Acrs are effective inhibitors of SpCas9 in zebrafish. These results indicate that at least three orthogonal CRISPR-Cas systems and two anti-CRISPR proteins are functional in zebrafish embryos. These CRISPR-Cas systems and Acr proteins will enable combinatorial and intersectional strategies for spatiotemporal control of genome editing in zebrafish.

scholarly journals Outcomes Comparison of SpCas9- and LbCas12a-mediated DNA Editing in Zebrafish Embryos

2020 ◽  
Author(s):  
Darya Meshalkina ◽  
Aleksei Glushchenko ◽  
Elana Kysil ◽  
Igor Mizgirev ◽  
Andrej Frolov
Keyword(s):  
Genome Editing ◽  
Mode Of Action ◽  
Target Gene ◽  
Gene Knockout ◽  
The Other ◽  
Zebrafish Embryos ◽  
Guide Rnas ◽  
Research Technology ◽  
Ribonucleoprotein Complexes ◽  
Dna Editing

CRISPR/Cas genome editing is a widely used research technology. Its simplest variant is gene knockout resulting from reparation errors after introduction of dsDNA breaks by Cas nuclease. We compared the outcomes of the break repair by two commonly used nucleases (SpCas9 and LbCas12a) in zebrafish embryos to reveal if application of one nuclease is advantageous in comparison to the other. To address this question, we injected ribonucleoprotein complexes of nucleases and corresponding guide RNAs in zebrafish zygotes and three days later sequenced the target gene regions. We found that LbCas12a breaks resulted in longer deletions and more rare inserts, in comparison to those generated by SpCas9, while the editing efficiencies of both nucleases were the same. On the other hand, overlapping protospacers were shown to lead to similarities in repair outcome, although they were cut by two different nucleases. Thus, our results indicate that the repair outcome depends both on the nuclease mode of action and on protospacer sequence.

scholarly journals Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells

2021 ◽  
Vol 2 ◽  
Author(s):  
Daniel Allen ◽  
Michael Rosenberg ◽  
Ayal Hendel
Keyword(s):  
Genome Editing ◽  
Mammalian Cells ◽  
Nuclear Staining ◽  
Two Component System ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Guide Rnas ◽  
Therapeutic Benefits ◽  
A Genome ◽  
Cas9 Protein

CRISPR-Cas9 is quickly revolutionizing the way we approach gene therapy. CRISPR-Cas9 is a complexed, two-component system using a short guide RNA (gRNA) sequence to direct the Cas9 endonuclease to the target site. Modifying the gRNA independent of the Cas9 protein confers ease and flexibility to improve the CRISPR-Cas9 system as a genome-editing tool. gRNAs have been engineered to improve the CRISPR system's overall stability, specificity, safety, and versatility. gRNAs have been modified to increase their stability to guard against nuclease degradation, thereby enhancing their efficiency. Additionally, guide specificity has been improved by limiting off-target editing. Synthetic gRNA has been shown to ameliorate inflammatory signaling caused by the CRISPR system, thereby limiting immunogenicity and toxicity in edited mammalian cells. Furthermore, through conjugation with exogenous donor DNA, engineered gRNAs have been shown to improve homology-directed repair (HDR) efficiency by ensuring donor proximity to the edited site. Lastly, synthetic gRNAs attached to fluorescent labels have been developed to enable highly specific nuclear staining and imaging, enabling mechanistic studies of chromosomal dynamics and genomic mapping. Continued work on chemical modification and optimization of synthetic gRNAs will undoubtedly lead to clinical and therapeutic benefits and, ultimately, routinely performed CRISPR-based therapies.

scholarly journals Assembly of mitochondrial ribonucleoprotein complexes involves specific guide RNA (gRNA)-binding proteins and gRNA domains but does not require preedited mRNA.

1994 ◽  
Vol 14 (4) ◽  
pp. 2629-2639 ◽  
Author(s):  
L K Read ◽  
H U Göringer ◽  
K Stuart
Keyword(s):  
Complex Formation ◽  
Rna Editing ◽  
Binding Proteins ◽  
Macromolecular Complex ◽  
Differential Distribution ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Recognition Elements

RNA editing in kinetoplastids probably employs a macromolecular complex, the editosome, that is likely to include the guide RNAs (gRNAs) which specify the edited sequence. Specific ribonucleoprotein (RNP) complexes which form in vitro with gRNAs (H. U. Göringer, D. J. Koslowsky, T. H. Morales, and K. D. Stuart, Proc. Natl. Acad. Sci. USA, in press) are potential editosomes or their precursors. We find that several factors are important for in vitro formation of these RNP complexes and identify specific gRNA-binding proteins present in the complexes. Preedited mRNA promotes the in vitro formation of the four major gRNA-containing RNP complexes under some conditions but is required for the formation of only a subcomponent of one complex. The 5' gRNA sequence encompassing the RYAYA and anchor regions and the 3' gRNA oligo(U) tail are both important in complex formation, since their deletion results in a dramatic decrease of some complexes and the absence of others. UV cross-linking experiments identify several proteins which are in contact with gRNA and preedited mRNA in mitochondrial extracts. Proteins of 25 and 90 kDa are highly specific for gRNAs, and the 90-kDa protein binds specifically to gRNA oligo(U) tails. The gRNA-binding proteins exhibit a differential distribution between the four in vitro-formed complexes. These experiments reveal several proteins potentially involved in RNA editing and indicate that multiple recognition elements in gRNAs are used for complex formation.

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