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Hardiyani, Wulan Arum, Ali Wafa, Wahyu Indra Duwi Fanata y Hardian Susilo Addy. "Design and construction of single guide RNA for CRISPR/Cas9 system based on the xa13 resistance gene in some varieties of rice (Oryza sativa)". Jurnal Hama dan Penyakit Tumbuhan Tropika 23, n.º 1 (18 de enero de 2023): 47–55. http://dx.doi.org/10.23960/jhptt.12347-55.
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The xa13 gene is a recessive resistance gene against Xanthomonas oryzae pv. oryzae (Xoo) found in several rice varieties. Activation of this gene will trigger the formation of sucrose as a nutrient supply to Xoo for their growth in the plant. The disruption of this recessive gene expression in the plant can affect the negative impact of the gene, and recently can be created using clustered regularly interspaced short palindromic repeats (CRISPR) system using CRISPR-associated protein-9 (CRISPR/Cas9) technology that requires gRNA to recognize the targeted-sequence. This study aimed to design and construct the gRNA-targeting xa13 gene in rice using bioinformatics tools. CHOPCHOP was used for generated the gRNA candidates according to the target gene sequence. Two candidates of gRNA-targeted xa13 have been selected based on the analysis of bioinformatics data. Each candidate of gRNA consisted of 20 nucleotides (nt) of the target sequence upstream 3 nt of the protospacer adjacent motif (PAM) sequence (5’-NGG) targeting two exons in the xa13 gene. The gRNA1 will target exon 1 and the gRNA2 will target exon 2, with an efficiency of 52.51% and 44.63% respectively. Data showed that the GC content of all gRNA candidates ranged from 55–70% with no target-off location in the whole genome of rice. The transformation and confirmation test based on the physiological and genomic characteristics of transformants confirmed that the design has been successfully constructed.
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Read, L. K., H. U. Göringer y K. Stuart. "Assembly of mitochondrial ribonucleoprotein complexes involves specific guide RNA (gRNA)-binding proteins and gRNA domains but does not require preedited mRNA". Molecular and Cellular Biology 14, n.º 4 (abril de 1994): 2629–39. http://dx.doi.org/10.1128/mcb.14.4.2629-2639.1994.
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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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Read, L. K., H. U. Göringer y K. Stuart. "Assembly of mitochondrial ribonucleoprotein complexes involves specific guide RNA (gRNA)-binding proteins and gRNA domains but does not require preedited mRNA." Molecular and Cellular Biology 14, n.º 4 (abril de 1994): 2629–39. http://dx.doi.org/10.1128/mcb.14.4.2629.
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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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Cruz-Reyes, Jorge, Alevtina Zhelonkina, Laura Rusche y Barbara Sollner-Webb. "Trypanosome RNA Editing: Simple Guide RNA Features Enhance U Deletion 100-Fold". Molecular and Cellular Biology 21, n.º 3 (1 de febrero de 2001): 884–92. http://dx.doi.org/10.1128/mcb.21.3.884-892.2001.
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ABSTRACT Trypanosome RNA editing is a massive processing of mRNA by U deletion and U insertion, directed by trans-acting guide RNAs (gRNAs). A U deletion cycle and a U insertion cycle have been reproduced in vitro using synthetic ATPase (A6) pre-mRNA and gRNA. Here we examine which gRNA features are important for this U deletion. We find that, foremost, this editing depends critically on the single-stranded character of a few gRNA and a few mRNA residues abutting the anchor duplex, a feature not previously appreciated. That plus any base-pairing sequence to tether the upstream mRNA are all the gRNA needs to direct unexpectedly efficient in vitro U deletion, using either the purified editing complex or whole extract. In fact, our optimized gRNA constructs support faithful U deletion up to 100 times more efficiently than the natural gRNA, and they can edit the majority of mRNA molecules. This is a marked improvement of in vitro U deletion, in which previous artificial gRNAs were no more active than natural gRNA and the editing efficiencies were at most a few percent. Furthermore, this editing is not stimulated by most other previously noted gRNA features, including its potential ligation bridge, 3′ OH moiety, any U residues in the tether, the conserved structure of the central region, or proteins that normally bind these regions. Our data also have implications about evolutionary forces active in RNA editing.
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Collins, Scott P., William Rostain, Chunyu Liao y Chase L. Beisel. "Sequence-independent RNA sensing and DNA targeting by a split domain CRISPR–Cas12a gRNA switch". Nucleic Acids Research 49, n.º 5 (22 de febrero de 2021): 2985–99. http://dx.doi.org/10.1093/nar/gkab100.
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Abstract CRISPR technologies increasingly require spatiotemporal and dosage control of nuclease activity. One promising strategy involves linking nuclease activity to a cell's transcriptional state by engineering guide RNAs (gRNAs) to function only after complexing with a ‘trigger’ RNA. However, standard gRNA switch designs do not allow independent selection of trigger and guide sequences, limiting gRNA switch application. Here, we demonstrate the modular design of Cas12a gRNA switches that decouples selection of these sequences. The 5′ end of the Cas12a gRNA is fused to two distinct and non-overlapping domains: one base pairs with the gRNA repeat, blocking formation of a hairpin required for Cas12a recognition; the other hybridizes to the RNA trigger, stimulating refolding of the gRNA repeat and subsequent gRNA-dependent Cas12a activity. Using a cell-free transcription-translation system and Escherichia coli, we show that designed gRNA switches can respond to different triggers and target different DNA sequences. Modulating the length and composition of the sensory domain altered gRNA switch performance. Finally, gRNA switches could be designed to sense endogenous RNAs expressed only under specific growth conditions, rendering Cas12a targeting activity dependent on cellular metabolism and stress. Our design framework thus further enables tethering of CRISPR activities to cellular states.
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Moniruzzaman, M., Yun Zhong, Zhifeng Huang y Guangyan Zhong. "Having a Same Type IIS Enzyme’s Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis". International Journal of Molecular Sciences 23, n.º 9 (28 de abril de 2022): 4889. http://dx.doi.org/10.3390/ijms23094889.
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Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a Cas12a nuclease and four gRNAs was used for Agrobacterium-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors.
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Foreman, Hui-Chen Chang, Varvara Kirillov, Gabrielle Paniccia, Demetra Catalano, Trevor Andrunik, Swati Gupta, Laurie T. Krug y Yue Zhang. "RNA-guided gene editing of the murine gammaherpesvirus 68 genome reduces infectious virus production". PLOS ONE 16, n.º 6 (4 de junio de 2021): e0252313. http://dx.doi.org/10.1371/journal.pone.0252313.
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Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) are cancer-causing viruses that establish lifelong infections in humans. Gene editing using the Cas9-guideRNA (gRNA) CRISPR system has been applied to decrease the latent load of EBV in human Burkitt lymphoma cells. Validating the efficacy of Cas9-gRNA system in eradicating infection in vivo without off-target effects to the host genome will require animal model systems. To this end, we evaluated a series of gRNAs against individual genes and functional genomic elements of murine gammaherpesvirus 68 (MHV68) that are both conserved with KSHV and important for the establishment of latency or reactivation from latency in the host. gRNA sequences against ORF50, ORF72 and ORF73 led to insertion, deletion and substitution mutations in these target regions of the genome in cell culture. Murine NIH3T3 fibroblast cells that stably express Cas9 and gRNAs to ORF50 were most resistant to replication upon de novo infection. Latent murine A20 B cell lines that stably express Cas9 and gRNAs against MHV68 were reduced in their reactivation by approximately 50%, regardless of the viral gene target. Lastly, co-transfection of HEK293T cells with the vector expressing the Cas9-MHV68 gRNA components along with the viral genome provided a rapid read-out of gene editing and biological impact. Combinatorial, multiplex MHV68 gRNA transfections in HEK293T cells led to near complete ablation of infectious particle production. Our findings indicate that Cas9-gRNA editing of the murine gammaherpesvirus genome has a deleterious impact on productive replication in three independent infection systems.
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Clement, Sandra L., Melissa K. Mingler y Donna J. Koslowsky. "An Intragenic Guide RNA Location Suggests a Complex Mechanism for Mitochondrial Gene Expression in Trypanosoma brucei". Eukaryotic Cell 3, n.º 4 (agosto de 2004): 862–69. http://dx.doi.org/10.1128/ec.3.4.862-869.2004.
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ABSTRACT In Trypanosoma brucei, two classes of transcripts are produced from two distinct mitochondrial genome components. Guide RNAs (gRNAs) are usually minicircle encoded and exist as primary transcripts, while the maxicircle-encoded rRNAs and mRNAs are processed from a polycistronic precursor. The genes for the gRNAs gMURF2-II and gCYb(560) each have uncommon kinetoplast DNA (kDNA) locations that are not typically associated with transcription initiation events. We demonstrate that the conserved maxicircle gRNA gMURF2-II has an unusual location within the ND4 gene. This is the first report of a completely intragenic gene in kDNA. In addition, the gMURF2-II and ND4 transcripts are generated by distinctly different events; the ND4 mRNA is processed from a polycistronic precursor, while transcription of the gRNA initiates downstream of the 5′ end of the ND4 gene. The gCYb(560) gene has an atypical minicircle location in that it is not flanked by the inverted repeat sequences that surround the majority of minicircle gRNA genes. Our data indicate that the mature gCYb(560) gRNA is also a primary transcript and that the 5′-end heterogeneity previously observed for this gRNA is a result of multiple transcription initiation sites and not of imprecise 5′-end processing. Together, these data indicate that gRNA genes represent individual transcription units, regardless of their genomic context, and suggest a complex mechanism for mitochondrial gene expression in T. brucei.
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Goulah, Christopher C. y Laurie K. Read. "Differential Effects of Arginine Methylation on RBP16 mRNA Binding, Guide RNA (gRNA) Binding, and gRNA-containing Ribonucleoprotein Complex (gRNP) Formation". Journal of Biological Chemistry 282, n.º 10 (17 de enero de 2007): 7181–90. http://dx.doi.org/10.1074/jbc.m609485200.
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Tylec, Brianna L., Rachel M. Simpson, Laura E. Kirby, Runpu Chen, Yijun Sun, Donna J. Koslowsky y Laurie K. Read. "Intrinsic and regulated properties of minimally edited trypanosome mRNAs". Nucleic Acids Research 47, n.º 7 (30 de enero de 2019): 3640–57. http://dx.doi.org/10.1093/nar/gkz012.
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Abstract Most mitochondrial mRNAs in kinetoplastids require extensive uridine insertion/deletion editing to generate translatable open reading frames. Editing is specified by trans-acting gRNAs and involves a complex machinery including basal and accessory factors. Here, we utilize high-throughput sequencing to analyze editing progression in two minimally edited mRNAs that provide a simplified system due their requiring only two gRNAs each for complete editing. We show that CYb and MURF2 mRNAs exhibit barriers to editing progression that differ from those previously identified for pan-edited mRNAs, primarily at initial gRNA usage and gRNA exchange. We demonstrate that mis-edited junctions arise through multiple pathways including mis-alignment of cognate gRNA, incorrect and sometimes promiscuous gRNA utilization and inefficient gRNA anchoring. We then examined the roles of accessory factors RBP16 and MRP1/2 in maintaining edited CYb and MURF2 populations. RBP16 is essential for initiation of CYb and MURF2 editing, as well as MURF2 editing progression. In contrast, MRP1/2 stabilizes both edited mRNA populations, while further promoting progression of MURF2 mRNA editing. We also analyzed the effects of RNA Editing Substrate Binding Complex components, TbRGG2 and GAP1, and show that both proteins modestly impact progression of editing on minimally edited mRNAs, suggesting a novel function for GAP1.
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Vichera, G., D. Viale, R. Olivera, V. Arnold, A. Grundnig, J. Baston, S. Miriuka y L. Moro. "20 Generation of myostatin knockout horse embryos using clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9 and somatic cell nuclear transfer". Reproduction, Fertility and Development 31, n.º 1 (2019): 136. http://dx.doi.org/10.1071/rdv31n1ab20.
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Historically, livestock improvement by selective breeding was the principal selection force in animal production and welfare, but the desired phenotype may involve more than 1 generation. Nowadays, new technologies such as CRISPR/Cas9 could overpass these limits and improve animal quality by insertion or modification of the desired genotype. In this work, we aim to knock out the myostatin (MSTN) gene, a negative regulator of muscle mass development, in equine cells and to generate equine cloned embryos with this modified genotype. To achieve this, 1×105 equine fibroblasts were nucleofected with 5, 2, or 1μg of the plasmid hspCas9-2A-Puro V2.0 that codified for the Cas 9 nuclease and a single guide RNA (gRNA). Two different gRNA (gRNA 1 and gRNA 2) complementary to the first exon of the MSTN gene were designed and evaluated. Cells were also nucleofected with the enhanced green fluorescent protein-N1 plasmid in order to determine the transfection efficiency, obtaining more than 90% of enhanced green fluorescent protein+ cells in the 3 conditions. Forty-eight hours after nucleofection, cells were treated with 2.5μg mL−1 of puromycin for 48h to isolate cells that incorporated the plasmid. After that, clonal culture was achieved by plating individual cells in 96-well plates. The clones were then expanded individually and genomic DNA was isolated from each one, genotyped for the MSTN exon 1 locus by PCR amplification, and Sanger sequenced. Both gRNA had mutational activity, with 96% efficiency (24/25 clones) for gRNA 1 and 55.5% mutation activity (10/18 clones) for gRNA2. We obtained different genotypes depending on the gRNA and the dose that was used-gRNA 1: 1μg=57% wt/wt, 14% wt/mutX, and 29% mutX/mutX; 2μg=33.3% wt/wt, 33.3% mutX/mutY, and 33.3% mutX/mutX; 5μg=40% wt/wt and 60% mutX/mutY; gRNA 2: 1μg=17% wt/wt, 17% wt/mutX, 50% mutX/mutY, and 17% mutX/mutX; 2μg=67% mutX/mutY and 34% mutX/mutX; 5μg=54% mutX/mutY and 46% mutX/mutX. Two of the gRNA2 mutated cell lines were used for embryo generation by NT, 1 wt/mutX line (gRNA2-1ug-C2, heterozygote clone) and 1 mutX/mutX line (gRNA2-5ug-C13, homozygote clone). Before that, to assess specificity, the first 2 highly ranked off-target sites of gRNA2 were checked by Sanger sequencing in the selected clones, not observing modifications in their sequences. In both cases, we could generate edited MSTN equine cloned blastocysts: 3/153 (2%), 3/155 (2%), 8/140 (6%), and 9/73 (12%) for gRNA2-1μg-C2, gRNA2-5μg-C13, control fibroblasts, and control mesenchymal cells, respectively. In conclusion, genome edition by CRISPR/Cas9 is an efficient method to edit the genome of horse fibroblasts in a dose-dependent manner with apparent high specificity. Moreover, equine embryos can be generated with these cells with lower blastocyst rates than control fibroblasts of the same cell line or mesenchymal cells, probably due to higher cell passages needed for cell clone isolation and expansion. To the best of our knowledge, this is the first report of genome edited horse embryos.
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Mekler, Vladimir, Konstantin Kuznedelov y Konstantin Severinov. "Quantification of the affinities of CRISPR–Cas9 nucleases for cognate protospacer adjacent motif (PAM) sequences". Journal of Biological Chemistry 295, n.º 19 (1 de abril de 2020): 6509–17. http://dx.doi.org/10.1074/jbc.ra119.012239.
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The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9–gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9–gRNA, SaCas9–gRNA, and FnCas9–gRNA, respectively) and of three engineered SpCas9–gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a “beacon” assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9–gRNA binding to fluorescently labeled target DNA derivatives called “Cas9 beacons.” We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9–gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.
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Espin Palazon, Raquel, Xiaoyi Cheng, Clyde A. Campbell, Liangdao Li, Bettina Schmid y David Traver. "Zebra "Fishing" the Role of Granulin in Hematopoiesis". Blood 134, Supplement_1 (13 de noviembre de 2019): 1194. http://dx.doi.org/10.1182/blood-2019-130781.
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Granulin (GRN) is a multifunctional protein with anti-inflammatory properties and involved in neurological diseases and tumorigenesis. It contains several cysteine-rich motifs that are unique to this protein, which are conserved from sponges to humans indicating their ancient evolutionary origin. Despite being highly expressed by certain hematopoietic cell lineages, the role that GRN plays in hematopoiesis has reminded elusive. The multifunctional nature of this protein, together with its wide expression in all mammalian cell types has challenged the characterization of its functional role in hematopoiesis due to its effects on other tissues. Therefore, we took advantage of the whole genomic duplication of the zebrafish (Danio rerio) and the high conservation of the cysteine-rich motifs among the zebrafish and human granulins to address this knowledge gap and explore their role in hematopoiesis in vivo. The whole genome duplication that separated teleost fish from mammals resulted in two copies of the granulin gene in the zebrafish (Granulin a and Granulin b, Grna and Grnb respectively). This has allowed us an unprecedented view into the function of this protein in hematopoiesis. We show that like mammals, grnb transcripts are found in all cell types, including hematopoietic cells. In contrast, grna is restricted to hematopoietic cells, including myeloid populations. The distinct cell expression of grna and grnb suggests that, in the zebrafish, grna evolved to specifically function in hematopoiesis, while grnb may have taken on the rest of the biological roles assigned to the mammalian granulin. The zebrafish is an animal model with unique advantages for in vivo studies. Its external development allows us to circumvent the challenges of in utero experimentation required using mammals, permitting the use of non-invasive imaging techniques to study developmental hematopoiesis. In addition, more than 70% of genes identified in the zebrafish are conserved in humans. These, together with its high conservation with the human hematopoietic system has led to a greater understanding and prevention of human hematologic diseases by using this elegant animal model. These unique advantages of the zebrafish, in addition to its genetic amenability allowed us to generate Grna and Grnb single mutants and identify their impact in the hematopoietic system in vivo. While the absence of Grnb did not affect the development of the hematopoietic system, lack of Grna led to decreased differentiation of myeloid precursors into neutrophils and macrophages. Therefore, Grna knockout allowed us to disrupt the hematopoietic function of granulin while keeping unaltered its function in the brain and other non-hematopoietic tissues. Although viable, adult Grna mutants developed kidney marrow (the fish analogous to the mammalian bone marrow) failure, with increased progenitors and decreased mature myeloid cells. Mechanistically, we found that pu.1, the main transcription factor that leads to myeloid differentiation, directly bound grna enhancers, upregulating its expression. We have demonstrated that Grna enhanced myeloid gene expression, and decreased gata1 expression thereby facilitating myeloid differentiation and inhibiting the erythroid genetic program. Finally, we show that these findings in the zebrafish are also conserved in humans. Altogether, we have identified the hematopoietic role of granulin without disturbing its biological functions in other tissues. We have unveiled a powerful and novel master regulator for myeloid differentiation that could potentially be utilized for the treatment of hematological disorders such as neutropenia and leukemia. Disclosures No relevant conflicts of interest to declare.
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Lapinaite, Audrone, Jennifer A. Doudna y Jamie H. D. Cate. "Programmable RNA recognition using a CRISPR-associated Argonaute". Proceedings of the National Academy of Sciences 115, n.º 13 (12 de marzo de 2018): 3368–73. http://dx.doi.org/10.1073/pnas.1717725115.
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Argonaute proteins (Agos) are present in all domains of life. Although the physiological function of eukaryotic Agos in regulating gene expression is well documented, the biological roles of many of their prokaryotic counterparts remain enigmatic. In some bacteria, Agos are associated with CRISPR (clustered regularly interspaced short palindromic repeats) loci and use noncanonical 5′-hydroxylated guide RNAs (gRNAs) for nucleic acid targeting. Here we show that using 5-bromo-2′-deoxyuridine (BrdU) as the 5′ nucleotide of gRNAs stabilizes in vitro reconstituted CRISPR-associated Marinitoga piezophila Argonaute–gRNA complexes (MpAgo RNPs) and significantly improves their specificity and affinity for RNA targets. Using reconstituted MpAgo RNPs with 5′-BrdU-modified gRNAs, we mapped the seed region of the gRNA and identified the nucleotides of the gRNA that play the most significant role in targeting specificity. We also show that these MpAgo RNPs can be programmed to distinguish between substrates that differ by a single nucleotide, using permutations at the sixth and seventh positions in the gRNA. Using these specificity features, we employed MpAgo RNPs to detect specific adenosine-to-inosine–edited RNAs in a complex mixture. These findings broaden our mechanistic understanding of the interactions of Argonautes with guide and substrate RNAs, and demonstrate that MpAgo RNPs with 5′-BrdU-modified gRNAs can be used as a highly specific RNA-targeting platform to probe RNA biology.
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Igo, Robert P., Sobomabo D. Lawson y Kenneth Stuart. "RNA Sequence and Base Pairing Effects on Insertion Editing in Trypanosoma brucei". Molecular and Cellular Biology 22, n.º 5 (1 de marzo de 2002): 1567–76. http://dx.doi.org/10.1128/mcb.22.5.1567-1576.2002.
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ABSTRACT RNA editing inserts and deletes uridylates (U's) in kinetoplastid mitochondrial pre-mRNAs by a series of enzymatic steps. Small guide RNAs (gRNAs) specify the edited sequence. Editing, though sometimes extensive, is precise. The effects of mutating pre-mRNA and gRNA sequences in, around, and upstream of the editing site on the specificity and efficiency of in vitro insertion editing were examined. U's could be added opposite guiding pyrimidines, but guiding purines, particularly A's, were required for efficient ligation. A base pair between mRNA and gRNA immediately upstream of the editing site was not required for insertion editing, although it greatly enhanced its efficiency and accuracy. In addition, a gRNA/mRNA duplex upstream of the editing site enhanced insertion editing when it was close to the editing site, but prevented cleavage, and hence editing, when immediately adjacent to the editing site. Thus, several aspects of mRNA-gRNA interaction, as well as gRNA base pairing with added U's, optimize editing efficiency, although they are not required for insertion editing.
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Kirillov, Bogdan, Ekaterina Savitskaya, Maxim Panov, Aleksey Y. Ogurtsov, Svetlana A. Shabalina, Eugene V. Koonin y Konstantin V. Severinov. "Uncertainty-aware and interpretable evaluation of Cas9–gRNA and Cas12a–gRNA specificity for fully matched and partially mismatched targets with Deep Kernel Learning". Nucleic Acids Research 50, n.º 2 (17 de noviembre de 2021): e11-e11. http://dx.doi.org/10.1093/nar/gkab1065.
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Abstract The choice of guide RNA (gRNA) for CRISPR-based gene targeting is an essential step in gene editing applications, but the prediction of gRNA specificity remains challenging. Lack of transparency and focus on point estimates of efficiency disregarding the information on possible error sources in the model limit the power of existing Deep Learning-based methods. To overcome these problems, we present a new approach, a hybrid of Capsule Networks and Gaussian Processes. Our method predicts the cleavage efficiency of a gRNA with a corresponding confidence interval, which allows the user to incorporate information regarding possible model errors into the experimental design. We provide the first utilization of uncertainty estimation in computational gRNA design, which is a critical step toward accurate decision-making for future CRISPR applications. The proposed solution demonstrates acceptable confidence intervals for most test sets and shows regression quality similar to existing models. We introduce a set of criteria for gRNA selection based on off-target cleavage efficiency and its variance and present a collection of pre-computed gRNAs for human chromosome 22. Using Neural Network Interpretation methods, we show that our model rediscovers an established biological factor underlying cleavage efficiency, the importance of the seed region in gRNA.
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Bringaud, F., R. Stripecke, G. C. Frech, S. Freedland, C. Turck, E. M. Byrne y L. Simpson. "Mitochondrial glutamate dehydrogenase from Leishmania tarentolae is a guide RNA-binding protein." Molecular and Cellular Biology 17, n.º 7 (julio de 1997): 3915–23. http://dx.doi.org/10.1128/mcb.17.7.3915.
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To identify specific proteins interacting with guide RNAs (gRNAs) in mitochondrial ribonucleoprotein complexes from Leishmania tarentolae, fractionated and unfractionated mitochondrial extracts were subjected to UV cross-linking with added labeled gRNA and also with [alpha-32P]UTP-labeled endogenous RNA. An abundant 110-kDa protein (p110) localized in the T-V complex, which sediments in glycerol gradients at the leading edge of the 10S terminal uridylyltransferase peak, was found to interact with both types of labeled RNAs. The p110 protein was gel isolated and subjected to microsequence analysis, and the gene was cloned. The sequence revealed significant similarity with mitochondrial glutamate dehydrogenases. A polyclonal antiserum was raised against a recombinant fragment of the p110 gene and was used to demonstrate a stable and specific gRNA-binding activity by coimmunoprecipitation and competitive gel shift analyses. Complex formation was strongly inhibited by competition with poly(U) or by deletion or substitution of the gRNA 3' oligo(U) tail. Also, addition of a 3' oligo(U) tail to an unrelated transcript was sufficient for p110 binding. Both the gRNA-binding activity of the p110 protein and in vitro gRNA-independent and gRNA-dependent uridine insertion activities in the mitochondrial extract were inhibited by high concentrations of dinucleotides.
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Jung, Soo Bin, Chae young Lee, Kwang-Ho Lee, Kyu Heo y Si Ho Choi. "A cleavage-based surrogate reporter for the evaluation of CRISPR–Cas9 cleavage efficiency". Nucleic Acids Research 49, n.º 15 (4 de junio de 2021): e85-e85. http://dx.doi.org/10.1093/nar/gkab467.
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Abstract CRISPR–Cas9 is a powerful tool for genome engineering, but its efficiency largely depends on guide RNA (gRNA). There are multiple methods available to evaluate the efficiency of gRNAs, including the T7E1 assay, surveyor nuclease assay, deep sequencing, and surrogate reporter systems. In the present study, we developed a cleavage-based surrogate that we have named the LacI-reporter to evaluate gRNA cleavage efficiency. The LacI repressor, under the control of the EF-1α promoter, represses luciferase or EGFP reporter expression by binding to the lac operator. Upon CRISPR–Cas9 cleavage at a target site located between the EF-1α promoter and the lacI gene, repressor expression is disrupted, thereby triggering luciferase or EGFP expression. Using this system, we can quantitate gRNA cleavage efficiency by assessing luciferase activity or EGFP expression. We found a strong positive correlation between the cleavage efficiency of gRNAs measured using this reporter and mutation frequency, measured using surveyor and deep sequencing. The genome-editing efficiency of gRNAs was validated in human liver organoids. Our LacI-reporter system provides a useful tool to select efficient gRNAs for genome editing.
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Darcis, Gilles, Caroline Binda, Bep Klaver, Elena Herrera-Carrillo, Ben Berkhout y Atze Das. "The Impact of HIV-1 Genetic Diversity on CRISPR-Cas9 Antiviral Activity and Viral Escape". Viruses 11, n.º 3 (13 de marzo de 2019): 255. http://dx.doi.org/10.3390/v11030255.
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The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely explored for sequence-specific attack on HIV-1 proviral DNA. We recently identified dual-guide RNA (dual-gRNA) combinations that can block HIV-1 replication permanently in infected cell cultures and prevent viral escape. Although the gRNAs were designed to target highly conserved viral sequences, their efficacy may be challenged by high genetic variation in the HIV-1 genome. We therefore evaluated the breadth of these dual-gRNA combinations against distinct HIV-1 isolates, including several subtypes. Replication of nearly all virus isolates could be prevented by at least one gRNA combination, which caused inactivation of the proviral genomes and the gradual loss of replication-competent virus over time. The dual-gRNA efficacy was not affected by most single nucleotide (nt) mismatches between gRNA and the viral target. However, 1-nt mismatches at the Cas9 cleavage site and two mismatches anywhere in the viral target sequence significantly reduced the inhibitory effect. Accordingly, sequence analysis of viruses upon breakthrough replication revealed the acquisition of escape mutations in perfectly matching and most 1-nt mismatching targets, but not in targets with a mismatch at the Cas9 cleavage site or with two mismatches. These results demonstrate that combinatorial CRISPR-Cas9 treatment can cure T cells infected by distinct HIV-1 isolates, but even minor sequence variation in conserved viral target sites can affect the efficacy of this strategy. Successful cure attempts against isolates with divergent target sequences may therefore require adaptation of the gRNAs.
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Easmin, Farhana, Naim Hassan, Yu Sasano, Keisuke Ekino, Hisataka Taguchi y Satoshi Harashima. "gRNA-transient expression system for simplified gRNA delivery in CRISPR/Cas9 genome editing". Journal of Bioscience and Bioengineering 128, n.º 3 (septiembre de 2019): 373–78. http://dx.doi.org/10.1016/j.jbiosc.2019.02.009.
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Yuan, Guoliang, Stanton Martin, Md Mahmudul Hassan, Gerald A. Tuskan y Xiaohan Yang. "PARA: A New Platform for the Rapid Assembly of gRNA Arrays for Multiplexed CRISPR Technologies". Cells 11, n.º 16 (9 de agosto de 2022): 2467. http://dx.doi.org/10.3390/cells11162467.
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Multiplexed CRISPR technologies have great potential for pathway engineering and genome editing. However, their applications are constrained by complex, laborious and time-consuming cloning steps. In this research, we developed a novel method, PARA, which allows for the one-step assembly of multiple guide RNAs (gRNAs) into a CRISPR vector with up to 18 gRNAs. Here, we demonstrate that PARA is capable of the efficient assembly of transfer RNA/Csy4/ribozyme-based gRNA arrays. To aid in this process and to streamline vector construction, we developed a user-friendly PARAweb tool for designing PCR primers and component DNA parts and simulating assembled gRNA arrays and vector sequences.
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Koreman, Gabriel T., Yineng Xu, Qinan Hu, Zijing Zhang, Sarah E. Allen, Mariana F. Wolfner, Bei Wang y Chun Han. "Upgraded CRISPR/Cas9 tools for tissue-specific mutagenesis inDrosophila". Proceedings of the National Academy of Sciences 118, n.º 14 (29 de marzo de 2021): e2014255118. http://dx.doi.org/10.1073/pnas.2014255118.
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CRISPR/Cas9 has emerged as a powerful technology for tissue-specific mutagenesis. However, tissue-specific CRISPR/Cas9 tools currently available inDrosophilaremain deficient in three significant ways. First, many existing gRNAs are inefficient, such that further improvements of gRNA expression constructs are needed for more efficient and predictable mutagenesis in both somatic and germline tissues. Second, it has been difficult to label mutant cells in target tissues with current methods. Lastly, application of tissue-specific mutagenesis at present often relies on Gal4-driven Cas9, which hampers the flexibility and effectiveness of the system. Here, we tackle these deficiencies by building upon our previous CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) tools. First, we significantly improved gRNA efficiency in somatic tissues by optimizing multiplexed gRNA design. Similarly, we also designed efficient dual-gRNA vectors for the germline. Second, we developed methods to positively and negatively label mutant cells in tissue-specific mutagenesis by incorporating co-CRISPR reporters into gRNA expression vectors. Lastly, we generated genetic reagents for convenient conversion of existing Gal4 drivers into tissue-specific Cas9 lines based on homology-assisted CRISPR knock-in. In this way, we expand the choices of Cas9 for CRISPR-TRiM analysis to broader tissues and developmental stages. Overall, our upgraded CRISPR/Cas9 tools make tissue-specific mutagenesis more versatile, reliable, and effective inDrosophila. These improvements may be also applied to other model systems.
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Igo, Robert P., Setareh S. Palazzo, Moffett L. K. Burgess, Aswini K. Panigrahi y Kenneth Stuart. "Uridylate Addition and RNA Ligation Contribute to the Specificity of Kinetoplastid Insertion RNA Editing". Molecular and Cellular Biology 20, n.º 22 (15 de noviembre de 2000): 8447–57. http://dx.doi.org/10.1128/mcb.20.22.8447-8457.2000.
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ABSTRACT RNA editing in Trypanosoma brucei inserts and deletes uridylates (U's) in mitochondrial pre-mRNAs under the direction of guide RNAs (gRNAs). We report here the development of a novel in vitro precleaved editing assay and its use to study the gRNA specificity of the U addition and RNA ligation steps in insertion RNA editing. The 5′ fragment of substrate RNA accumulated with the number of added U's specified by gRNA, and U addition products with more than the specified number of U's were rare. U addition up to the number specified occurred in the absence of ligation, but accumulation of U addition products was slowed. The 5′ fragments with the correct number of added U's were preferentially ligated, apparently by adenylylated RNA ligase since exogenously added ATP was not required and since ligation was eliminated by treatment with pyrophosphate. gRNA-specified U addition was apparent in the absence of ligation when the pre-mRNA immediately upstream of the editing site was single stranded and more so when it was base paired with gRNA. These results suggest that both the U addition and RNA ligation steps contributed to the precision of RNA editing.
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Igo, Robert P., David S. Weston, Nancy Lewis Ernst, Aswini K. Panigrahi, Reza Salavati y Kenneth Stuart. "Role of Uridylate-Specific Exoribonuclease Activity in Trypanosoma brucei RNA Editing". Eukaryotic Cell 1, n.º 1 (febrero de 2002): 112–18. http://dx.doi.org/10.1128/ec.1.1.112-118.2002.
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ABSTRACT Editing of mitochondrial mRNAs in kinetoplastid protozoa occurs by a series of enzymatic steps that insert and delete uridylates (U's) as specified by guide RNAs (gRNAs). The characteristics of the 3" exonuclease activity that removes the U's following cleavage during deletion editing were determined by using an in vitro precleaved deletion assay that is based on ATPase subunit 6 pre-mRNA and gA6[14] gRNA. The exonuclease in partially purified editing complexes is specific for U's. The specificity occurs in the absence of gRNA, but its activity is enhanced by the presence of gRNA. The 3" pre-mRNA fragment enhances the specificity, but not the efficiency, of U removal. The activity is sensitive to the 5" phosphate of the 3" fragment, which is not required for U removal. The ability of the 3" U's to base pair with purines in the gRNA protects them from removal, suggesting that the U-specific 3" exonuclease (exoUase) is specific for U's which are not base paired. ExoUase is stereospecific and cannot remove (Rp )α-thio-U. The specificity of the exoUase activity thus contributes to the precision of RNA editing.
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Prajapati, Archana. "Towards the diagnosis of dengue virus and its serotypes using designed CRISPR/Cas13 gRNAs". Bioinformation 18, n.º 8 (31 de agosto de 2022): 661–68. http://dx.doi.org/10.6026/97320630018661.
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Dengue Virus (DENV) is a mosquito-borne virus that is prevalent in the world's tropical and subtropical regions. Therefore, early detection and surveillance can help in the management of this disease. Current diagnostic methods rely primarily on ELISA, PCR, and RT-PCR, among others, which can only be performed in specialized laboratories and require sophisticated instruments and technical expertise. CRISPR-based technologies on the other hand have field-deployable viral diagnostics capabilities that could be used in the development of point-of-care molecular diagnostics. The first step in the field of CRISPR-based virus diagnosis is to design and screen gRNAs for high efficiency and specificity. In the present study, we employed a bioinformatics approach to design and screen DENV CRISPR/Cas13 gRNAs for conserved and serotype-specific variable genomic regions in the DENV genome. We identified one gRNA sequence specific for each of the lncRNA and NS5 regions and identified one gRNA against each of DENV1, DENV2, DENV3, and DENV4 to distinguish the four DENV serotypes. These CRISPR/Cas13 gRNA sequences will be useful in diagnosing the dengue virus and its serotypes for in vitro validation and diagnostics.
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Khanal, Sushant, Dechao Cao, Jinyu Zhang, Yi Zhang, Madison Schank, Xindi Dang, Lam Ngoc Thao Nguyen et al. "Synthetic gRNA/Cas9 Ribonucleoprotein Inhibits HIV Reactivation and Replication". Viruses 14, n.º 9 (28 de agosto de 2022): 1902. http://dx.doi.org/10.3390/v14091902.
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The current antiretroviral therapy (ART) for human immunodeficiency virus (HIV) can halt viral replication but cannot eradicate HIV infection because proviral DNA integrated into the host genome remains genetically silent in reservoir cells and is replication-competent upon interruption or cessation of ART. CRISPR/Cas9-based technology is widely used to edit target genes via mutagenesis (i.e., nucleotide insertion/deletion and/or substitution) and thus can inactivate integrated proviral DNA. However, CRISPR/Cas9 delivery systems often require viral vectors, which pose safety concerns for therapeutic applications in humans. In this study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a non-viral formulation to develop a novel HIV gene therapy. We designed a series of gRNAs targeting different HIV genes crucial for HIV replication and tested their antiviral efficacy and cellular cytotoxicity in lymphoid and monocytic latent HIV cell lines. Compared with the scramble gRNA control, HIV-gRNA/Cas9 RNP-treated cells exhibited efficient viral suppression with no apparent cytotoxicity, as evidenced by the significant inhibition of latent HIV DNA reactivation and RNA replication. Moreover, HIV-gRNA/Cas9 RNP inhibited p24 antigen expression, suppressed infectious viral particle production, and generated specific DNA cleavages in the targeted HIV genes that are confirmed by DNA sequencing. Because of its rapid DNA cleavage, low off-target effects, low risk of insertional mutagenesis, easy production, and readiness for use in clinical application, this study provides a proof-of-concept that synthetic gRNA/Cas9 RNP drugs can be utilized as a novel therapeutic approach for HIV eradication.
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Zimmer, Sara L., Sarah M. McEvoy, Jun Li, Jun Qu y Laurie K. Read. "A Novel Member of the RNase D Exoribonuclease Family Functions in Mitochondrial Guide RNA Metabolism in Trypanosoma brucei". Journal of Biological Chemistry 286, n.º 12 (20 de enero de 2011): 10329–40. http://dx.doi.org/10.1074/jbc.m110.152439.
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RNA turnover and RNA editing are essential for regulation of mitochondrial gene expression in Trypanosoma brucei. RNA turnover is controlled in part by RNA 3′ adenylation and uridylation status, with trans-acting factors also impacting RNA homeostasis. However, little is known about the mitochondrial degradation machinery or its regulation in T. brucei. We have identified a mitochondrial exoribonuclease, TbRND, whose expression is highly up-regulated in the insect proliferative stage of the parasite. TbRND shares sequence similarity with RNase D family enzymes but differs from all reported members of this family in possessing a CCHC zinc finger domain. In vitro, TbRND exhibits 3′ to 5′ exoribonuclease activity, with specificity toward uridine homopolymers, including the 3′ oligo(U) tails of guide RNAs (gRNAs) that provide the sequence information for RNA editing. Several lines of evidence generated from RNAi-mediated knockdown and overexpression cell lines indicate that TbRND functions in gRNA metabolism in vivo. First, TbRND depletion results in gRNA tails extended by 2–3 nucleotides on average. Second, overexpression of wild type but not catalytically inactive TbRND results in a substantial decrease in the total gRNA population and a consequent inhibition of RNA editing. The observed effects on the gRNA population are specific as rRNAs, which are also 3′-uridylated, are unaffected by TbRND depletion or overexpression. Finally, we show that gRNA binding proteins co-purify with TbRND. In summary, TbRND is a novel 3′ to 5′ exoribonuclease that appears to have evolved a function highly specific to the mitochondrion of trypanosomes.
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Champer, Samuel E., Suh Yeon Oh, Chen Liu, Zhaoxin Wen, Andrew G. Clark, Philipp W. Messer y Jackson Champer. "Computational and experimental performance of CRISPR homing gene drive strategies with multiplexed gRNAs". Science Advances 6, n.º 10 (marzo de 2020): eaaz0525. http://dx.doi.org/10.1126/sciadv.aaz0525.
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The rapid evolution of resistance alleles poses a major obstacle for genetic manipulation of populations with CRISPR homing gene drives. One proposed solution is using multiple guide RNAs (gRNAs), allowing a drive to function even if some resistant target sites are present. Here, we develop a model of homing mechanisms parameterized by experimental studies. Our model incorporates several factors affecting drives with multiple gRNAs, including timing of cleavage, reduction in homology-directed repair efficiency due to imperfect homology, Cas9 activity saturation, gRNA activity level variance, and incomplete homology-directed repair. We find that homing drives have an optimal number of gRNAs, usually between two and eight, depending on the specific drive type and performance parameters. These results contradict the notion that resistance rates can be reduced to arbitrarily low levels by gRNA multiplexing and highlight the need for combined approaches to counter resistance evolution in CRISPR homing drives.
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Xie, Kabin, Bastian Minkenberg y Yinong Yang. "Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system". Proceedings of the National Academy of Sciences 112, n.º 11 (2 de marzo de 2015): 3570–75. http://dx.doi.org/10.1073/pnas.1420294112.
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The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA–gRNA architecture were efficiently and precisely processed into gRNAs with desired 5′ targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits.
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Cooper, Sinclair, Elizabeth S. Wadsworth, Torsten Ochsenreiter, Alasdair Ivens, Nicholas J. Savill y Achim Schnaufer. "Assembly and annotation of the mitochondrial minicircle genome of a differentiation-competent strain of Trypanosoma brucei". Nucleic Acids Research 47, n.º 21 (30 de octubre de 2019): 11304–25. http://dx.doi.org/10.1093/nar/gkz928.
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Abstract Kinetoplastids are protists defined by one of the most complex mitochondrial genomes in nature, the kinetoplast. In the sleeping sickness parasite Trypanosoma brucei, the kinetoplast is a chain mail-like network of two types of interlocked DNA molecules: a few dozen ∼23-kb maxicircles (homologs of the mitochondrial genome of other eukaryotes) and thousands of ∼1-kb minicircles. Maxicircles encode components of respiratory chain complexes and the mitoribosome. Several maxicircle-encoded mRNAs undergo extensive post-transcriptional RNA editing via addition and deletion of uridines. The process is mediated by hundreds of species of minicircle-encoded guide RNAs (gRNAs), but the precise number of minicircle classes and gRNA genes was unknown. Here we present the first essentially complete assembly and annotation of the kinetoplast genome of T. brucei. We have identified 391 minicircles, encoding not only ∼930 predicted ‘canonical’ gRNA genes that cover nearly all known editing events (accessible via the web at http://hank.bio.ed.ac.uk), but also ∼370 ‘non-canonical’ gRNA genes of unknown function. Small RNA transcriptome data confirmed expression of the majority of both categories of gRNAs. Finally, we have used our data set to refine definitions for minicircle structure and to explore dynamics of minicircle copy numbers.
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McManus, Michael T., Brian K. Adler, Victoria W. Pollard y Stephen L. Hajduk. "Trypanosoma brucei Guide RNA Poly(U) Tail Formation Is Stabilized by Cognate mRNA". Molecular and Cellular Biology 20, n.º 3 (1 de febrero de 2000): 883–91. http://dx.doi.org/10.1128/mcb.20.3.883-891.2000.
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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.
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Chameettachal, Akhil, Valérie Vivet-Boudou, Fathima Nuzra Nagoor Pitchai, Vineeta N. Pillai, Lizna Mohamed Ali, Anjana Krishnan, Serena Bernacchi, Farah Mustafa, Roland Marquet y Tahir A. Rizvi. "A purine loop and the primer binding site are critical for the selective encapsidation of mouse mammary tumor virus genomic RNA by Pr77Gag". Nucleic Acids Research 49, n.º 8 (9 de abril de 2021): 4668–88. http://dx.doi.org/10.1093/nar/gkab223.
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Abstract Retroviral RNA genome (gRNA) harbors cis-acting sequences that facilitate its specific packaging from a pool of other viral and cellular RNAs by binding with high-affinity to the viral Gag protein during virus assembly. However, the molecular intricacies involved during selective gRNA packaging are poorly understood. Binding and footprinting assays on mouse mammary tumor virus (MMTV) gRNA with purified Pr77Gag along with in cell gRNA packaging study identified two Pr77Gag binding sites constituting critical, non-redundant packaging signals. These included: a purine loop in a bifurcated stem-loop containing the gRNA dimerization initiation site, and the primer binding site (PBS). Despite these sites being present on both unspliced and spliced RNAs, Pr77Gag specifically bound to unspliced RNA, since only that could adopt the native bifurcated stem–loop structure containing looped purines. These results map minimum structural elements required to initiate MMTV gRNA packaging, distinguishing features that are conserved amongst divergent retroviruses from those perhaps unique to MMTV. Unlike purine-rich motifs frequently associated with packaging signals, direct involvement of PBS in gRNA packaging has not been documented in retroviruses. These results enhance our understanding of retroviral gRNA packaging/assembly, making it not only a target for novel therapeutic interventions, but also development of safer gene therapy vectors.
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Xu, Jianyong, Wenlei Li, MD Munnaf Hossen, Yuning Jia, Lingyun Li y Zhong Huang. "Optimized Plasmid Construction Strategy for Cas9". Cellular Physiology and Biochemistry 48, n.º 1 (2018): 131–37. http://dx.doi.org/10.1159/000491669.
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Background/Aims: The target genome editing technology not only plays an important role in basic biology studies but also holds a great promise for potential clinical applications. The new generation of engineered nuclease RGEN (RNA Guided EndoNuclease) is much easier to construct and modify, and attracts more attentions. In the current study, we compared different plasmid construction strategies of Cas9-gRNA (guide RNA). Methods: Different plasmid construction strategies of Cas9-gRNA were compared. And more modifications were introduced into the plasmid construction strategy. Results: The plasmid construction efficiency of expressing the gRNA and Cas9 in one plasmid was lower than expressing them in two separate plasmids. However, they showed the similar genome editing efficiency. We further introduced the Golden-gate assembly and blue-white screening approaches into the Cas9-gRNA construction procedures, without the process of vector digestion and gel purification. Conclusions: Combing with the optimized gRNA structure (gRNA-BL) we identified before, we established one more cost-effective, time-saving and efficient plasmid construction strategy for Cas9-gRNA.
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Simon, Ian D., Nico van Rooijen y John K. Rose. "Vesicular Stomatitis Virus Genomic RNA Persists In Vivo in the Absence of Viral Replication". Journal of Virology 84, n.º 7 (23 de diciembre de 2009): 3280–86. http://dx.doi.org/10.1128/jvi.02052-09.
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ABSTRACT Our previous studies using intranasal inoculation of mice with vesicular stomatitis virus (VSV) vaccine vectors showed persistence of vector genomic RNA (gRNA) for at least 60 days in lymph nodes in the absence of detectable infectious virus. Here we show high-level concentration of virus and gRNA in lymph nodes after intramuscular inoculation of mice with attenuated or single-cycle VSV vectors as well as long-term persistence of gRNA in the lymph nodes. To determine if the persistence of gRNA was due to ongoing viral replication, we developed a tagged-primer approach that was critical for detection of VSV mRNA specifically. Our results show that VSV gRNA persists long-term in the lymph nodes while VSV mRNA is present only transiently. Because VSV transcription is required for replication, our results indicate that persistence of gRNA does not result from continuing viral replication. We also performed macrophage depletion studies that are consistent with initial trapping of VSV gRNA largely in lymph node macrophages and subsequent persistence elsewhere in the lymph node.
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Ke, Yuqing, Behafarid Ghalandari, Shiyi Huang, Sijie Li, Chengjie Huang, Xiao Zhi, Daxiang Cui y Xianting Ding. "2′-O-Methyl modified guide RNA promotes the single nucleotide polymorphism (SNP) discrimination ability of CRISPR–Cas12a systems". Chemical Science 13, n.º 7 (2022): 2050–61. http://dx.doi.org/10.1039/d1sc06832f.
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This study illustrates that 2′-O-methyl modified gRNAs improve the specificity of the CRISPR–Cas12a system (mg-CRISPR) via suppressing the Cas12a's affinity to off-target DNA and provides an efficient strategy for high-specificity gRNA design.
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Jameel, Mohd Rizwan. "From design to validation of CRISPR/gRNA primers towards genome editing". Bioinformation 18, n.º 5 (31 de mayo de 2022): 471–77. http://dx.doi.org/10.6026/97320630018471.
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CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated system) is used to edit specific genomic sequences with precision and efficacy. There are many online platforms/software for the design of gRNAs and related primers. However, there are concerns in design regarding off-site deletions besides knocking out sequences in the target genes. Nonetheless, a well known robust platform for CRISPR/gRNA primers design is CRISPRdirect. We demonstrate the use of this tool in the design of CRISPR/gRNA primers for soluble starch synthases (SSS) II-1, 2, and 3 genes in the Oryza sativa genome followed by the PCR-mediated amplification of SSS genes with corresponding confirmation towards genome editing having improved phenotype features.
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Roberson, Elisha D. "Identification of high-efficiency 3′GG gRNA motifs in indexed FASTA files with ngg2". PeerJ Computer Science 1 (18 de noviembre de 2015): e33. http://dx.doi.org/10.7717/peerj-cs.33.
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CRISPR/Cas9 is emerging as one of the most-used methods of genome modification in organisms ranging from bacteria to human cells. However, the efficiency of editing varies tremendously site-to-site. A recent report identified a novel motif, called the 3′GG motif, which substantially increases the efficiency of editing at all sites tested inC. elegans. Furthermore, they highlighted that previously published gRNAs with high editing efficiency also had this motif. I designed a Python command-line tool, ngg2, to identify 3′GG gRNA sites from indexed FASTA files. As a proof-of-concept, I screened for these motifs in six model genomes:Saccharomyces cerevisiae,Caenorhabditis elegans,Drosophila melanogaster,Danio rerio,Mus musculus, andHomo sapiens. I also scanned the genomes of pig (Sus scrofa) and African elephant (Loxodonta africana) to demonstrate the utility in non-model organisms. I identified more than 60 million single match 3′GG motifs in these genomes. Greater than 61% of all protein coding genes in the reference genomes had at least one unique 3′GG gRNA site overlapping an exon. In particular, more than 96% of mouse and 93% of human protein coding genes have at least one unique, overlapping 3′GG gRNA. These identified sites can be used as a starting point in gRNA selection, and the ngg2 tool provides an important ability to identify 3′GG editing sites in any species with an available genome sequence.
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Kawashima, Nozomu, Yusuke Okuno, Yuko Sekiya, Xinan Wang, Yinyan Xu, Atsushi Narita, Sayoko Doisaki et al. "Generation of Cell Lines Harboring SETBP1 Mutations By the Crispr/Cas9 System". Blood 124, n.º 21 (6 de diciembre de 2014): 4622. http://dx.doi.org/10.1182/blood.v124.21.4622.4622.
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Abstract Introduction Recent advances in cancer genetics have led to the identification of somatic mutations in SET-binding protein 1 (SETBP1) in myeloid malignancies categorized as myeloproliferative neoplasm (MPN) and myelodysplastic syndromes (MDS). Heterozygous point mutations in SETBP1 are essentially found at a genomic level in myeloid malignancies, and the frequency of the mutated allele in cDNA suggests somatic heterozygosity without substantial imbalance in allelic expression. Thus, mutant SETBP1 presumably has a dominant altered biological activity. Most mutations in SETBP1 are located in the SKI homologous region. This region is suggested to include regions critical for ubiquitin binding and SETBP1 degradation. SETBP1 binds to SET, which protects against protease cleavage, and thus may result in PP2A inhibition and cell proliferation. Overexpression of SETBP1 resulting from a p.G870S alteration showed higher levels of the protein compared with wild-type (WT), indicating a prolonged halftime of SETBP1, which led to reduced PP2A activity and a higher cell proliferation rate. To date, however, our molecular biological understanding of SETBP1 mutations has been obtained only through observations of exogenous overexpression in cell lines. This may result in bias, considering the predicted dominant-negative function of SETBP1 mutations. Therefore, we used an RNA-guided endonuclease (RGEN), the CRISPR/Cas9 system, to generate a cell line harboring point mutations resulting in only relevant single amino acid substitutions in SETBP1. We analyzed cell signaling using the cell line thus established. Methods pSpCas9(BB) (PX330) was used to express humanized S. pyogenes Cas9 and gRNAs of interest. The gRNAs were designed by searching for NGG protospacer adjacent motif (PAM) sequences near the point mutation target sites. The candidate gRNAs were gRNA#1, 5′-TAGGGAGCCAATCTCGCAC-3′; gRNA#2, 5′-TGTCCCAATGCCGCTGTCGC-3′; gRNA#4, 5′-GTCCCAATGCCGCTGTCGCT-3′; and gRNA#7, 5′-GAGACGATCCCCAGCGACAG-3′. pCAG-EGxxFP harboring the 500 bp target region of WT SETBP1 was constructed for gRNA selection. For homology-dependent repair (HDR), we synthesized 70 mer single-stranded oligonucleotides (ssODN) having both the SETBP1 c.2602G>A, p.D868N mutation and synonymous mutation in the PAM. HEK293T cells were cultured in DMEM with 10% FBS. For cell signaling analysis, the cells were serum-depleted for 16 h prior to western blotting. Anti-SETBP1 antibody (ab98222), anti-phospho-Y307 PP2A antibody (E155), and anti phospho-p44/42 MAPK antibody (CST#4370) were used for cell signaling analysis. Results To validate an efficient sgRNA for DNA scission, we cotransfected pCAG-EGxxFP-SETBP1 and pSpCas9(BB)-SETBP1-gRNA plasmids into HEK293T cells. EGFP fluorescence, whose intensity is correlated with the efficacy of HDR, was observed 48 h later, and we determined that gRNA#2 was the most efficient. Next we cotransfected 293T cells with pCAG-EGxxFP-SETBP1, pSpCas9(BB)-SETBP1-gRNA#2, and ssODN for mutagenesis. Five days after transfection, single EGFP-positive clones were isolated using the FACSAria cell sorting system. Sanger sequencing confirmed that 293T cells harboring the SETBP1 p.D868N homozygous mutation were established. A clone with WT SETBP1 was also maintained as a control. To elucidate the effects of the SETBP1 mutation in 293T cells, we performed cell signaling analysis by western blotting. 293T-SETBP1 p.D868N cells showed higher levels of SETBP1 protein with lower molecular weight compared with WT, indicating a prolonged halftime, possibly due to loss of ubiquitination. In addition, 293T-SETBP1 p.D868N cells showed a higher phosphorylation level of PP2A (Y307, C subunit), a marker of PP2A inactivation. Finally, the phosphorylation level of p44/42 MAPK (ERK1/2) was increased in 293T-SETBP1 p.D868N cells. Conclusions We confirmed that the SETBP1 p.D868N mutation caused a prolonged halftime, resulting in PP2A inactivation and p44/42 MAPK activation in 293T cell lines. Our data suggest a potential therapy target for malignancies harboring SETBP1 mutations. More generally, this work illustrates the utility of RGEN technology for studying hematological malignancies. Disclosures No relevant conflicts of interest to declare.
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Mullally, Grace, Kara van Aelst, Mohsin M. Naqvi, Fiona M. Diffin, Tautvydas Karvelis, Giedrius Gasiunas, Virginijus Siksnys y Mark D. Szczelkun. "5′ modifications to CRISPR–Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage". Nucleic Acids Research 48, n.º 12 (4 de junio de 2020): 6811–23. http://dx.doi.org/10.1093/nar/gkaa477.
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Abstract A key aim in exploiting CRISPR–Cas is gRNA engineering to introduce additional functionalities, ranging from individual nucleotide changes that increase efficiency of on-target binding to the inclusion of larger functional RNA aptamers or ribonucleoproteins (RNPs). Cas9–gRNA interactions are crucial for complex assembly, but several distinct regions of the gRNA are amenable to modification. We used in vitro ensemble and single-molecule assays to assess the impact of gRNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that RNP formation was unaffected by any of our modifications. R-loop formation and DNA cleavage activity were also essentially unaffected by modification of the Upper Stem, first Hairpin and 3′ end. In contrast, we found that 5′ additions of only two or three nucleotides could reduce R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5′ end of a gRNA still supported RNP formation but produced a stable ∼9 bp R-loop that could not activate DNA cleavage. Consideration of these observations will assist in successful gRNA design.
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Bhagwat, Aditya M., Johannes Graumann, Rene Wiegandt, Mette Bentsen, Jordan Welker, Carsten Kuenne, Jens Preussner, Thomas Braun y Mario Looso. "multicrispr: gRNA design for prime editing and parallel targeting of thousands of targets". Life Science Alliance 3, n.º 11 (9 de septiembre de 2020): e202000757. http://dx.doi.org/10.26508/lsa.202000757.
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Targeting the coding genome to introduce nucleotide deletions/insertions via the CRISPR/Cas9 technology has become a standard procedure. It has quickly spawned a multitude of methods such as prime editing, APEX proximity labeling, or homology directed repair, for which supporting bioinformatics tools are, however, lagging behind. New CRISPR/Cas9 applications often require specific gRNA design functionality, and a generic tool is critically missing. Here, we introduce multicrispr, an R/bioconductor tool, intended to design individual gRNAs and complex gRNA libraries. The package is easy to use; detects, scores, and filters gRNAs on both efficiency and specificity; visualizes and aggregates results per target or CRISPR/Cas9 sequence; and finally returns both genomic ranges and sequences of gRNAs. To be generic, multicrispr defines and implements a genomic arithmetic framework as a basis for facile adaptation to techniques recently introduced such as prime editing or yet to arise. Its performance and design concepts such as target set–specific filtering render multicrispr a tool of choice when dealing with screening-like approaches.
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Binda, Caroline S., Bep Klaver, Ben Berkhout y Atze T. Das. "CRISPR-Cas9 Dual-gRNA Attack Causes Mutation, Excision and Inversion of the HIV-1 Proviral DNA". Viruses 12, n.º 3 (18 de marzo de 2020): 330. http://dx.doi.org/10.3390/v12030330.
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Although several studies demonstrated that the HIV proviral DNA can be effectively targeted and inactivated by the CRISPR-Cas9 system, the precise inactivation mechanism has not yet been analyzed. Whereas some studies suggested efficient proviral DNA excision upon dual-gRNA/Cas9 treatment, we previously demonstrated that hypermutation of the target sites correlated with permanent virus inactivation. To better understand the mechanism underlying HIV inactivation, we analyzed the proviral DNA upon Cas9 attack with gRNA pairs. We observed that dual-gRNA targeting resulted more frequently in target site mutation than fragment excision, while fragment inversion was rarely observed. The frequencies varied for different gRNA combinations without an obvious relationship with the distance between the target sites, indicating that other gRNA and target DNA characteristics influence the DNA cleavage and repair processes.
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Menees, Thomas M. "RNA Lariat Debranching Enzyme as a Retroviral and Long-Terminal-Repeat Retrotransposon Host Factor". Annual Review of Virology 7, n.º 1 (29 de septiembre de 2020): 189–202. http://dx.doi.org/10.1146/annurev-virology-012720-094902.
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Host cell factors are integral to viral replication. Human immunodeficiency virus 1 (HIV-1), the retroviral agent of acquired immune deficiency syndrome, requires several host factors for reverse transcription of the viral genomic RNA (gRNA) into DNA shortly after viral entry. One of these host factors is the RNA lariat debranching enzyme (Dbr1), which cleaves the 2′–5′ bond of branched and lariat RNAs. A recent study has revealed that Dbr1 cleaves HIV-1 gRNA lariats that form early after viral entry. Without Dbr1 activity, HIV-1 reverse transcription stalls, consistent with blockage of viral reverse transcriptase at gRNA branch points. These findings echo an earlier study with the long-terminal-repeat retrotransposon of Saccharomyces cerevisiae, Ty1, which is a retrovirus model. Currently, branching and debranching of viral gRNA are not widely recognized as features of HIV-1 replication, and the role of a gRNA lariat is not known. Future studies will determine whether these gRNA dynamics represent fundamental features of retroviral biology and whether they occur for other positive-sense RNA viruses.
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Breunig, Christopher T., Tamara Durovic, Andrea M. Neuner, Valentin Baumann, Maximilian F. Wiesbeck, Anna Köferle, Magdalena Götz, Jovica Ninkovic y Stefan H. Stricker. "One step generation of customizable gRNA vectors for multiplex CRISPR approaches through string assembly gRNA cloning (STAgR)". PLOS ONE 13, n.º 4 (27 de abril de 2018): e0196015. http://dx.doi.org/10.1371/journal.pone.0196015.
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Arakawa, Hiroshi. "A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism". Science Advances 2, n.º 8 (agosto de 2016): e1600699. http://dx.doi.org/10.1126/sciadv.1600699.
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The clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system is a powerful tool for genome editing that can be used to construct a guide RNA (gRNA) library for genetic screening. For gRNA design, one must know the sequence of the 20-mer flanking the protospacer adjacent motif (PAM), which seriously impedes experimentally making gRNA. I describe a method to construct a gRNA library via molecular biology techniques without relying on bioinformatics. Briefly, one synthesizes complementary DNA from the mRNA sequence using a semi-random primer containing a PAM complementary sequence and then cuts out the 20-mer adjacent to the PAM using type IIS and type III restriction enzymes to create a gRNA library. The described approach does not require prior knowledge about the target DNA sequences, making it applicable to any species.
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Rice, Breanna L., Timothy L. Lochmann y Leslie J. Parent. "RNA-Binding Domains of Heterologous Viral Proteins Substituted for Basic Residues in the RSV Gag NC Domain Restore Specific Packaging of Genomic RNA". Viruses 12, n.º 4 (27 de marzo de 2020): 370. http://dx.doi.org/10.3390/v12040370.
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The Rous sarcoma virus Gag polyprotein transiently traffics through the nucleus, which is required for efficient incorporation of the viral genomic RNA (gRNA) into virus particles. Packaging of gRNA is mediated by two zinc knuckles and basic residues located in the nucleocapsid (NC) domain in Gag. To further examine the role of basic residues located downstream of the zinc knuckles in gRNA encapsidation, we used a gain-of-function approach. We replaced a basic residue cluster essential for gRNA packaging with heterologous basic residue motif (BR) with RNA-binding activity from either the HIV-1 Rev protein (Rev BR) or the HSV ICP27 protein (ICP27 BR). Compared to wild-type Gag, the mutant ICP27 BR and Rev BR Gag proteins were much more strongly localized to the nucleus and released significantly lower levels of virus particles. Surprisingly, both the ICP27 BR and Rev BR mutants packaged normal levels of gRNA per virus particle when examined in the context of a proviral vector, yet both mutants were noninfectious. These results support the hypothesis that basic residues located in the C-terminal region of NC are required for selective gRNA packaging, potentially by binding non-specifically to RNA via electrostatic interactions.
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Garbitt-Hirst, Rachel, Scott P. Kenney y Leslie J. Parent. "Genetic Evidence for a Connection between Rous Sarcoma Virus Gag Nuclear Trafficking and Genomic RNA Packaging". Journal of Virology 83, n.º 13 (15 de abril de 2009): 6790–97. http://dx.doi.org/10.1128/jvi.00101-09.
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ABSTRACT The packaging of retroviral genomic RNA (gRNA) requires cis-acting elements within the RNA and trans-acting elements within the Gag polyprotein. The packaging signal ψ, at the 5′ end of the viral gRNA, binds to Gag through interactions with basic residues and Cys-His box RNA-binding motifs in the nucleocapsid. Although specific interactions between Gag and gRNA have been demonstrated previously, where and when they occur is not well understood. We discovered that the Rous sarcoma virus (RSV) Gag protein transiently localizes to the nucleus, although the roles of Gag nuclear trafficking in virus replication have not been fully elucidated. A mutant of RSV (Myr1E) with enhanced plasma membrane targeting of Gag fails to undergo nuclear trafficking and also incorporates reduced levels of gRNA into virus particles compared to those in wild-type particles. Based on these results, we hypothesized that Gag nuclear entry might facilitate gRNA packaging. To test this idea by using a gain-of-function genetic approach, a bipartite nuclear localization signal (NLS) derived from the nucleoplasmin protein was inserted into the Myr1E Gag sequence (generating mutant Myr1E.NLS) in an attempt to restore nuclear trafficking. Here, we report that the inserted NLS enhanced the nuclear localization of Myr1E.NLS Gag compared to that of Myr1E Gag. Also, the NLS sequence restored gRNA packaging to nearly wild-type levels in viruses containing Myr1E.NLS Gag, providing genetic evidence linking nuclear trafficking of the retroviral Gag protein with gRNA incorporation.
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Seraj, Zeba I. y Sabrina Hque. "Remodelling of a bacterial immune system as the simple gene editing tool, Crispr-Cas, for food security and human health". Journal of Bangladesh Academy of Sciences 45, n.º 2 (27 de enero de 2022): 131–45. http://dx.doi.org/10.3329/jbas.v45i2.57208.
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The CRISPR system consists of a guide RNA (gRNA) complementary to a target editable DNA sequence and a CRISPR-associated endonuclease (Cas). The gRNA and Cas together form a ribonucleoprotein (RNP) complex. The gRNA guides the Cas enzyme to the precise site for cutting the target DNA. In bacteria, the gRNA leads the endonuclease to the viral DNA for destruction. This ingenious bacterial immune principle has been used to design gRNA to target an organism’s genome at precise locations for gene editing purposes. Gene edits may include deletion or insertion, repression or activation, base, and epigenome editing, or nucleotide replacement. Therefore the CRISPR-Cas system has created the potential for altering genomes of microbes, plants, and animals. The CRISPR-Cas system has now been developed for use in many applications like finding functions of genes, searching for drug targets, diagnostics, crop improvement, and gene therapy. J. Bangladesh Acad. Sci. 45(2); 131-145: December 2021
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Mayes, Cathryn Michelle y Joshua Santarpia. "Evaluating the Impact of gRNA SNPs in CasRx Activity for Reducing Viral RNA in HCoV-OC43". Cells 11, n.º 12 (7 de junio de 2022): 1859. http://dx.doi.org/10.3390/cells11121859.
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Viruses within a given family often share common essential genes that are highly conserved due to their critical role for the virus’s replication and survival. In this work, we developed a proof-of-concept for a pan-coronavirus CRISPR effector system by designing CRISPR targets that are cross-reactive among essential genes of different human coronaviruses (HCoV). We designed CRISPR targets for both the RNA-dependent RNA polymerase (RdRp) gene as well as the nucleocapsid (N) gene in coronaviruses. Using sequencing alignment, we determined the most highly conserved regions of these genes to design guide RNA (gRNA) sequences. In regions that were not completely homologous among HCoV species, we introduced mismatches into the gRNA sequence and tested the efficacy of CasRx, a Cas13d type CRISPR effector, using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in HCoV-OC43. We evaluated the effect that mismatches in gRNA sequences has on the cleavage activity of CasRx and found that this CRISPR effector can tolerate up to three mismatches while still maintaining its nuclease activity in HCoV-OC43 viral RNA. This work highlights the need to evaluate off-target effects of CasRx with gRNAs containing up to three mismatches in order to design safe and effective CRISPR experiments.
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Cifuentes-Rojas, Catherine, Paula Pavia, Alfredo Hernandez, Daniel Osterwisch, Concepcion Puerta y Jorge Cruz-Reyes. "Substrate Determinants for RNA Editing and Editing Complex Interactions at a Site for Full-round U Insertion". Journal of Biological Chemistry 282, n.º 7 (7 de diciembre de 2006): 4265–76. http://dx.doi.org/10.1074/jbc.m605554200.
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Multisubunit RNA editing complexes catalyze uridylate insertion/deletion RNA editing directed by complementary guide RNAs (gRNAs). Editing in trypanosome mitochondria is transcript-specific and developmentally controlled, but the molecular mechanisms of substrate specificity remain unknown. Here we used a minimal A6 pre-mRNA/gRNA substrate to define functional determinants for full-round insertion and editing complex interactions at the editing site 2 (ES2). Editing begins with pre-mRNA cleavage within an internal loop flanked by upstream and downstream duplexes with gRNA. We found that substrate recognition around the internal loop is sequence-independent and that completely artificial duplexes spanning a single helical turn are functional. Furthermore, after our report of cross-linking interactions at the deletion ES1 (35), we show for the first time editing complex contacts at an insertion ES. Our studies using site-specific ribose 2′ substitutions defined 2′-hydroxyls within the (a) gRNA loop region and (b) flanking helixes that markedly stimulate both pre-mRNA cleavage and editing complex interactions at ES2. Modification of the downstream helix affected scissile bond specificity. Notably, a single 2′-hydroxyl at ES2 is essential for cleavage but dispensable for editing complex cross-linking. This study provides new insights on substrate recognition during full-round editing, including the relevance of secondary structure and the first functional association of specific (pre-mRNA and gRNA) riboses with both endonuclease cleavage and cross-linking activities of editing complexes at an ES. Importantly, most observed cross-linking interactions are both conserved and relatively stable at ES2 and ES1 in hybrid substrates. However, they were also detected as transient low-stability contacts in a non-edited transcript.
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Sarni, Samantha, Banhi Biswas, Shuohui Liu, Erik D. Olson, Jonathan P. Kitzrow, Alan Rein, Vicki H. Wysocki y Karin Musier-Forsyth. "HIV-1 Gag protein with or without p6 specifically dimerizes on the viral RNA packaging signal". Journal of Biological Chemistry 295, n.º 42 (13 de agosto de 2020): 14391–401. http://dx.doi.org/10.1074/jbc.ra120.014835.
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The HIV-1 Gag protein is responsible for genomic RNA (gRNA) packaging and immature viral particle assembly. Although the presence of gRNA in virions is required for viral infectivity, in its absence, Gag can assemble around cellular RNAs and form particles resembling gRNA-containing particles. When gRNA is expressed, it is selectively packaged despite the presence of excess host RNA, but how it is selectively packaged is not understood. Specific recognition of a gRNA packaging signal (Psi) has been proposed to stimulate the efficient nucleation of viral assembly. However, the heterogeneity of Gag–RNA interactions renders capturing this transient nucleation complex using traditional structural biology approaches challenging. Here, we used native MS to investigate RNA binding of wild-type (WT) Gag and Gag lacking the p6 domain (GagΔp6). Both proteins bind to Psi RNA primarily as dimers, but to a control RNA primarily as monomers. The dimeric complexes on Psi RNA require an intact dimer interface within Gag. GagΔp6 binds to Psi RNA with high specificity in vitro and also selectively packages gRNA in particles produced in mammalian cells. These studies provide direct support for the idea that Gag binding to Psi specifically promotes nucleation of Gag–Gag interactions at the early stages of immature viral particle assembly in a p6-independent manner.