DOCSLIB.ORG

Crispr/Cas9 Target Prediction with Deep Learning

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Crispr/Cas9 Target Prediction with Deep Learning Research Article ISSN: 2574 -1241 DOI: 10.26717/BJSTR.2019.21.003652 Crispr/Cas9 Target Prediction with Deep Learning Özlem Aktaş, Elif Doğan* and Tolga Ensari Department of Computer Engineering, Faculty of Engineering, Turkey *Corresponding author: Elif Doğan, Department of Computer Engineering, Faculty of Engineering, Dokuz Eylül University, Turkey ARTICLE INFO Abstract Received: Published: September 24, 2019 The CRISPR/CAS9 system is a powerful tool for regulating damaged genome October 03, 2019 sequences. Nucleases that are damaged in their sequence are called miRNAs (micro RNAs). The miRNAs targeted by multiple promoter sgRNA (single guide RNA) are Citation: cut or regulated from RNA by the CRISPR/CAS9 method. The sgRNAs targeted to the Özlem Aktaş, Elif Doğan, Tolga incorrect miRNAs may provoke undesired genome abnormalities. In this study, in Ensari. Crispr/Cas9 Target Prediction with order to minimize these genome distortions, sgRNA target estimation was performed Deep Learning. Biomed J Sci & Tech Res for CRISPR/CAS9 with deep learning in this study. In this article, convolutional neural 21(5)-2019. BJSTR. MS.ID.003652. networks (Convolutional Neural Networks-CNN) and multilayer perceptron (Multi- Layer Perceptron-MLP) algorithms are used for experimental analysis. We also compare theAbbreviations: performance of CRISPR/CAS9 system for three algorithms. CNN: Convolutional Neural Networks; BLSTM: Bidirectional Long-Short Term Memory; MLP: Multi Layer Perceptron Introduction E. Coli In in silico silico the genomes to be studied in this respect. Various miRNA target In an Esherichia Coli ( ) bacteria that investigated by “ data sets used in “ ” studies will be used [7]. The aim of this ” (simulation) method which is important role nowadays study, it is made mirRNA target estimation with machine learning has been discovered an immune system named CRISPR (clustered E.Coli algorithms. The result of wrong targeted miRNA may be cause of regularly interspaced short palindromic repeats)/Cas9 [1]. undesirable gene mutations [8]. It is aimed to minimize the Type According to this system an bacteria which is infected by 1 and Type 2 errors of miRNAs by applying machine learning and any virus, add the virus DNA its memory and recognize when any deep learning method of Type 1 and Type 2 errors as output. Thus, other virus attack accrues. This defined virus DNA cuts this virus the wrong target estimate will be minimized. In this way, it will be DNA from its own DNA through the CAS9 enzyme. Thus, DNA repair possible to reliably repair gene damage by correctly targeting mis- will occur. According to recent research the leading factor in gene targeted miRNAs. Genetic diseases will be eliminated by repair of regulation has been observed the “microRNA (miRNA)” targeted gene damage. in silico by the “single-guide RNA (sGRNA)” s. MiRNAs are small and non- coding RNA molecules [2]. In diseases, for example, different The purpose of the “ ” technique is to increase the miRNAs involved in all stages of cancer make cancer diagnosis and accuracy of disease prevention by single pointing with the help of treatment available [3]. CRISPR/CAS9 is used to destroy targeted mechanization. The large base readings provided by mechanization miRNAs in cells [4]. It uses “guideRNA (gRNA)” as a guide to target also contribute greatly. Studies for miRNA targeting, support vector the CRISPR/CAS9 nucleus to the DNA sequence and trigger the machines (Support Vector Machine -SVM) [9], deep learning, double-strand break at the desired location. Breaking and repair of constrained logic programming (Constraint Logic Programming) these threads can cause random addition and alteration of DNA [5]. [10] and a class classification (One Class Classification-OCC) [11] methods were used. Besides, in the future big data may be MiRNA activities are different in each cell type [6]. So, the decreased to its most valuable parts with digital data forgetting miRNA activities of a human cell and a bacterial cell will not be concepts. It makes computations faster and the machines will store the same. From this point, It is important to find a dataset about less data in disks [37]. Copyright@ 16131 Elif Doğan | Biomed J Sci & Tech Res | BJSTR. MS.ID.003652. Volume 21- Issue 5 DOI: 10.26717/BJSTR.2019.21.003652 Materials and Methods CRISPR [22], CRISPOR [23], CRISPR Local [24]. In the algorithm studies performed with these data sets, estimation tools such as mirWalk [25], TargetScan (ID2 PPI analysis network) [26], miRanda [27], CRISPR/CAS9 system is a strong genome editing mechanism mirBase [28], mirTarget [29], TarBase [30] have been developed. used in many biotechnology applications [12]. MiRNAs are RNA molecules that play an important role in gene regulation in In this study, CRISPR Local data set has been used for the animals and plants [13]. Damaged miRNAs are targeted by sgRNAs. analysis [31]. The source of CRISPR Local dataset is ENSEMBL However, the effectiveness of sgRNAs have not been defined firmly Plants. There are approximately 854.610 lines of CRISPR data in to target area [14]. In the systems of CRISPR/CAS9 (clustered the original. In this study, we used 34.200 lines of data because of regularly interspaced short palindromic repeats), Cas9 nuclease technical issues. There are 11 column features in this dataset which creates short array (about 20 nucleotide) with RNA guideline two are examples of “Cyanidioschyzon merolae” alga. This features; the stranded in the determined region at DNA. CRISPR/CAS9 has been gene in which the sgRNA, on target estimated chromosome and a unique technology that allows genetic and medical researchers its coordinate, sgRNA sequence with 23’nt., on target prediction to add, subtract or modify DNA sequences in various parts of the score, off target prediction gene which has the greatest CFD score, genome. In contrast to ZFN (Zinc-Finger Nuclease) and TALENs the chromosome on target prediction, its coordinate and beginning (Transcription Activator-Like Effector Nuclease), CRISPR/CAS9 is position, off-target prediction sequence, the number of sgRNA not man-made; currently the system is part of the bacterial immune and mismatch on the off-target sequence, axon name, axon start system, which helps to protect from invasive phages. Originality in position, all off-target and sgRNA having the highest CFD score. The CRISPR is achieved using an RNA molecule that is complementary sequences having 4 channels like Adenin (A), Sitozin (C), Guanin to the gene of interest. After binding, this RNA molecule (also called (G) ve Timin (T) are used as [1,0,0,0], [0,1,0,0], [0,0,1,0], [0,0,0,11]. guide RNA) traps a CAS9 nuclease, a double-strand break that According these channels, the process has been realized with causes a frame shift when repaired by NHEJ (Non-Homologous End converted RNA sequence to binary system. These sequences were Joining). CRISPR is the most effective process to date in gene repair used as binary in the data set. In this example, each base in the and editing tools. Efficiency varies depending on the organism and sequence is considered as a separate column and feature. The 23nt. the target site. Since it is the simplest, versatile and sensitive method sgRNA sequence, on-target prediction score features were used. of genetic manipulation that is currently available, it attracts great Figure 1 shows an illustration of the sample data set. used. Figure 1 attention in the world of science. In summary, CRISPR/CAS9 differs shows an illustration of the sample data set. from ZFN and TALENs in an important aspect that makes them superior to genomic regulation applications: The ZFN and TALENs bind to DNA through a direct protein-DNA interaction that requires DATAredesigning Set the protein for each new target. Applied “in silico” research and review, BLAST (Basic Local Figure 1: Sample Data Set. Alignment Search Tool) was used for CRISPR [15]. BLAST is a search tool that analyzes the amino acids and DNA sequences of proteins and finds similarities between them. Besides BLAST, Experimental Studies the data set resources have been used such as National Human Genome Research Institute (NHGRI) [16], miRBase [17], Genome In this study, CNN, MLP and BLSTM models were constituted Crispr [18], CrisprInc [19], ENSEMBL [20], ENCODE [21], CRISPRz Multilayer Perceptron -MLP and compared. Figure 2: MLP Model. Copyright@ 16132 Elif Doğan | Biomed J Sci & Tech Res | BJSTR. MS.ID.003652. Volume 21- Issue 5 DOI: 10.26717/BJSTR.2019.21.003652 A 4-layer MLP model has been developed according to the MLP of the matrices. In order to prevent over fitting, maxpooling layer algorithm run using the Google Colab GPU (https://colab.research. was used. It selects the elements with the maximum value from the google.com). In the MLP model a fully connected structure of dense matrix pool of the specified size in the maxpooling layer. Accordingly, layers was formed. Information on the model used is shown in the information obtained when the CNN model is generated can be Figure 2. The rates of logistic regression and accuracy according seen in Figure 6. The rate of accuracy according to the CNN model to the MLP model are shown in Figure 3 and Figure 4. Accuracy can be seen from Figure 7. obtained 80.38% according to MLP model. Figure 5: Convolution Process. Figure 3: Model Accuracy. Figure 4: Model loss. Convolutional Neural Network - CNN Figure 6: CNN Model. Convolutional Neural Networks are a model of artificial neutral network which is used successfully in image processing, bioinformatics, robotics, data mining, finance and many other areas. However, except for image analysis, surprisingly high accuracy ratio was obtained in emotion analysis, text classification and question answering applications. According to this model, it is applied to nxn matrix with nxn filtering method (dot product), with acceptation of n>m.
Load more

Recommended publications
  • Engineering Nucleic Acid Chemistry for Precise and Controllable CRISPR
    Science Bulletin 64 (2019) 1841–1849 Contents lists available at ScienceDirect Science Bulletin journal homepage: www.elsevier.com/locate/scib Review Engineering nucleic acid chemistry for precise and controllable CRISPR/Cas9 genome editing ⇑ Weiqi Cai a,b, Ming Wang a,b, a Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, CAS Research/Education Center for Excellence in Molecule Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China b University of Chinese Academy of Sciences, Beijing 100049, China article info abstract Article history: The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/ Received 21 June 2019 Cas9) genome editing technology is revolutionizing our approach and capability to precisely manipulate Received in revised form 16 July 2019 the genetic flow of mammalians. The facile programmability of Cas9 protein and guide RNA (gRNA) Accepted 23 July 2019 sequence has recently expanded biomedical application of CRISPR/Cas9 technology from editing mam- Available online 30 July 2019 malian genome to various genetic manipulations. The therapeutic and clinical translation potential of CRISPR/Cas9 genome editing, however, are challenged by its off-target effect and low genome editing effi- Keywords: ciency. In this regard, developing new Cas9 variants and conditional control of Cas9/gRNA activity are of CRISPR/Cas9 genome editing great potential for improving genome editing accuracy and on-target efficiency. In this review, we sum- RNA engineering Nucleic acid chemistry marize chemical strategies that have been developed recently to engineer the nucleic acid chemistry of Gene therapy gRNA to enhance CRISPR/Cas9 genome editing efficacy, specificity and controllability.
    [Show full text]
  • Tissue-Specific Delivery of CRISPR Therapeutics
    International Journal of Molecular Sciences Review Tissue-Specific Delivery of CRISPR Therapeutics: Strategies and Mechanisms of Non-Viral Vectors Karim Shalaby 1 , Mustapha Aouida 1,* and Omar El-Agnaf 1,2,* 1 Division of Biological and Biomedical Sciences (BBS), College of Health & Life Sciences (CHLS), Hamad Bin Khalifa University (HBKU), Doha 34110, Qatar; [email protected] 2 Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Doha 34110, Qatar * Correspondence: [email protected] (M.A.); [email protected] (O.E.-A.) Received: 12 September 2020; Accepted: 27 September 2020; Published: 5 October 2020 Abstract: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing system has been the focus of intense research in the last decade due to its superior ability to desirably target and edit DNA sequences. The applicability of the CRISPR-Cas system to in vivo genome editing has acquired substantial credit for a future in vivo gene-based therapeutic. Challenges such as targeting the wrong tissue, undesirable genetic mutations, or immunogenic responses, need to be tackled before CRISPR-Cas systems can be translated for clinical use. Hence, there is an evident gap in the field for a strategy to enhance the specificity of delivery of CRISPR-Cas gene editing systems for in vivo applications. Current approaches using viral vectors do not address these main challenges and, therefore, strategies to develop non-viral delivery systems are being explored. Peptide-based systems represent an attractive approach to developing gene-based therapeutics due to their specificity of targeting, scale-up potential, lack of an immunogenic response and resistance to proteolysis.
    [Show full text]
  • Ribozyme-Mediated, Multiplex CRISPR Gene Editing and Crispri in Plasmodium Yoelii
    bioRxiv preprint doi: https://doi.org/10.1101/481416; this version posted November 29, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Ribozyme-Mediated, Multiplex CRISPR Gene Editing and CRISPRi in Plasmodium yoelii Michael P. Walker1 and Scott E. Lindner1 * 1Department of Biochemistry and Molecular Biology, the Huck Center for Malaria Research, Pennsylvania State University, University Park, PA. *Correspondence: Scott E. Lindner, [email protected] Running Title: CRISPR-RGR in Plasmodium yoelii Keywords: Plasmodium, CRISPR, CRISPRi, Ribozyme, HDR, ALBA bioRxiv preprint doi: https://doi.org/10.1101/481416; this version posted November 29, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Abstract 2 Functional characterization of genes in Plasmodium parasites often relies on genetic 3 manipulations to disrupt or modify a gene-of-interest. However, these approaches are limited by 4 the time required to generate transgenic parasites for P. falciparum and the availability of a 5 single drug selectable marker for P. yoelii. In both cases, there remains a risk of disrupting native 6 gene regulatory elements with the introduction of exogenous sequences. To address these 7 limitations, we have developed CRISPR-RGR, a SpCas9-based gene editing system for 8 Plasmodium that utilizes a Ribozyme-Guide-Ribozyme (RGR) sgRNA expression strategy. 9 Using this system with P. yoelii, we demonstrate that both gene disruptions and coding sequence 10 insertions are efficiently generated, producing marker-free and scar-free parasites with homology 11 arms as short as 80-100bp.
    [Show full text]
  • CRISPR/Cas9 with Single Guide RNA Expression Driven by Small Trna Promoters Showed Reduced Editing Efficiency Compared to a U6 Promoter
    Downloaded from rnajournal.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press LETTER TO THE EDITOR CRISPR/Cas9 with single guide RNA expression driven by small tRNA promoters showed reduced editing efficiency compared to a U6 promoter YUDA WEI,1,2 YAN QIU,1,2 YANHAO CHEN,1,2 GAIGAI LIU,1,2 YONGXIAN ZHANG,1,2 LUWEI XU,3 and QIURONG DING1,2 1CAS Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 2University of Chinese Academy of Sciences, Shanghai 200031, China 3Taizhou Hospital of Traditional Chinese Medicine, Taizhou 225300, China ABSTRACT Multiplex genome engineering in vivo with CRISPR/Cas9 shows great promise as a potential therapeutic approach. The ability to incorporate multiple single guide RNA (sgRNA) cassettes together with Cas9 gene expression in one AAV vector could greatly enhance the efficiency. In a recent Method article, Mefferd and coworkers indicated that small tRNA promoters could be used to drive sgRNA expression to facilitate the construction of a more effective AAV vector. In contrast, we found that when targeting endogenous genomic loci, CRISPR/Cas9 with tRNA promoter-driven sgRNA expression showed much reduced genome editing activity, compared with significant cleavage with U6 promoter-driven sgRNA expression. Though the underlying mechanisms are still under investigation, our study suggests that the CRISPR/Cas9 system with tRNA promoter- driven sgRNA expression needs to be reevaluated before it can be used for therapeutic genome editing. Keywords: CRISPR/Cas9; genome editing; tRNA promoter The use of clustered regularly interspaced short palindromic have a DNA packaging limit of ∼5 kb, and the standard repeats (CRISPR)/CRISPR-associated (Cas) systems for in AAV–SaCRISPR system almost fills the cargo limitation, it vivo genome editing has emerged as a potential treatment would be helpful to further reduce the total length of the for human diseases (Cox et al.
    [Show full text]
  • RNA Interference in the Age of CRISPR: Will CRISPR Interfere with Rnai?
    International Journal of Molecular Sciences Review RNA Interference in the Age of CRISPR: Will CRISPR Interfere with RNAi? Unnikrishnan Unniyampurath 1, Rajendra Pilankatta 2 and Manoj N. Krishnan 1,* 1 Program on Emerging Infectious Diseases, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore; [email protected] 2 Department of Biochemistry and Molecular Biology, School of Biological Sciences, Central University of Kerala, Nileshwar 671328, India; [email protected] * Correspondence: [email protected]; Tel.: +65-6516-2666 Academic Editor: Michael Ladomery Received: 2 November 2015; Accepted: 15 February 2016; Published: 26 February 2016 Abstract: The recent emergence of multiple technologies for modifying gene structure has revolutionized mammalian biomedical research and enhanced the promises of gene therapy. Over the past decade, RNA interference (RNAi) based technologies widely dominated various research applications involving experimental modulation of gene expression at the post-transcriptional level. Recently, a new gene editing technology, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system, has received unprecedented acceptance in the scientific community for a variety of genetic applications. Unlike RNAi, the CRISPR/Cas9 system is bestowed with the ability to introduce heritable precision insertions and deletions in the eukaryotic genome. The combination of popularity and superior capabilities of CRISPR/Cas9 system raises the possibility that this technology may occupy the roles currently served by RNAi and may even make RNAi obsolete. We performed a comparative analysis of the technical aspects and applications of the CRISPR/Cas9 system and RNAi in mammalian systems, with the purpose of charting out a predictive picture on whether the CRISPR/Cas9 system will eclipse the existence and future of RNAi.
    [Show full text]
  • Snorna Guide Activities – Real and Ambiguous Svetlana Deryusheva, Gaëlle JS Talross
    Downloaded from rnajournal.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Deryusheva, Talross and Gall SnoRNA guide activities – real and ambiguous Svetlana Deryusheva, Gaëlle J.S. Talross 1, Joseph G. Gall Department of Embryology, Carnegie Institution for Science, Baltimore, Maryland 21218, USA 1 Present address: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06510, USA Corresponding authors: e-mail [email protected] [email protected] Running title: Testing snoRNA activities Keywords: modification guide RNA activity, 2’-O-methylation, pseudouridylation, Pus7p, snoRNA 1 Downloaded from rnajournal.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Deryusheva, Talross and Gall ABSTRACT In eukaryotes, rRNAs and spliceosomal snRNAs are heavily modified posttranscriptionally. Pseudouridylation and 2’-O-methylation are the most abundant types of RNA modifications. They are mediated by modification guide RNAs, also known as small nucleolar (sno)RNAs and small Cajal body-specific (sca)RNAs. We used yeast and vertebrate cells to test guide activities predicted for a number of snoRNAs, based on their regions of complementarity with rRNAs. We showed that human SNORA24 is a genuine guide RNA for 18S-Ψ609, despite some non- canonical base-pairing with its target. At the same time, we found quite a few snoRNAs that have the ability to base-pair with rRNAs and can induce predicted modifications in artificial substrate RNAs, but do not modify the same target sequence within endogenous rRNA molecules. Furthermore, certain fragments of rRNAs can be modified by the endogenous yeast modification machinery when inserted into an artificial backbone RNA, even though the same sequences are not modified in endogenous yeast rRNAs.
    [Show full text]
  • CRISPR/Cas9 Genome Editing Brochure
    mirusbio.com Cas9 Target Sequence Guide RNA GENOME EDITING: CRISPR/CAS9 DELIVERY METHODS GENOME EDITING: CRISPR/CAS9 DELIVERY What is CRISPR/Cas9 Genome Editing? The CRISPR/Cas9 system is a powerful tool for genome editing in mammalian cells that allows researchers to generate genetic variants at lower cost and with higher throughput than alternative methods like zinc finger nuclease (ZFN) or transcription activator-like effector nuclease (TALEN) genome editing. Cas9 PAM Genomic DNA Target Sequence Guide RNA crRNA tracrRNA The CRISPR/Cas9 RNP Complex. The CRISPR associated protein 9 (Cas9) endonuclease (blue) is targeted to DNA by a guide RNA (gRNA), which can be supplied as a two-part system consisting of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) or as a single guide RNA (sgRNA), where the crRNA and tracrRNA are connected by a linker (dotted line). Target recognition is facilitated by the protospacer-adjacent motif (PAM). A double strand break (DSB) occurs 3 bp upstream of the PAM. CRISPR Facilitates Multiple Types of Genome Modification Cleavage of Target DNA By Cas9 Deletion Modication Insertion Multiple Genomic Alterations are Possible Following Cleavage of Target DNA by Cas9. Variable length insertions and/ or deletions (indels) can result near the DNA break due to mistakes in DNA repair by the endogenous non-homologous end joining (NHEJ) pathway. These indels frequently result in disruption of gene function. Alternatively, by supplying a DNA repair template, researchers can leverage the homology-directed repair (HDR) pathway to create defined deletions, insertions or other modifications. 2 TO ORDER | Toll Free 888.530.0801 | Direct 608.441.2852 | www.mirusbio.com Glossary of CRISPR Terms Term Definition CRISPR Associated Protein 9 - Cas9 is an RNA-guided DNA endonuclease from the type Cas9 II CRISPR system of Streptococcus pyogenes that has been adapted for use in genome editing applications.
    [Show full text]
  • Advances in Oligonucleotide Drug Delivery
    REVIEWS Advances in oligonucleotide drug delivery Thomas C. Roberts 1,2 ✉ , Robert Langer 3 and Matthew J. A. Wood 1,2 ✉ Abstract | Oligonucleotides can be used to modulate gene expression via a range of processes including RNAi, target degradation by RNase H-mediated cleavage, splicing modulation, non-coding RNA inhibition, gene activation and programmed gene editing. As such, these molecules have potential therapeutic applications for myriad indications, with several oligonucleotide drugs recently gaining approval. However, despite recent technological advances, achieving efficient oligonucleotide delivery, particularly to extrahepatic tissues, remains a major translational limitation. Here, we provide an overview of oligonucleotide-based drug platforms, focusing on key approaches — including chemical modification, bioconjugation and the use of nanocarriers — which aim to address the delivery challenge. Oligonucleotides are nucleic acid polymers with the In addition to their ability to recognize specific tar- potential to treat or manage a wide range of diseases. get sequences via complementary base pairing, nucleic Although the majority of oligonucleotide therapeutics acids can also interact with proteins through the for- have focused on gene silencing, other strategies are being mation of three-dimensional secondary structures — a pursued, including splice modulation and gene activa- property that is also being exploited therapeutically. For tion, expanding the range of possible targets beyond example, nucleic acid aptamers are structured
    [Show full text]
  • Ribonucleic Acid (RNA)
    AccessScience from McGraw-Hill Education Page 1 of 11 www.accessscience.com Ribonucleic acid (RNA) Contributed by: Michael W. Gray, Ann L. Beyer Publication year: 2014 One of the two major classes of nucleic acid, mainly involved in translating the genetic information carried in deoxyribonucleic acid (DNA) into proteins. Various types of ribonucleic acids (RNAs) [see table] function in protein synthesis: transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) function in the synthesis of all proteins, whereas messenger RNAs (mRNAs) are a diverse set, with each member specifically directing the synthesis of one protein. Messenger RNA is the intermediate in the usual biological pathway of DNA → RNA → protein. However, RNA is a very versatile molecule. Other types of RNA serve other important functions for cells and viruses, such as the involvement of small nuclear RNAs (snRNAs) in mRNA splicing. In some cases, RNA performs functions typically considered DNA-like, such as serving as the genetic material for certain viruses, or roles typically carried out by proteins, such as RNA enzymes or ribozymes. See also: DEOXYRIBONUCLEIC ACID (DNA); NUCLEIC ACID. Structure and synthesis RNA is a linear polymer of four different nucleotides (Fig. 1). Each nucleotide is composed of three parts: a five-carbon sugar known as ribose; a phosphate group; and one of four bases attached to each ribose, that is, adenine (A), cytosine (C), guanine (G), or uracil (U). The combination of base and sugar constitutes a nucleoside. The structure of RNA is basically a repeating chain of ribose and phosphate moieties, with one of the four bases attached to each ribose.
    [Show full text]
  • Conditional Deoxyribozyme−Nanoparticle Conjugates For
    www.acsami.org Research Article Conditional Deoxyribozyme−Nanoparticle Conjugates for miRNA- Triggered Gene Regulation Jiahui Zhang, Rong Ma, Aaron Blanchard, Jessica Petree, Hanjoong Jo,* and Khalid Salaita* Cite This: ACS Appl. Mater. Interfaces 2020, 12, 37851−37861 Read Online ACCESS Metrics & More Article Recommendations *sı Supporting Information ABSTRACT: DNA−nanoparticle (NP) conjugates have been used to knockdown gene expression transiently and effectively, making them desirable tools for gene regulation therapy. Because DNA−NPs are constitutively active and are rapidly taken up by most cell types, they offer limited control in terms of tissue or cell type specificity. To take a step toward solving this issue, we incorporate toehold-mediated strand exchange, a versatile molec- ular programming modality, to switch the DNA−NPs from an inactive state to an active state in the presence of a specific RNA input. Because many transcripts are unique to cell subtype or disease state, this approach could one day lead to responsive nucleic acid therapeutics with enhanced specificity. As a proof of concept, we designed conditional deoxyribozyme−nanoparticles (conditional DzNPs) that knockdown tumor necrosis factor α (TNFα) mRNA upon miR-33 triggering. We demonstrate toehold- mediated strand exchange and restoration of TNFα DNAzyme activity in the presence of miR-33 trigger, with optimization of the preparation, configuration, and toehold length of conditional DzNPs. Our results indicate specific and strong ON/OFF response of conditional DzNPs to the miR-33 trigger in buffer. Furthermore, we demonstrate endogenous miR-33-triggered knockdown of TNFα mRNA in mouse macrophages, implying the potential of conditional gene regulation applications using these DzNPs.
    [Show full text]
  • Perfectly Matched 20-Nucleotide Guide RNA Sequences Enable Robust
    Zhang et al. Genome Biology (2017) 18:191 DOI 10.1186/s13059-017-1325-9 METHOD Open Access Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases Dingbo Zhang1,2†, Huawei Zhang1†, Tingdong Li1,2, Kunling Chen1, Jin-Long Qiu3 and Caixia Gao1* Abstract High-fidelity SpCas9 variants (eSpCas9 and SpCas9-HF1) have been engineered to reduce off-target effects. We found that changes in guide RNA length induced significant reductions in the editing activities of SpCas9 variants in plant cells. Single guide RNAs harboring precise, perfectly matched 20-nucleotide guide sequences are necessary for high on-target editing activities of eSpCas9 and SpCas9-HF1. Precise 20-nucleotide guide sequences derived from tRNA– sgRNA precursors enable robust on-target editing by these variants with enhanced specificity. Our work reveals an effective way of enhancing the use of the high-fidelity SpCas9 nucleases for efficient and precise genome engineering. Background Here, we evaluated the efficiency and specificity of The CRISPR-Cas9 system recognizes genomic sites via eSpCas9 and SpCas9-HF1 in plant genome editing pro- Watson-Crick base pairing by virtue of 20-nucleotide (nt) cesses, and found that precise perfectly matched 20-nt guide sequences in the guide RNAs (gRNAs) that direct guide sequences could ensure the high efficiencies of these Cas9 for targeted cleavage [1–4]. The CRISPR-Cas9 SpCas9 variants and maintain their high fidelity. methodology is revolutionizing genome engineering and genetic therapy [5, 6]. However, Cas9 can also target DNA sequences that harbor one or multiple mismatches with Results gRNAs.
    [Show full text]
  • Boosting CRISPR/Cas9 Multiplex Editing Capability with the Endogenous Trna-Processing System
    Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system Kabin Xie, Bastian Minkenberg, and Yinong Yang1 Department of Plant Pathology and Environmental Microbiology and the Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA 16802 Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved February 3, 2015 (received for review October 22, 2014) The clustered regularly interspaced short palindromic repeat (CRISPR)/ III (Pol III) promoter, gRNA, and the Pol III terminator. Due to CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed the limitation of delivery method and plasmid vector capacity, as a powerful tool for genome engineering in basic research, molec- simultaneous production of numerous gRNAs would be a chal- ular therapy, and crop improvement. This system uses a small guide lenge with such a gRNA-expressing strategy for most organisms. RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, Moreover, eukaryotic Pol III-transcribed RNA is obligated to its targeting capability is largely constrained by the gRNA-expressing start with a specific ribonucleotide, which may reduce the Cas9/ device. In this study, we developed a general strategy to produce gRNA targetable sites. An advanced strategy would be to com- numerous gRNAs from a single polycistronic gene. The endogenous pact multiple gRNAs in one synthetic gene and engineer an tRNA-processing system, which precisely cleaves both ends of the RNA-processing system to excise individual gRNAs from the tRNA precursor, was engineered as a simple and robust platform to primary transcript. The only proven method based on such a boost the targeting and multiplex editing capability of the CRISPR/ strategy is the expression of Csy4 endoribonuclease (endo-RNase) Cas9 system.
    [Show full text]