Global quantification exposes abundant low-level off-target activity by base editors

2021 ◽  
pp. gr.275770.121
Author(s):  
Ilana Buchumenski ◽  
Shalom Hillel Roth ◽  
Eli Kopel ◽  
Efrat Katsman ◽  
Ariel Feiglin ◽  
...  
Keyword(s):  
Computational Tool ◽  
Efficient Manner ◽  
Genomic Variations ◽  
Guide Rnas ◽  
New Class ◽  
Complementary Approach ◽  
Powerful Approach ◽  
Single Base Resolution ◽  
Target Rna ◽  
Target Activity

Base editors are dedicated engineered deaminases that enable directed conversion of specific bases in the genome or transcriptome in a precise and efficient manner, and hold promise for correcting pathogenic mutations. A major concern limiting application of this powerful approach is the issue of off-target edits. Several recent studies have shown substantial off-target RNA activity induced by base editors and demonstrated that off-target mutations may be suppressed by improved deaminases versions or optimized guide RNAs. Here we describe a new class of off-target events that are invisible to the established methods for detection of genomic variations, and were thus far overlooked. We show that much of the off-target activity of the deaminases is nonspecific, seemingly stochastic, affecting a large number of sites throughout the genome or the transcriptome and accounting for the majority of off-target activity. We develop and employ a different, complementary, approach that is sensitive to the stochastic off-targets activity, and use it to quantify the abundant off-target RNA mutations due to current optimized deaminase editors. Engineered base editors enable directed manipulation of the genome or transcriptome at single-base resolution. We believe that implementation of this computational approach would facilitate design of more specific base editors. We provide a computational tool to quantify global off-target activity, which can be used to optimize future base editors.

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

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

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

Approximation of Crossing Intensities for Non Linear Responses Subjected to Non Gaussian Loadings

2010 ◽  
Author(s):  
Thomas Galtier ◽  
Sayan Gupta ◽  
Igor Rychlik
Keyword(s):  
Heavy Tails ◽  
Moving Average ◽  
Ocean Waves ◽  
Response Characteristic ◽  
Second Order ◽  
Computationally Efficient ◽  
Marine Structures ◽  
Efficient Manner ◽  
New Class ◽  
Non Gaussian

Crossing intensity constitute an important response characteristic for randomly vibrating structures, especially if one is interested in estimating the risk against failures. This paper focusses on developing approximations by which estimates of the crossing intensities for response of marine structures can be obtained in a computationally efficient manner, when the loads are modeled as a special class of non-Gaussian processes, namely as LMA processes. Ocean waves exhibit considerable non-Gaussianity as marked by their skewed marginal distributions and heavy tails. Here, a new class of processes-the Laplace driven Moving Average (LMA) processes are used to model the ocean waves. LMA processes are non-Gaussian, strictly stationary, can model in principle any spectrum and have the additional flexibility to model the skewness and the kurtosis of the marginal distribution. The structure behavior assumed is limited to quadratic systems characterized by second order kernels, which is common for marine structures. Thus, an estimation of the crossing intensities of the response involves studying the crossing characteristics of a LMA process passing through a second order filter. A new computationally efficient hybrid method, which uses the saddle point approximations along with limited Monte Carlo simulations, is developed to compute crossing intensity of the response. The proposed method is illustrated through numerical examples.

scholarly journals METTL4 catalyzes m6Am methylation in U2 snRNA to regulate pre-mRNA splicing

2020 ◽  
Vol 48 (16) ◽  
pp. 9250-9261
Author(s):  
Yeek Teck Goh ◽  
Casslynn W Q Koh ◽  
Donald Yuhui Sim ◽  
Xavier Roca ◽  
W S Sho Goh
Keyword(s):  
Rna Processing ◽  
Human Cells ◽  
Mrna Splicing ◽  
U2 Snrna ◽  
Mrna Transcripts ◽  
Single Base Resolution ◽  
Accelerated Studies ◽  
Rna Motif ◽  
Target Rna

Abstract N 6-methylation of 2′-O-methyladenosine (Am) in RNA occurs in eukaryotic cells to generate N6,2′-O-dimethyladenosine (m6Am). Identification of the methyltransferase responsible for m6Am catalysis has accelerated studies on the function of m6Am in RNA processing. While m6Am is generally found in the first transcribed nucleotide of mRNAs, the modification is also found internally within U2 snRNA. However, the writer required for catalyzing internal m6Am formation had remained elusive. By sequencing transcriptome-wide RNA methylation at single-base-resolution, we identified human METTL4 as the writer that directly methylates Am at U2 snRNA position 30 into m6Am. We found that METTL4 localizes to the nucleus and its conserved methyltransferase catalytic site is required for U2 snRNA methylation. By sequencing human cells with overexpressed Mettl4, we determined METTL4’s in vivo target RNA motif specificity. In the absence of Mettl4 in human cells, U2 snRNA lacks m6Am thereby affecting a subset of splicing events that exhibit specific features such as 3′ splice-site weakness and an increase in exon inclusion. These findings suggest that METTL4 methylation of U2 snRNA regulates splicing of specific pre-mRNA transcripts.

scholarly journals METTL4 catalyzes m6Am methylation in U2 snRNA to regulate pre-mRNA splicing

2020 ◽  
Author(s):  
Yeek Teck Goh ◽  
Casslynn W. Q. Koh ◽  
Donald Yuhui Sim ◽  
Xavier Roca ◽  
W. S. Sho Goh
Keyword(s):  
Rna Processing ◽  
Human Cells ◽  
Mrna Splicing ◽  
U2 Snrna ◽  
Mrna Transcripts ◽  
Single Base Resolution ◽  
Accelerated Studies ◽  
Rna Motif ◽  
Target Rna

AbstractN6-methylation of 2’-O-methyladenosine (Am) in RNA occurs in eukaryotic cells to generate N6,2’-O-dimethyladenosine (m6Am). Identification of the methyltransferase responsible for m6Am catalysis has accelerated studies on the function of m6Am in RNA processing. While m6Am is generally found in the first transcribed nucleotide of mRNAs, the modification is also found internally within U2 snRNA. However, the writer required for catalyzing internal m6Am formation had remained elusive. By sequencing transcriptome-wide RNA methylation at single-base-resolution, we identified human METTL4 as the writer that directly methylates Am at U2 snRNA position 30 into m6Am. We found that METTL4 localizes to the nucleus and its conserved methyltransferase catalytic site is required for U2 snRNA methylation. By sequencing human cells with overexpressed Mettl4, we determined METTL4’s in vivo target RNA motif specificity. In the absence of Mettl4 in human cells, U2 snRNA lacks m6Am thereby affecting a subset of splicing events that exhibit specific features such as overall 3’ splice-site weakness with certain motif positions more affected than others. This study establishes that METTL4 methylation of U2 snRNA regulates splicing of specific pre-mRNA transcripts.

scholarly journals CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations

2021 ◽  
Author(s):  
Ida Höijer ◽  
Anastasia Emmanouilidou ◽  
Rebecka Östlund ◽  
Robin van Schendel ◽  
Selma Bozorgpana ◽  
...  
Keyword(s):  
Juvenile Fish ◽  
Structural Variants ◽  
Mosaic Pattern ◽  
Guide Rnas ◽  
Target Sites ◽  
Long Read ◽  
Target Activity ◽  
F1 Generation

To investigate the extent and distribution of unintended mutations induced by CRISPR-Cas9 in vivo, we edited the genome of fertilized zebrafish eggs and investigated DNA from >1100 larvae, juvenile and adult fish in the F0 and F1 generations. Four guide RNAs (gRNAs) were used, selected from 23 gRNAs with high on-target efficiency in vivo in previous functional experiments. CRISPR-Cas9 outcomes were analyzed by long-read sequencing of on-target sites and off-target sites detected in vitro. In founder larvae, on-target editing of the four gRNAs was 93-97% efficient, and three sites across two gRNAs were identified with in vivo off-target editing. Seven percent of the CRISPR-Cas9 editing outcomes correspond to structural variants (SVs), i.e., insertions and deletions ≥50 bp. The adult founder fish displayed a mosaic pattern of editing events in somatic and germ cells. The F1 generation contained high levels of genome editing, with all alleles of 46 examined F1 juvenile fish affected by on-target mutations, including four cases of SVs. In addition, 26% of the juvenile F1 fish (n=12) carried off-target mutations. These CRISPR-induced off-target mutations in F1 fish were successfully validated in pooled larvae from the same founder parents. In conclusion, we demonstrate that large SVs and off-target mutations can be introduced in vivo and passed through the germline to the F1 generation. The results have important consequences for the use of CRISPR-Cas9 in clinical applications, where pre-testing for off-target activity and SVs on patient material is advisable to reduce the risk of unanticipated effects with potentially large implications.

scholarly journals CovProfile: profiling the viral genome and gene expressions of SARS-COV-2

2020 ◽  
Author(s):  
Yonghan Yu ◽  
Zhengtu Li ◽  
Yinhu Li ◽  
Le Yu ◽  
Wenlong Jia ◽  
...  
Keyword(s):  
Viral Genome ◽  
Terminally Ill ◽  
N Gene ◽  
Viral Gene ◽  
Computational Tool ◽  
M Gene ◽  
Gene Expressions ◽  
Genomic Variations ◽  
Viral Genes ◽  
Multiple Viral Strains

AbstractThe SARS-CoV-2 virus has infected more than one million people worldwide to date. Knowing its genome and gene expressions is essential to understand the virus’ mechanism. Here, we propose a computational tool CovProfile to detect the viral genomic variations as well as viral gene expressions from the sequences obtained from Nanopore devices. We applied CovProfile to 11 samples, each from a terminally ill patient, and discovered that all the patients are infected by multiple viral strains, which might affect the reliability of phylogenetic analysis. Moreover, the expression of viral genes ORF1ab gene, S gene, M gene, and N gene are high among most of the samples. While performing the tests, we noticed a consistent abundance of transcript segments of MUC5B, presumably from the host, across all the samples.

scholarly journals Specificity Principles in RNA-Guided Targeting

2016 ◽  
Author(s):  
Namita Bisaria ◽  
Inga Jarmoskaite ◽  
Daniel Herschlag
Keyword(s):  
Gene Regulation ◽  
Rna Interference ◽  
Binding Affinity ◽  
Small Rna ◽  
Kinetic Models ◽  
Specific Gene ◽  
Rational Engineering ◽  
Target Rna ◽  
Target Activity ◽  
Derived Rules

ABSTRACTRNA-guided nucleases (RGNs) provide sequence-specific gene regulation through base-pairing interactions between a small RNA guide and target RNA or DNA. RGN systems, which include CRISPR-Cas9 and RNA interference (RNAi), hold tremendous promise as programmable tools for engineering and therapeutic purposes. However, pervasive targeting of sequences that closely resemble the intended target has remained a major challenge, limiting the reliability and interpretation of RGN activity and the range of possible applications. Efforts to reduce off-target activity and enhance RGN specificity have led to a collection of empirically derived rules, which often paradoxically include decreased binding affinity of the RNA-guided nuclease to its target. Here we demonstrate that simple kinetic considerations of the targeting reaction can explain these and other literature observations. The kinetic models described provide a foundation for understanding RGN systems and a necessary physical and functional framework for their rational engineering.

scholarly journals Structural basis for the RNA-guided ribonuclease activity of CRISPR-Cas13d

2018 ◽  
Author(s):  
Cheng Zhang ◽  
Silvana Konermann ◽  
Nicholas J. Brideau ◽  
Peter Lotfy ◽  
Scott J. Novick ◽  
...  
Keyword(s):  
Conformational Dynamics ◽  
Enzyme Activation ◽  
Structural Basis ◽  
Binary Complex ◽  
Molecular Architecture ◽  
Guide Rna ◽  
Guide Rnas ◽  
Rna Targeting ◽  
Hydrogen Deuterium ◽  
Target Rna

AbstractCRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function, we resolved cryo-electron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, explain the compact molecular architecture of Cas13d and provide insights into the structural transitions required for enzyme activation. Our comprehensive analysis of Cas13d in diverse enzymatic states facilitated site-specific truncations for minimal size and delineates a blueprint for improving biomolecular applications of RNA targeting.

scholarly journals Structural basis for Cas9 off-target activity

2021 ◽  
Author(s):  
Martin Pacesa ◽  
Chun-Han Lin ◽  
Antoine Clery ◽  
Katja Bargsten ◽  
Matthew J. Irby ◽  
...  
Keyword(s):  
Rational Design ◽  
Target Prediction ◽  
Structural Basis ◽  
Guide Rna ◽  
Guide Rnas ◽  
Target Dna ◽  
Bona Fide ◽  
Dna Specificity ◽  
Target Binding ◽  
Target Activity

The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of non- canonical base pairing interactions and preservation of base stacking within the guide-off-target heteroduplex. Off-target sites containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping rather than RNA bulge formation. Additionally, PAM-distal mismatches result in duplex unpairing and induce a conformational change of the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.

scholarly journals Managing Technological Change in the Digital Age: The Role of Architectural Frames

2014 ◽  
Vol 29 (1) ◽  
pp. 27-43 ◽  
Author(s):  
Ola Henfridsson ◽  
Lars Mathiassen ◽  
Fredrik Svahn
Keyword(s):  
Technological Change ◽  
Digital Technology ◽  
Design Patterns ◽  
Digital Age ◽  
Future Research ◽  
Loosely Coupled ◽  
Complementary Approach ◽  
Powerful Approach

Inspired by Herbert Simon's notion of nearly decomposable systems, researchers have examined modularity as a powerful approach to manage technological change in product innovation. We articulate this approach as the hierarchy-of-parts architecture and explain how it emphasizes decomposition of a design into loosely coupled parts and subsequent aggregation of these into an industrial product. To realize the scale benefits of modularity, firms successively freeze design specifications before production and therefore only allow limited windows of functionality design and redesign. This makes it difficult to take advantage of the increased speed by which digitized products can be developed and modified. To address this problem, we draw on Christopher Alexander's notion of design patterns to introduce a complementary approach to manage technological change that is resilient to digital technology. We articulate this approach as the network-of-patterns architecture and explain how it emphasizes generalization of ideas into patterns and subsequent specialization of patterns for different design purposes. In response to the increased digitization of industrial products, we demonstrate the value of complementing hierarchy-of-parts thinking with network-of-patterns thinking through a case study of infotainment architecture at an automaker. As a result, we contribute to the literature on managing products in the digital age: we highlight the properties of digital technology that increase the speed by which digitized products can be redesigned; we offer the notion of architectural frames and propose hierarchy-of-parts and network-of-patterns as frames to support innovation of digitized products; and, we outline an agenda for future research that reconsiders the work of Simon and Alexander as well as their followers to address key challenges in innovating digitized products.

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