Ribonucleoprotein Complexes
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2022 ◽  
Thomas Swartjes ◽  
Peng Shang ◽  
Dennis van den Berg ◽  
Tim A. Kunne ◽  
Niels Geijsen ◽  

CRISPR-Cas has revolutionized genome editing and has a great potential for applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. Here we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides down-regulate Cas9 activity in human cells, reducing both on and off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: the length of the complementary region, and the presence of a PAM-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.

BMC Genomics ◽  
2022 ◽  
Vol 23 (1) ◽  
Chenna Swetha ◽  
Anushree Narjala ◽  
Awadhesh Pandit ◽  
Varsha Tirumalai ◽  
P. V. Shivaprasad

Abstract Background Small non-coding (s)RNAs are involved in the negative regulation of gene expression, playing critical roles in genome integrity, development and metabolic pathways. Targeting of RNAs by ribonucleoprotein complexes of sRNAs bound to Argonaute (AGO) proteins results in cleaved RNAs having precise and predictable 5` ends. While tools to study sliced bits of RNAs to confirm the efficiency of sRNA-mediated regulation are available, they are sub-optimal. In this study, we provide an improvised version of a tool with better efficiency to accurately validate sRNA targets. Results Here, we improvised the CleaveLand tool to identify additional micro (mi)RNA targets that belong to the same family and also other targets within a specified free energy cut-off. These additional targets were otherwise excluded during the default run. We employed these tools to understand the sRNA targeting efficiency in wild and cultivated rice, sequenced degradome from two rice lines, O. nivara and O. sativa indica Pusa Basmati-1 and analyzed variations in sRNA targeting. Our results indicate the existence of multiple miRNA-mediated targeting differences between domesticated and wild species. For example, Os5NG4 was targeted only in wild rice that might be responsible for the poor secondary wall formation when compared to cultivated rice. We also identified differential mRNA targets of secondary sRNAs that were generated after miRNA-mediated cleavage of primary targets. Conclusions We identified many differentially targeted mRNAs between wild and domesticated rice lines. In addition to providing a step-wise guide to generate and analyze degradome datasets, we showed how domestication altered sRNA-mediated cascade silencing during the evolution of indica rice.

2022 ◽  
Vol 8 (1) ◽  
pp. 1
Tom Dielforder ◽  
Christina Maria Braun ◽  
Fabian Hölzgen ◽  
Shuang Li ◽  
Mona Thiele ◽  

The synthesis of ribosomes involves the correct folding of the pre-ribosomal RNA within pre-ribosomal particles. The first ribosomal precursor or small subunit processome assembles stepwise on the nascent transcript of the 35S gene. At the earlier stages, the pre-ribosomal particles undergo structural and compositional changes, resulting in heterogeneous populations of particles with highly flexible regions. Structural probing methods are suitable for resolving these structures and providing evidence about the architecture of ribonucleoprotein complexes. Our approach used MNase tethered to the assembly factors Nan1/Utp17, Utp10, Utp12, and Utp13, which among other factors, initiate the formation of the small subunit processome. Our results provide dynamic information about the folding of the pre-ribosomes by elucidating the relative organization of the 5′ETS and ITS1 regions within the 35S and U3 snoRNA around the C-terminal domains of Nan1/Utp17, Utp10, Utp12, and Utp13.

2021 ◽  
Manuel Albanese ◽  
Adrian Ruhle ◽  
Jennifer Mittermaier ◽  
Ernesto Mejías-Pérez ◽  
Madeleine Gapp ◽  

AbstractCD4+ T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4+ T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4+ T cells using optimized cell cultivation and nucleofection conditions of Cas9–guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4+ T cells.

RNA ◽  
2021 ◽  
pp. rna.079025.121
Joshua J Black ◽  
Arlen W Johnson

Ribosomes are the universally conserved ribonucleoprotein complexes that synthesize proteins. The two subunits of the eukaryotic ribosome are produced through a quasi-independent assembly-line-like pathway involving the hierarchical actions of numerous trans-acting biogenesis factors and the incorporation of ribosomal proteins. The factors work together to shape the nascent subunits through a series of intermediate states into their functional architectures. The earliest intermediate of the small subunit (SSU or 40S) is the SSU Processome which is subsequently transformed into the pre-40S intermediate. This transformation is, in part, facilitated by the binding of the methyltransferase Bud23. How Bud23 is released from the resultant pre-40S is not known. The ribosomal proteins Rps0, Rps2, and Rps21, termed the Rps0-cluster proteins, and several biogenesis factors are known to bind the pre-40S around the time that Bud23 is released, suggesting that one or more of these factors induce Bud23 release. Here, we systematically examined the requirement of these factors for the release of Bud23 from pre-40S particles. We found that the Rps0-cluster proteins are needed but not sufficient for Bud23 release. The atypical kinase/ATPase Rio2 shares a binding site with Bud23 and is thought to be recruited to pre-40S after the Rps0-cluster proteins. Depletion of Rio2 prevented the release of Bud23 from the pre-40S. More importantly, the addition of recombinant Rio2 to pre-40S particles affinity-purified from Rio2-depleted cells was sufficient for Bud23 release in vitro. The ability of Rio2 to displace Bud23 was independent of nucleotide hydrolysis. We propose a novel role for Rio2 in which its binding to the pre-40S actively displaces Bud23 from the pre-40S, and we suggest a model in which the binding of the Rps0-cluster proteins and Rio2 promote the release of Bud23.

2021 ◽  
Christian Shema Mugisha ◽  
Tung Dinh ◽  
Kasyap Tenneti ◽  
Jenna Eve Eschbach ◽  
Keanu Davis ◽  

Independent of its catalytic activity, HIV-1 integrase (IN) enzyme regulates proper particle maturation by binding to and packaging the viral RNA genome (gRNA) inside the mature capsid lattice. Allosteric integrase inhibitors (ALLINIs) and class II IN substitutions inhibit the binding of IN to the gRNA and cause the formation of non-infectious virions characterized by mislocalization of the viral ribonucleoprotein complexes between the translucent conical capsid lattice and the viral lipid envelope. To gain insight into the molecular nature of IN-gRNA interactions, we have isolated compensatory substitutions in the background of a class II IN (R269A/K273A) variant that directly inhibits IN binding to the gRNA. We found that additional D256N and D270N substitutions in the C-terminal domain (CTD) of IN restored its ability to bind gRNA and led to the formation of infectious particles with correctly matured morphology. Furthermore, reinstating the overall positive electrostatic potential of the CTD through individual D256R or D256K substitutions was sufficient to restore IN-RNA binding and infectivity for the R269A/K273A as well as the R262A/R263A class II IN mutants. The compensatory mutations did not impact functional IN oligomerization, suggesting that they directly contributed to IN binding to the gRNA. Interestingly, HIV-1 IN R269A/K273A, but not IN R262A/R263A, bearing compensatory mutations was more sensitive to ALLINIs providing key genetic evidence that specific IN residues required for RNA binding also influence ALLINI activity. Structural modeling provided further insight into the molecular nature of IN-gRNA interactions and ALLINI mechanism of action. Taken together, our findings highlight an essential role of IN-gRNA interactions for proper virion maturation and reveal the importance of electrostatic interactions between the IN CTD and the gRNA.

2021 ◽  
Vol 11 (1) ◽  
Maki Hirata ◽  
Manita Wittayarat ◽  
Zhao Namula ◽  
Quynh Anh Le ◽  
Qingyi Lin ◽  

AbstractThe specificity and efficiency of CRISPR/Cas9 gene-editing systems are determined by several factors, including the mode of delivery, when applied to mammalian embryos. Given the limited time window for delivery, faster and more reliable methods to introduce Cas9-gRNA ribonucleoprotein complexes (RNPs) into target embryos are needed. In pigs, somatic cell nuclear transfer using gene-modified somatic cells and the direct introduction of gene editors into the cytoplasm of zygotes/embryos by microinjection or electroporation have been used to generate gene-edited embryos; however, these strategies require expensive equipment and sophisticated techniques. In this study, we developed a novel lipofection-mediated RNP transfection technique that does not require specialized equipment for the generation of gene-edited pigs and produced no detectable off-target events. In particular, we determined the concentration of lipofection reagent for efficient RNP delivery into embryos and successfully generated MSTN gene-edited pigs (with mutations in 7 of 9 piglets) after blastocyst transfer to a recipient gilt. This newly established lipofection-based technique is still in its early stages and requires improvements, particularly in terms of editing efficiency. Nonetheless, this practical method for rapid and large-scale lipofection-mediated gene editing in pigs has important agricultural and biomedical applications.

2021 ◽  
Rajika Arora ◽  
Maxime Bodak ◽  
Laura Penouty ◽  
Cindy Hackmann ◽  
Constance Ciaudo

LINE-1 (L1) are autonomous retroelements that have retained their ability to mobilize. Mechanisms regulating L1 mobility include DNA methylation in somatic cells and the Piwi-interacting RNA pathway in the germline. During pre-implantation stages of mouse embryonic development, however, both pathways are inactivated leading to a critical window necessitating alternate means of L1 regulation. We previously reported an increase in L1 levels in Dicer_KO mouse embryonic stem cells (mESCs). Intriguingly this was accompanied by only a marginal increase in retrotransposition, suggestive of additional mechanisms suppressing L1 mobility. Here, we demonstrate that L1 Ribonucleoprotein complexes (L1 RNP) accumulate as aggregates in Dicer_KO cytoplasm along with the RNA helicase MOV10. The combined overexpression of L1 RNAs and MOV10 is sufficient to create L1 RNP aggregates in stem cells. In Dicer_KO mESCs, MOV10 is upregulated due to the loss of its direct regulation by miRNAs. The newly discovered post-transcriptional regulation of Mov10 expression, and its role in preventing L1 retrotransposition by driving novel cytosolic aggregation affords alternate routes to explore for therapy and disease progression.

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A697-A697
Ronal Peralta ◽  
Greg Delgoffe

BackgroundUpon infiltration into tumors, T cells experiencing persistent antigen stimulation progressively differentiate into a state of dysfunction, known as exhaustion. Exhausted T cells are characterized by the sustained upregulation of co-inhibitory molecules and reduced effector cytokine production. Additionally, exhausted T cells exist in a state of metabolic dysfunction in the tumor microenvironment (TME), due to disrupted mitochondrial biogenesis, hypoxia and lack of metabolites. Highly glycolytic tumor and stromal cells outcompete T cells for glucose, and secrete lactate into the TME, acidifying the extracellular space. Recent studies have shown lactate can be metabolized by tumor infiltrating Tregs and macrophages. We hypothesized that CD8+ tumor-infiltrating lymphocytes (TIL) may also take up lactate as an alternative carbon source to meet their metabolic demands.MethodsFor lactate uptake experiments, B16 melanoma single cell suspensions from B6 mice were loaded with the pH sensitive dye pHrodo, then pulsed with 5µM lactic acid. MCT11 KO OT-I T cells were generated via transfection of Slc16a11 sgRNA-Cas9 ribonucleoprotein complexes, and adoptively transferred into B16-OVA bearing mice.ResultsRNA sequencing and flow cytometry data from CD8+ T cell subsets in the TME revealed MCT11 (encoded by Slc16a11), a monocarboxylate transporter (MCT) only recently discovered, to be highly and uniquely expressed in terminally exhausted T cells (Tex). As lactate is an abundant monocarboxylate in tumors, we asked whether MCT11 supports lactate uptake into Tex cells. Antibody blockade of MCT11 resulted in reduced lactic acid uptake, but whether lactic acid promoted or inhibited effector function. Intriguingly, overexpression of MCT11 in OT-I T cells adoptively transferred into B16-OVA bearing mice resulted in accelerated exhaustion: increased co-inhibitory marker expression and decreased TNFa and IFN production. Conversely, knockdown of MCT11 in the same model resulted in decreased co-inhibitory marker expression and increased TNFa and IFN production. Further, MCT11 KO OT-I T cells used therapeutically had decreased tumor burden over mice treated with control OT-I T cells. As MCT11's uptake function was blocked with an antibody, we also used the antibody therapeutically, revealing that single-agent MCT11 antibody treatment reduced tumor burden and increased survival in B16 melanoma bearing mice.ConclusionsOur data support a model where exhausted CD8+ T cells upregulate MCT11, which renders them sensitive to toxic lactic acid in the TME. Our data suggest MCT11 could be deleted on therapeutic T cells or blocked using an antibody on endogenous T cells to render exhausted T cells impervious to lactic acid such and promote tumor eradication.

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