CRISPR guides induce gene silencing in plants in the absence of Cas

Abstract Background RNA-targeting CRISPR-Cas can provide potential advantages over DNA editing, such as avoiding pleiotropic effects of genome editing, providing precise spatiotemporal regulation, and expanded function including antiviral immunity. Results Here, we report the use of CRISPR-Cas13 in plants to reduce both viral and endogenous RNA. Unexpectedly, we observe that crRNA designed to guide Cas13 could, in the absence of the Cas13 protein, cause substantial reduction in RNA levels as well. We demonstrate Cas13-independent guide-induced gene silencing (GIGS) in three plant species, including stable transgenic Arabidopsis. Small RNA sequencing during GIGS identifies the production of small RNA that extend beyond the crRNA expressed sequence in samples expressing multi-guide crRNA. Additionally, we demonstrate that mismatches in guide sequences at position 10 and 11 abolish GIGS. Finally, we show that GIGS is elicited by guides that lack the Cas13 direct repeat and can extend to Cas9 designed crRNA of at least 28 base pairs, indicating that GIGS can be elicited through a variety of guide designs and is not dependent on Cas13 crRNA sequences or design. Conclusions Collectively, our results suggest that GIGS utilizes endogenous RNAi machinery despite the fact that crRNA are unlike canonical triggers of RNAi such as miRNA, hairpins, or long double-stranded RNA. Given similar evidence of Cas13-independent silencing in an insect system, it is likely GIGS is active across many eukaryotes. Our results show that GIGS offers a novel and flexible approach to RNA reduction with potential benefits over existing technologies for crop improvement and functional genomics.

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RNA silencing by CRISPR in plants does not require Cas13

10.1101/2021.05.20.445036 ◽

2021 ◽

Author(s):

Veerendra Sharma ◽

Sandeep R Marla ◽

Wenguang Zheng ◽

Divya Mishra ◽

Jun Huang ◽

...

Keyword(s):

Plant Species ◽

Crop Improvement ◽

Substantial Reduction ◽

Viral Immunity ◽

Rnai Machinery ◽

Double Stranded Rna ◽

Spatiotemporal Regulation ◽

Rna Targeting ◽

Potential Benefits ◽

Dna Editing

RNA-targeting CRISPR-Cas can provide potential advantages over DNA editing, such as avoiding pleiotropic effects of genome editing, providing precise spatiotemporal regulation and expanded function including anti-viral immunity. Here, we report the use of CRISPR-Cas13 in plants to reduce both viral and endogenous RNA. Unexpectedly, we discovered that crRNA designed to guide Cas13 could, in the absence of the Cas13 protein, cause substantial reduction in RNA levels as well. We demonstrate Cas13-independent guide-induced gene silencing (GIGS) in three plant species, including stable transgenic Arabidopsis. We determined that GIGS utilizes endogenous RNAi machinery despite the fact that crRNA are unlike canonical triggers of RNAi such as miRNA, hairpins or long double-stranded RNA. These results suggest that GIGS offers a novel and flexible approach to RNA reduction with potential benefits over existing technologies for crop improvement. Our results demonstrate that GIGS is active across a range of plant species, evidence similar to recent findings in an insect system, which suggests that GIGS is potentially active across many eukaryotes.

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Expression and processing of polycistronic artificial microRNAs and trans-acting siRNAs in Solanum lycopersicum and Nicotiana benthamiana

10.1101/2021.01.06.425596 ◽

2021 ◽

Author(s):

Alice Lunardon ◽

Samwel Muiruri Kariuki ◽

Michael J. Axtell

Keyword(s):

Gene Silencing ◽

Solanum Lycopersicum ◽

Rna Sequencing ◽

Small Rna ◽

Nicotiana Benthamiana ◽

Small Rna Sequencing ◽

Artificial Microrna ◽

Single Target ◽

Small Regulatory Rnas ◽

Interfering Rna

AbstractTargeted gene silencing using small regulatory RNAs is a widely used technique for genetic studies in plants. Artificial microRNAs are one common approach; they have the advantage of producing just a single functional small RNA which can be designed for high target specificity and low off-target effects. Simultaneous silencing of multiple targets with artificial microRNAs can be achieved by producing polycistronic microRNA precursors. Alternatively, specialized trans-acting short interfering RNA (tasiRNA) precursors can be designed to produce several specific tasiRNAs at once. Here we tested several artificial microRNA- and tasiRNA-based methods for multiplexed gene silencing in Solanum lycopersicum (tomato) and Nicotiana benthamiana. Small RNA sequencing analyses revealed that many previously described approaches resulted in poor small RNA processing. The 5’-most microRNA precursor hairpins on polycistronic artificial microRNA precursors were generally processed more accurately than precursors at the 3’ end. Polycistronic artificial microRNAs where the hairpin precursors were separated by transfer RNAs had the best processing precision. Strikingly, artificial tasiRNA precursors failed to be processed in the expected phased manner in our system. These results highlight the need for further development of multiplexed artificial microRNA and tasiRNA strategies. The importance of small RNA sequencing, as opposed to single-target assays such as RNA blots or real-time PCR, is also discussed.Significance statementSeveral strategies for multiplexed gene silencing using artificial microRNAs or tasiRNAs have been described. We find that many result in imprecise processing, and thus low accumulation of the intended small RNAs. Our findings highlight the importance of small RNA sequencing to fully analyze gene silencing experiments, and also the need for continued methodological development of these methods.

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Faculty Opinions recommendation of Identification of nutrient-responsive Arabidopsis and rapeseed microRNAs by comprehensive real-time polymerase chain reaction profiling and small RNA sequencing.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1161246.621733 ◽

2009 ◽

Author(s):

Alain Gojon

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Rna Sequencing ◽

Small Rna ◽

Small Rna Sequencing ◽

Chain Reaction ◽

Polymerase Chain

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Faculty Opinions recommendation of E-cadherin is transcriptionally activated via suppression of ZEB1 transcriptional repressor by small RNA-mediated gene silencing.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13436964.14811067 ◽

2012 ◽

Author(s):

Marjori Matzke

Keyword(s):

Gene Silencing ◽

Small Rna ◽

Transcriptional Repressor ◽

E Cadherin

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Analysis of miRNAs Targeted Storage Regulatory Genes during Soybean Seed Development Based on Transcriptome Sequencing

Genes ◽

10.3390/genes10060408 ◽

2019 ◽

Vol 10 (6) ◽

pp. 408 ◽

Cited By ~ 2

Author(s):

Jing-Yao Yu ◽

Zhan-Guo Zhang ◽

Shi-Yu Huang ◽

Xue Han ◽

Xin-Yu Wang ◽

...

Keyword(s):

Expression Pattern ◽

Seed Development ◽

Small Rna ◽

Target Genes ◽

Soybean Seed ◽

Regulatory Genes ◽

Small Rna Sequencing ◽

Grain Development ◽

Regulatory Relationship ◽

Novel Mirna

Soybeans are an important cash crop and are widely used as a source of vegetable protein and edible oil. MicroRNAs (miRNA) are endogenous small RNA that play an important regulatory role in the evolutionarily conserved system of gene expression. In this study, we selected four lines with extreme phenotypes, as well as high or low protein and oil content, from the chromosome segment substitution line (CSSL) constructed from suinong (SN14) and ZYD00006, and planted and sampled at three stages of grain development for small RNA sequencing and expression analysis. The sequencing results revealed the expression pattern of miRNA in the materials, and predicted miRNA-targeted regulatory genes, including 1967 pairs of corresponding relationships between known-miRNA and their target genes, as well as 597 pairs of corresponding relationships between novel-miRNA and their target genes. After screening and annotating genes that were targeted for regulation, five specific genes were identified to be differentially expressed during seed development and subsequently analyzed for their regulatory relationship with miRNAs. The expression pattern of the targeted gene was verified by Real-time Quantitative PCR (RT-qPCR). Our research provides more information about the miRNA regulatory network in soybeans and further identifies useful genes that regulate storage during soy grain development, providing a theoretical basis for the regulation of soybean quality traits.

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Small RNA Sequencing to Discover Circulating MicroRNA Biomarkers of Testicular Toxicity in Dogs

International Journal of Toxicology ◽

10.1177/1091581820961515 ◽

2020 ◽

pp. 109158182096151

Author(s):

Jennifer C. Shing ◽

Kai Schaefer ◽

Shaun E. Grosskurth ◽

Andy H. Vo ◽

Tatiana Sharapova ◽

...

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

Exploratory Study ◽

Quantitative Polymerase Chain Reaction ◽

Small Rna Sequencing ◽

Circulating Microrna ◽

Testicular Toxicity ◽

Potential Candidate ◽

Drug Induced ◽

Organ Systems

Predictive indicators of testicular toxicity could improve drug development by allowing early in-life screening for this adverse effect before it becomes severe. We hypothesized that circulating microRNAs (miRNAs) could serve as testicular toxicity biomarkers in dogs. Herein, we describe the results of an exploratory study conducted to discover biomarkers of drug-induced testicular injury. Following a dose-selection study using the testicular toxicant ethylene glycol monomethyl ether (EGME), we chose a dose of 50 mg/kg/d EGME to avoid systemic toxicity and treated 2 groups of dogs (castrated, non-castrated) for 14 to 28 days. Castrated animals were used as negative controls to identify biomarkers specific for testicular toxicity because EGME can cause toxicity to organ systems in addition to the testis. Blood was collected daily during the dosing period, followed by recovery for 29 to 43 days with less frequent sampling. Dosing was well tolerated, resulting in mild-to-moderate degeneration in testes and epididymides. Global profiling of serum miRNAs at selected dosing and recovery time points was completed by small RNA sequencing. Bioinformatics data analysis using linear modeling demonstrated several circulating miRNAs that were differentially abundant during the dosing period compared with baseline and/or castrated control samples. Confirmatory reverse transcription quantitative polymerase chain reaction data in these animals was unable to detect sustained alterations of miRNAs in serum, except for 1 potential candidate cfa-miR-146b. Taken together, we report the results of a comprehensive exploratory study and suggest future directions for follow-up research to address the challenge of developing diagnostic biomarkers of testicular toxicity.

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Small RNA sequencing reveals distinct nuclear microRNAs in pig granulosa cells during ovarian follicle growth

Journal of Ovarian Research ◽

10.1186/s13048-021-00802-3 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Derek Toms ◽

Bo Pan ◽

Yinshan Bai ◽

Julang Li

Keyword(s):

Granulosa Cells ◽

Small Rna ◽

High Throughput Sequencing ◽

Ovarian Function ◽

Ovarian Follicle ◽

Small Rna Sequencing ◽

Cell Fractionation ◽

Steroid Synthesis ◽

Non Coding Rna ◽

Dna Binding Sites

AbstractNuclear small RNAs have emerged as an important subset of non-coding RNA species that are capable of regulating gene expression. A type of small RNA, microRNA (miRNA) have been shown to regulate development of the ovarian follicle via canonical targeting and translational repression. Little has been done to study these molecules at a subcellular level. Using cell fractionation and high throughput sequencing, we surveyed cytoplasmic and nuclear small RNA found in the granulosa cells of the pig ovarian antral preovulatory follicle. Bioinformatics analysis revealed a diverse network of small RNA that differ in their subcellular distribution and implied function. We identified predicted genomic DNA binding sites for nucleus-enriched miRNAs that may potentially be involved in transcriptional regulation. The small nucleolar RNA (snoRNA) SNORA73, known to be involved in steroid synthesis, was also found to be highly enriched in the cytoplasm, suggesting a role for snoRNA species in ovarian function. Taken together, these data provide an important resource to study the small RNAome in ovarian follicles and how they may impact fertility.

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Extensive Analysis of miRNA Trimming and Tailing Indicates that AGO1 Has a Complex Role in miRNA Turnover

Plants ◽

10.3390/plants10020267 ◽

2021 ◽

Vol 10 (2) ◽

pp. 267

Author(s):

Axel J. Giudicatti ◽

Ariel H. Tomassi ◽

Pablo A. Manavella ◽

Agustin L. Arce

Keyword(s):

Small Rna ◽

Stress Responses ◽

Cellular Localization ◽

Mirna Biogenesis ◽

Small Rna Sequencing ◽

Sequencing Data ◽

Extensive Analysis ◽

Small Regulatory Rnas ◽

Regulatory Rnas ◽

Argonaute 1

MicroRNAs are small regulatory RNAs involved in several processes in plants ranging from development and stress responses to defense against pathogens. In order to accomplish their molecular functions, miRNAs are methylated and loaded into one ARGONAUTE (AGO) protein, commonly known as AGO1, to stabilize and protect the molecule and to assemble a functional RNA-induced silencing complex (RISC). A specific machinery controls miRNA turnover to ensure the silencing release of targeted-genes in given circumstances. The trimming and tailing of miRNAs are fundamental modifications related to their turnover and, hence, to their action. In order to gain a better understanding of these modifications, we analyzed Arabidopsis thaliana small RNA sequencing data from a diversity of mutants, related to miRNA biogenesis, action, and turnover, and from different cellular fractions and immunoprecipitations. Besides confirming the effects of known players in these pathways, we found increased trimming and tailing in miRNA biogenesis mutants. More importantly, our analysis allowed us to reveal the importance of ARGONAUTE 1 (AGO1) loading, slicing activity, and cellular localization in trimming and tailing of miRNAs.

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Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis

Diagnostics ◽

10.3390/diagnostics11060964 ◽

2021 ◽

Vol 11 (6) ◽

pp. 964

Author(s):

Sarka Benesova ◽

Mikael Kubista ◽

Lukas Valihrach

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

High Sensitivity ◽

Small Rna Sequencing ◽

Rna Seq ◽

Liquid Biopsies ◽

Comprehensive Overview ◽

Rna Molecules ◽

Novel Mirna ◽

The Many

MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol’s performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.

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