Assessing Sufficiency and Necessity of Enhancer Activities for Gene Expression and the Mechanisms of Transcription Activation
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Structure of the Cpf1 Endonuclease R-Loop Complex After Target DNA Cleavage
bioRxiv preprint doi: https://doi.org/10.1101/122648; this version posted April 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage Stefano Stella*+, Pablo Alcón+ and Guillermo Montoya* Protein Structure & Function Programme, Macromolecular Crystallography Group, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, Copenhagen, 2200, Denmark. +Joint first author. *To whom correspondence should be addressed Email: [email protected], [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/122648; this version posted April 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract Cpf1 is a single RNA-guided endonuclease of class 2 type V CRISPR-Cas system, emerging as a powerful genome editing tool 1,2. To provide insight into its DNA targeting mechanism, we have determined the crystal structure of Francisella novicida Cpf1 (FnCpf1) in complex with the triple strand R-loop formed after target DNA cleavage. The structure reveals a unique machinery for target DNA unwinding to form a crRNA-DNA hybrid and a displaced DNA strand inside FnCpf1. -
Optimized CRISPR-Cpf1 System for Genome Editing in Zebrafish
Accepted Manuscript Optimized CRISPR-Cpf1 system for genome editing in zebrafish Juan P. Fernandez, Charles E. Vejnar, Antonio J. Giraldez, Romain Rouet, Miguel A. Moreno-Mateos PII: S1046-2023(18)30021-5 DOI: https://doi.org/10.1016/j.ymeth.2018.06.014 Reference: YMETH 4513 To appear in: Methods Received Date: 9 March 2018 Revised Date: 15 June 2018 Accepted Date: 26 June 2018 Please cite this article as: J.P. Fernandez, C.E. Vejnar, A.J. Giraldez, R. Rouet, M.A. Moreno-Mateos, Optimized CRISPR-Cpf1 system for genome editing in zebrafish, Methods (2018), doi: https://doi.org/10.1016/j.ymeth. 2018.06.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Optimized CRISPR-Cpf1 system for genome editing in zebrafish Juan P. Fernandez a,*, Charles E. Vejnar a,*, Antonio J. Giraldez a,b,c‡, d,‡ a,1,‡ Romain Rouet and Miguel A. Moreno-Mateos a Department of Genetics, b Yale Stem Cell Center, c Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06510, USA, d Garvan Institute of Medical Research, Sydney, NSW, Australia, *Co-first Authors ‡To whom correspondence should be addressed: E-mail: [email protected], [email protected], [email protected] 1 Present address: Department of Regeneration and Cell Therapy, Andalusian Molecular Biology and Regenerative Medicine Centre (CABIMER), Seville, Spain. -
Robustly Improved Base Editing Efficiency of Cpf1 Base Editor Using
Chen et al. Cell Discovery (2020) 6:62 Cell Discovery https://doi.org/10.1038/s41421-020-00195-5 www.nature.com/celldisc CORRESPONDENCE Open Access Robustly improved base editing efficiency of Cpf1 base editor using optimized cytidine deaminases Siyu Chen1, Yingqi Jia1, Zhiquan Liu1,HuanhuanShan1,MaoChen1,HaoYu1, Liangxue Lai1,2,3,4 and Zhanjun Li1 Dear Editor, reconstructed dLbCpf1-BE3 (dCpf1-BE3)9 with three dis- Programmable clustered regularly interspaced short tinctive deaminases (evoAPOBEC1, evoCDA110,11, human – palindromic repeats (CRISPR) associated Cpf1 endonu- APOBEC3A (A3A)12 14) to produce optimized dCpf1- cleases, also known as Cas12a, are single RNA-guided based BEs. Here, we demonstrated that there is no sig- (crRNA) effectors1 that have been commonly utilized in nificantly improved base editing frequency observed by various species to manipulate genome for their remarkable using engineering of crRNAs, while the dramatically – specificity2 4 and concise structures1. Cpf1 can recognize increased base editing efficiency was perceived by using thymidine-rich (TTTV (V = A/G/C)) protospacer-adjacent cytidine deaminase optimized Cpf1 BE (dCpf1-eCDA1). motif (PAM) sequences and generates sticky breaks5, Firstly, the three crRNA configurations were con- which enables it to be a complement to Cas9 in genome structed and tested at six genomic sites (Fig. 1a, Supple- editing and broadens the genomic targeting scope. How- mentary Fig. S1a). The results showed that U-rich crRNA ever, Cpf1-based base editors (BEs) generate lower editing slightly improved editing efficiency at all target sites efficiencies than SpCas9-based BE systems, due to the fact ranging from 1.05- to 1.69-fold and significantly improved that the binding of Cpf1 nuclease to corresponding DNA base editing efficiency at the EMX1 site. -
Advances in the Delivery of RNA Therapeutics: from Concept to Clinical Reality
Advances in the delivery of RNA therapeutics: from concept to clinical reality The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Kaczmarek, James C., Piotr S. Kowalski, and Daniel G. Anderson. “Advances in the Delivery of RNA Therapeutics: From Concept to Clinical Reality.” Genome Medicine 9, no. 1 (June 27, 2017). As Published http://dx.doi.org/10.1186/s13073-017-0450-0 Publisher BioMed Central Version Final published version Citable link http://hdl.handle.net/1721.1/110582 Terms of Use Creative Commons Attribution Detailed Terms http://creativecommons.org/licenses/by/4.0/ Kaczmarek et al. Genome Medicine (2017) 9:60 DOI 10.1186/s13073-017-0450-0 REVIEW Open Access Advances in the delivery of RNA therapeutics: from concept to clinical reality James C. Kaczmarek1,2†, Piotr S. Kowalski2† and Daniel G. Anderson1,2,3,4* Abstract The rapid expansion of the available genomic data continues to greatly impact biomedical science and medicine. Fulfilling the clinical potential of genetic discoveries requires the development of therapeutics that can specifically modulate the expression of disease-relevant genes. RNA-based drugs, including short interfering RNAs and antisense oligonucleotides, are particularly promising examples of this newer class of biologics. For over two decades, researchers have been trying to overcome major challenges for utilizing such RNAs in a therapeutic context, including intracellular delivery, stability, and immune response activation. This research is finally beginning to bear fruit as the first RNA drugs gain FDA approval and more advance to the final phases of clinical trials. -
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. -
Molecular Basis of the Function of Transcriptional Enhancers
cells Review Molecular Basis of the Function of Transcriptional Enhancers 1,2, 1, 1,3, Airat N. Ibragimov y, Oleg V. Bylino y and Yulii V. Shidlovskii * 1 Laboratory of Gene Expression Regulation in Development, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., 119334 Moscow, Russia; [email protected] (A.N.I.); [email protected] (O.V.B.) 2 Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., 119334 Moscow, Russia 3 I.M. Sechenov First Moscow State Medical University, 8, bldg. 2 Trubetskaya St., 119048 Moscow, Russia * Correspondence: [email protected]; Tel.: +7-4991354096 These authors contributed equally to this study. y Received: 30 May 2020; Accepted: 3 July 2020; Published: 5 July 2020 Abstract: Transcriptional enhancers are major genomic elements that control gene activity in eukaryotes. Recent studies provided deeper insight into the temporal and spatial organization of transcription in the nucleus, the role of non-coding RNAs in the process, and the epigenetic control of gene expression. Thus, multiple molecular details of enhancer functioning were revealed. Here, we describe the recent data and models of molecular organization of enhancer-driven transcription. Keywords: enhancer; promoter; chromatin; transcriptional bursting; transcription factories; enhancer RNA; epigenetic marks 1. Introduction Gene transcription is precisely organized in time and space. The process requires the participation of hundreds of molecules, which form an extensive interaction network. Substantial progress was achieved recently in our understanding of the molecular processes that take place in the cell nucleus (e.g., see [1–9]). -
Optimizing a CRISPR-Cpf1-Based Genome Engineering System For
Zhang et al. Microb Cell Fact (2019) 18:60 https://doi.org/10.1186/s12934-019-1109-x Microbial Cell Factories RESEARCH Open Access Optimizing a CRISPR-Cpf1-based genome engineering system for Corynebacterium glutamicum Jiao Zhang1, Fayu Yang1, Yunpeng Yang1,2, Yu Jiang3 and Yi‑Xin Huo1,2* Abstract Background: Corynebacterium glutamicum is an important industrial strain for the production of a diverse range of chemicals. Cpf1 nucleases are highly specifc and programmable, with efciencies comparable to those of Cas9. Although the Francisella novicida (Fn) CRISPR‑Cpf1 system has been adapted for genome editing in C. glutamicum, the editing efciency is currently less than 15%, due to false positives caused by the poor targeting efciency of the crRNA. Results: To address this limitation, a screening strategy was developed in this study to systematically evaluate crRNA targeting efciency in C. glutamicum. We quantitatively examined various parameters of the C. glutamicum CRISPR‑ Cpf1 system, including the protospacer adjacent motif (PAM) sequence, the length of the spacer sequence, and the type of repair template. We found that the most efcient C. glutamicum crRNA contained a 5′‑NYTV‑3′ PAM and a 21 bp spacer sequence. Moreover, we observed that linear DNA could be used to repair double strand breaks. Conclusions: Here, we identifed optimized PAM‑related parameters for the CRISPR‑Cpf1 system in C. glutamicum. Our study sheds light on the function of the FnCpf1 endonuclease and Cpf1‑based genome editing. This optimized system, with higher editing efciency, could be used to increase the production of bulk chemicals, such as isobu‑ tyrate, in C. -
Enhancer Rnas Are an Important Regulatory Layer of the Epigenome
REVIEW ARTICLE https://doi.org/10.1038/s41594-020-0446-0 Enhancer RNAs are an important regulatory layer of the epigenome Vittorio Sartorelli 1 and Shannon M. Lauberth 2 ✉ Noncoding RNAs (ncRNAs) direct a remarkable number of diverse functions in development and disease through their regula- tion of transcription, RNA processing and translation. Leading the charge in the RNA revolution is a class of ncRNAs that are synthesized at active enhancers, called enhancer RNAs (eRNAs). Here, we review recent insights into the biogenesis of eRNAs and the mechanisms underlying their multifaceted functions and consider how these findings could inform future investigations into enhancer transcription and eRNA function. he explosion of high-throughput sequencing data has Many different models for how enhancers function in gene con- revealed the complexity and diversity of the transcriptome. trol have been proposed since their initial discovery nearly four TThese data have also unexpectedly revealed that only 1–2% decades ago19–21. Specifically, there is considerable evidence demon- of the transcriptome provides instructions for the synthesis of strating that looping of distal enhancers to their target promoters is functional proteins, while the remaining 98–99% gives rise to a required for enhancer function (reviewed in ref. 22). For example, plethora of ncRNAs, including transfer RNAs (tRNAs), ribosomal a key study revealed that experimental induction of chromatin RNAs (rRNAs), intronic RNAs, small nuclear (sn)RNAs, small looping between the mouse β-globin (Hbb) promoter and its asso- nucleolar (sno)RNAs, microRNAs (miRNAs) and long noncoding ciated enhancer region results in transcriptional activation of the RNAs (lncRNAs). A recent addition to the expanding list of regu- Hbb gene23. -
Base Editing: the Ever Expanding Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Tool Kit for Precise Genome Editing in Plants
G C A T T A C G G C A T genes Review Base Editing: The Ever Expanding Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Tool Kit for Precise Genome Editing in Plants Mahmuda Binte Monsur y, Gaoneng Shao y, Yusong Lv, Shakeel Ahmad , Xiangjin Wei, Peisong Hu and Shaoqing Tang * State Key Laboratory of Rice Biology and China National Center for Rice Improvement, China National Rice Research Institute, Hangzhou 310006, China; [email protected] (M.B.M.); [email protected] (G.S.); [email protected] (Y.L.); [email protected] (S.A.); [email protected] (X.W.); [email protected] (P.H.) * Correspondence: [email protected] These authors contributed equally to this work. y Received: 11 February 2020; Accepted: 21 April 2020; Published: 24 April 2020 Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9), a newly developed genome-editing tool, has revolutionized animal and plant genetics by facilitating modification of target genes. This simple, convenient base-editing technology was developed to improve the precision of genome editing. Base editors generate precise point mutations by permanent base conversion at a specific point, with very low levels of insertions and deletions. Different plant base editors have been established by fusing various nucleobase deaminases with Cas9, Cas13, or Cas12a (Cpf1), proteins. Adenine base editors can efficiently convert adenine (A) to guanine (G), whereas cytosine base editors can convert cytosine (C) to thymine (T) in the target region. RNA base editors can induce a base substitution of A to inosine (I) or C to uracil (U). In this review, we describe the precision of base editing systems and their revolutionary applications in plant science; we also discuss the limitations and future perspectives of this approach. -
Intron-Based Single Transcript Unit CRISPR Systems for Plant Genome
Zhong et al. Rice (2020) 13:8 https://doi.org/10.1186/s12284-020-0369-8 ORIGINAL ARTICLE Open Access Intron-Based Single Transcript Unit CRISPR Systems for Plant Genome Editing Zhaohui Zhong1†, Shishi Liu1†, Xiaopei Liu1, Binglin Liu1, Xu Tang1, Qiurong Ren1, Jianping Zhou1, Xuelian Zheng1, Yiping Qi3,4* and Yong Zhang1,2* Abstract Background: Expression of either Cas9 or Cas12a and guide RNAs by a single Polymerase II (Pol II) promoter represents a compact CRISPR expression system and has many advantages for different applications. In order to make this system routine in plant biology, engineering efforts are needed for developing and optimizing such single transcript unit (STU) systems for plant genome editing. Results: To develop novel intron-based STU (iSTU) CRISPR system (STU CRISPR 3.0), we first evaluated three introns from three plant species for carrying guide RNAs by using an enhanced green fluorescence protein (eGFP) system in rice. After validation of proper intron slicing, we inserted these gRNA-containing introns into the open reading frames (ORFs) of Cas9 and Cas12a for testing their genome editing capability. Different guide RNA processing strategies have been tested for Cas9 and Cas12a. We demonstrated singular genome editing and multiplexed genome editing with these iSTU-Cas9 and iSTU-Cas12a systems. Conclusion: We developed multiple iSTU-CRISPR/Cas9 and Cas12a systems for plant genome editing. Our results shed light on potential directions for further improvement of the iSTU systems. Keywords: STU CRISPR 3.0, Cas9, Cas12a, iSTU, Rice Background editing (Zhang et al. 2019). Application of CRISPR tech- Sequence-specific nucleases (SSNs), such as zinc finger nologies in plants often requires the co-expression of the nucleases (ZFNs), TAL effector nucleases (TALENs) and Cas protein and guide RNAs (gRNAs). -
Enhancer Rnas: Transcriptional Regulators and Workmates of Namirnas in Myogenesis
Odame et al. Cell Mol Biol Lett (2021) 26:4 https://doi.org/10.1186/s11658-021-00248-x Cellular & Molecular Biology Letters REVIEW Open Access Enhancer RNAs: transcriptional regulators and workmates of NamiRNAs in myogenesis Emmanuel Odame , Yuan Chen, Shuailong Zheng, Dinghui Dai, Bismark Kyei, Siyuan Zhan, Jiaxue Cao, Jiazhong Guo, Tao Zhong, Linjie Wang, Li Li* and Hongping Zhang* *Correspondence: [email protected]; zhp@sicau. Abstract edu.cn miRNAs are well known to be gene repressors. A newly identifed class of miRNAs Farm Animal Genetic Resources Exploration termed nuclear activating miRNAs (NamiRNAs), transcribed from miRNA loci that and Innovation Key exhibit enhancer features, promote gene expression via binding to the promoter and Laboratory of Sichuan enhancer marker regions of the target genes. Meanwhile, activated enhancers pro- Province, College of Animal Science and Technology, duce endogenous non-coding RNAs (named enhancer RNAs, eRNAs) to activate gene Sichuan Agricultural expression. During chromatin looping, transcribed eRNAs interact with NamiRNAs University, Chengdu 611130, through enhancer-promoter interaction to perform similar functions. Here, we review China the functional diferences and similarities between eRNAs and NamiRNAs in myogen- esis and disease. We also propose models demonstrating their mutual mechanism and function. We conclude that eRNAs are active molecules, transcriptional regulators, and partners of NamiRNAs, rather than mere RNAs produced during enhancer activation. Keywords: Enhancer RNA, NamiRNAs, MicroRNA, Myogenesis, Transcriptional regulator Introduction Te identifcation of lin-4 miRNA in Caenorhabditis elegans in 1993 [1] triggered research to discover and understand small microRNAs’ (miRNAs) mechanisms. Recently, some miRNAs are reported to activate target genes during transcription via base pairing to the 3ʹ or 5ʹ untranslated regions (3ʹ or 5ʹ UTRs), the promoter [2], and the enhancer regions [3]. -
Alt-R CRISPR-Cpf1—RNP Electroporation, Amaxa
genome editing protocol Alt-R® CRISPR-Cas12a (Cpf1) System: Delivery of ribonucleoprotein complexes into HEK-293 cells using the Amaxa® Nucleofector® System See what more we can do for you at www.idtdna.com. For Research Use Only Version 2.1 protocol genome editing Table of contents Introduction 3 Important considerations 3 Required materials 4 Protocol 5 A. Culture cells [1] 5 B. Form the RNP complex 5 C. Prepare Nucleofector system 6 D. Perform electroporation of cells with Nucleofector system [1] 6 References 8 Revision history 8 2 See what we can do for you at www.idtdna.com. genome editing protocol Introduction This protocol describes the delivery of a CRISPR-Cas12a (Cpf1) ribonucleoprotein (RNP) complex, containing Alt-R CRISPR-Cpf1 crRNA and Alt-R A.s. Cpf1 Nuclease 2NLS, into HEK-293 cells using electroporation with the Amaxa Nucleofector system (Lonza) [1]. To detect on-target mutations and estimate editing efficiency, we recommend using the Alt-R Genome Editing Detection Kit [2]. The CRISPR-Cas12a (Cpf1) system is distinct from the more commonly used CRISPR-Cas9 system. For example, Cas12a nuclease does not require a tracrRNA; recognizes a T-rich, protospacer-adjacent motif (PAM: TTTV, where V is an A, C, or G base); and creates a staggered double-stranded DNA cut with a 5’ overhang. For additional information, visit www.idtdna.com/CRISPR-Cpf1. Important considerations 1. Use low-passage, healthy cells. A critical factor affecting the success of electroporation is the health of the cells. It is important to: • Use the lowest passage number cells available • Subculture cells for at least 2–3 days before the electroporation procedure • Replace the media the day before electroporation • Determine the optimal confluency for your cell type Optimal confluency for HEK-293 cells is 80–90% at the time of Nucleofection.