DOCSLIB.ORG

The CRISPR-Cas System

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The CRISPR-Cas System Linköping University - Department of Physics, Chemistry and Biology Bachelor thesis, 16 hp - Educational program: Physics, Chemistry and Biology Spring term 2020 - LITH - IFM - G - EX -- 20/3902 -- SE The CRISPR-Cas system Can CRISPR bring about a brighter future? An extensive and up-to-date CRISPR overview, focusing on the CRISPR-Cas9 technology including a suggestion of a CRISPR-Cas9 laboratory assignment for University students. Sara Berggren, Isabella Enoksson and Cassandra Stens Examiner: Assoc. Prof. Lars-Göran Mårtensson 1 Abstract Derived from and inspired by the adaptive immune system of bacteria, CRISPR has gone from basic biology knowledge to a revolutionizing biotechnological tool, applicable in many research areas such as medicine, industry and agriculture. The full mechanism of CRISPR-Cas9 was first published in 2012 and various CRISPR-Cas systems have already passed the first stages of clinical trials as new gene therapies. The immense research has resulted in continuously growing knowledge of CRISPR systems and the technique seems to have the potential to greatly impact all life on our planet. Therefore, this literature study aims to thoroughly describe the CRISPR-Cas system, and further suggest an undergraduate laboratory exercise involving gene editing with the CRISPR-Cas9 tool. In this paper, we describe the fundamental technical background of the CRISPR-Cas system, especially emphasizing the most studied CRISPR-Cas9 system, its development and applications areas, as well as highlighting its current limitations and ethical concerns. The history of genetic engineering and the discovery of the CRISPR system is also described, along with a comparison with other established gene editing techniques. This study concludes that a deeper knowledge about CRISPR is important and required since the technique is applicable in many research areas. A laboratory exercise will not only inspire but also provide extended theoretical and practical knowledge for undergraduate students. Acknowledgements We are forever grateful for all of the guidance our examiner, Assoc. Prof. Lars-Göran Mårtensson at Linköping University, has given us during these ten weeks of intense CRISPR-Cas exploration. You have shared your valuable knowledge and experience from years of research and publications, giving us a great starting point and potentiality through the paper. Thank you for your critical reading, thoughtful recommendations and many laughs - you have made our bachelor fun and memorable! Further appreciations and thanks goes to the BioRender website, enabling quick and artistic illustrations for our report. While writing this paper, we have tried to cite original articles as much as possible, but we apologize in advance if there are mistakes unintentionally citing other articles and researchers than the originals. 2 Terminology, abbreviations and acronyms Abbreviations and acronyms AdV adenovirus AAV adeno-associated virus bp base pairs Cas CRISPR associated protein CRISPR clustered regularly interspaced short palindromic repeats CRISPRa CRISPR activation CRISPRi CRISPR interference crRNA CRISPR RNA cr:tracrRNA a crRNA-tracrRNA hybrid dCas9 dead Cas9 (at times called deactivated Cas9) dsDNA double stranded DNA DSB double stranded break HDR homology directed repair Indels insertions or deletions NHEJ nonhomologous end joining nt nucleotides PAMs protospacer adjacent motifs pre-crRNA precursor CRISPR RNA repeats direct repeats RNP ribonucleoprotein ssDNA single stranded DNA SpCas9 Streptococcus pyogenes​ CRISPR associated (Cas) protein tracrRNA trans-activating crRNA WT wild type Terminology Anti-CRISPR protein: ​inhibitors of the CRISPR-Cas system which can be applied to both mammalian and prokaryotic cells for more precise and controllable editing. Cas9​: the most common and used type of Cas protein. Discovered in ​Streptococcus pyogenes​. Cas9 complex​: the Cas9 protein attached to gRNA (cr:tracrRNA or sgRNA). Cas protein, CRISPR associated protein​: is a non-specific endonuclease, i.e. capable of cleaving DNA. Requires a guide RNA to find where in the genome to execute the cut. There are multiple types of Cas proteins isolated from various bacteria and archaea. 3 CRISPR array​: a series of palindromic, repeated sequences called ​direct repeats​ interspaced by different target-specific sequences called ​spacers​. CRISPR locus​: consists of a clustered set of tracrRNA genes, Cas genes and a CRISPR array at fixed regions in chromosomes. A single genome can incorporate multiple CRISPR loci. CRISPR-Cas9 system​: relies on two components, a Cas9 protein and a guide RNA. The guide RNA can be in the form of a two-component crRNA and tracrRNA hybrid (cr:tracrRNA), or as a one-component sgRNA (crRNA and tracrRNA linked via a ​linker loop​). CRISPR RNA, crRNA​: a nucleotide sequence consisting of a ​spacer ​and a ​repeat ​sequence. The spacer is the customizable part of the gRNA and can be programmed to recognize particular target DNA sequences. crRNAs are transcribed from the CRISPR array. cr:tracrRNA​: a gRNA configuration where crRNA and tracrRNA are bound to each other. Pronunciation: crispr tracr RNA. dCas9​: a mutated (dead or deactivated) Cas9 protein without cleavage activity. Direct repeats​: identical, repetitive and palindromic sequences interspaced between spacer fragments. Called repeats ​for short. The direct repeats are complementary to sequences in tracrRNAs. Electroporation​: a microbiology technique in which an electric field is applied on a sample in order to increase the permeability of cell membranes, thereby supporting drugs, chemicals and genetic material to be introduced into target cells. Endonucleases​: catalyse the cleavage of DNA by cutting the phosphodiester bonds in nucleic acid strands. Guide RNA, gRNA​: can be in two conformations; a two-component cr:tracrRNA, or a one-component sgRNA. A gRNA in the shape of a cr:tracrRNA consists of a crRNA and a tracrRNA bound to each other. A gRNA in the form of an sgRNA consists of crRNA and tracrRNA linked together by a sequence called the linker loop. Despite configuration, gRNAs are capable of binding to Cas proteins and are used to recognize DNA regions of interest, thereby guiding the Cas protein to a certain target site. HNH:​ an endonuclease domain in the Cas9 protein responsible to cut the target strand. Leader sequence​: a sequence ahead of the CRISPR array, includes the promoter. The leader sequence has an important role in the process of spacer acquisition. Linker loop​: a sequence connecting the crRNA and tracrRNA together. 4 Motif​: a sequence motif is a short, recurring sequence pattern in DNA presumed to have a biochemical function, as for example to indicate binding sites for proteins such as nucleases (e.g. Cas proteins). A sequence motif is distinguished from a structural motif; the latter describing a specific part of a protein’s 3D structure. Multiplex​: a quality of CRISPR-Cas9 systems, where several genome sites in a cell are targeted and cut simultaneously, with the aid of multiple gRNAs. Palindromic repeats​: sequences that are read the same forward as backwards on two complementary strands. PAM​: a short, recurring sequence (motif) required for target site recognition of Cas proteins. Different Cas proteins are specific for different PAMs, for instance, the Cas9 PAM motif is NGG. PAMs are located immediately adjacent to the target protospacer in various organism’s DNA. pre-crRNA​: a single spacer sequence flanked by repeat fragments. The CRISPR array is transcribed into a long precursor CRISPR RNA (pre-crRNA), prior to being cleaved into matured crRNAs. In the case of CRISPR Type II, assistance of tracrRNA, Cas9 and RNase III is required to process pre-crRNA into matured crRNA. Protospacer​: a short DNA sequence derived and collected from foreign genomes. Protospacers are directly connected to PAM motifs in foreign genomes. When the protospacer is inserted into the CRISPR array in the bacterial genome it is termed a spacer. RNP complex​: the Cas9 protein in complex with gRNA (cr:tracrRNA or sgRNA). RuvC:​ a nuclease domain, common in multiple types of Cas proteins, capable to cut the non-target strand. Seed sequence​: a 8-12 bp sequence of the guide RNA that must be strictly complementary to the target DNA. Single guide RNA, sgRNA​: a single RNA sequence composed of a crRNA linked to a tracrRNA via a ​linker loop​. Spacers​: ~20 bp sequences of various foreign DNA that is inserted and found within the CRISPR array. Spacers are small, unique fragments of DNA gathered from viruses that previously have tried to infect the bacterial cell. The incorporated spacers are used to express short guide RNAs, called crRNAs. By means of comparing the spacer with the complementary foreign DNA (the protospacers), CRISPR-Cas systems can identify and eliminate foreign DNA. Spacer acquisition​: the insertion process of new foreign DNA fragments into the CRISPR array. tracrRNA, trans-activating crispr RNA​: serves as a binding scaffold for the Cas protein and the crRNA. The tracrRNA is fixed and not programmable (in contrast to the case of crRNAs). 5 Contents 1. Introduction 9 1.1 Aim 9 1.2 Background 9 2. History 11 2.1 Genetic engineering in historical perspective 11 3. Theory 13 3.1 The CRISPR-Cas system 13 3.1.1 Basic technical background of CRISPR-Cas9 14 3.1.2 The protein structure of CRISPR-Cas9 18 3.1.3 The DNA repair mechanisms of eukaryotic cells 22 3.1.4 Cas classes and types 24 3.1.5 Transcription of the CRISPR locus 26 3.1.6 Spacer acquisition into the CRISPR array 29 3.1.7 Introduction of CRISPR-Cas9 into cells 32 3.1.7.1 DNA plasmid delivery 34 3.1.7.2 Viral vector delivery 35 3.1.7.3 RNA delivery 37 3.1.7.4 Protein delivery (RNP delivery) 38 3.1.7.5 Physical and chemical delivery methods (non-viral methods)38 3.1.7.6 Cytosolic vs nucleic insertion 40 3.1.7.7 In vivo​ and ​ex vivo​ delivery 41 3.1.8 Modifications of the CRISPR-Cas9 system 42 3.1.8.1 dCas9, CRISPRa and CRISPRi 43 3.1.8.2 Nickases 44 3.1.8.3 Base editing and prime editing 46 6 4.
Load more

Recommended publications
  • Multiple Origins of Viral Capsid Proteins from Cellular Ancestors
    Multiple origins of viral capsid proteins from PNAS PLUS cellular ancestors Mart Krupovica,1 and Eugene V. Kooninb,1 aInstitut Pasteur, Department of Microbiology, Unité Biologie Moléculaire du Gène chez les Extrêmophiles, 75015 Paris, France; and bNational Center for Biotechnology Information, National Library of Medicine, Bethesda, MD 20894 Contributed by Eugene V. Koonin, February 3, 2017 (sent for review December 21, 2016; reviewed by C. Martin Lawrence and Kenneth Stedman) Viruses are the most abundant biological entities on earth and show genome replication. Understanding the origin of any virus group is remarkable diversity of genome sequences, replication and expres- possible only if the provenances of both components are elucidated sion strategies, and virion structures. Evolutionary genomics of (11). Given that viral replication proteins often have no closely viruses revealed many unexpected connections but the general related homologs in known cellular organisms (6, 12), it has been scenario(s) for the evolution of the virosphere remains a matter of suggested that many of these proteins evolved in the precellular intense debate among proponents of the cellular regression, escaped world (4, 6) or in primordial, now extinct, cellular lineages (5, 10, genes, and primordial virus world hypotheses. A comprehensive 13). The ability to transfer the genetic information encased within sequence and structure analysis of major virion proteins indicates capsids—the protective proteinaceous shells that comprise the that they evolved on about 20 independent occasions, and in some of cores of virus particles (virions)—is unique to bona fide viruses and these cases likely ancestors are identifiable among the proteins of distinguishes them from other types of selfish genetic elements cellular organisms.
    [Show full text]
  • Como Citar Este Artigo Número Completo Mais Informações Do
    Encontros Bibli: revista eletrônica de biblioteconomia e ciência da informação ISSN: 1518-2924 Programa de Pós-graduação em Ciência da Informação - Universidade Federal de Santa Catarina STANFORD, Jailiny Fernanda Silva; SILVA, Fábio Mascarenhas e Prêmio Nobel como fator de influência nas citações dos pesquisadores: uma análise dos laureados de Química e Física (2005 - 2015) Encontros Bibli: revista eletrônica de biblioteconomia e ciência da informação, vol. 26, e73786, 2021, Janeiro-Abril Programa de Pós-graduação em Ciência da Informação - Universidade Federal de Santa Catarina DOI: https://doi.org/10.5007/1518-2924.2021.e73786 Disponível em: https://www.redalyc.org/articulo.oa?id=14768130002 Como citar este artigo Número completo Sistema de Informação Científica Redalyc Mais informações do artigo Rede de Revistas Científicas da América Latina e do Caribe, Espanha e Portugal Site da revista em redalyc.org Sem fins lucrativos acadêmica projeto, desenvolvido no âmbito da iniciativa acesso aberto Artigo Original Prêmio Nobel como fator de influência nas citações dos pesquisadores: uma análise dos laureados de Química e Física (2005 - 2015) Nobel Prize as an influencing factor in researchers' citations: an analysis of Chemistry and Physics laureates (2005 to 2015) Jailiny Fernanda Silva STANFORD Mestre em Ciência da Informação (PPGCI/UFPE) Bibliotecária-chefe Seminário Teológico Batista do Norte do Brasil (STBNB), Recife, Brasil [email protected] https://orcid.org/0000-0003-2112-6561 Fábio Mascarenhas e SILVA Doutor em Ciência da Informação (USP), Professor Associado Universidade Federal de Pernambuco, Departamento de Ciência da Informação, Recife, Brasil [email protected] https://orcid.org/0000-0001-5566-5120 A lista completa com informações dos autores está no final do artigo RESUMO Objetivo: Analisa a influência nos índices de citação por parte dos pesquisadores que foram contemplados pelo prêmio Nobel nas áreas da Física e Química no período de 2005 a 2015.
    [Show full text]
  • Biochemistrystanford00kornrich.Pdf
    University of California Berkeley Regional Oral History Office University of California The Bancroft Library Berkeley, California Program in the History of the Biosciences and Biotechnology Arthur Kornberg, M.D. BIOCHEMISTRY AT STANFORD, BIOTECHNOLOGY AT DNAX With an Introduction by Joshua Lederberg Interviews Conducted by Sally Smith Hughes, Ph.D. in 1997 Copyright 1998 by The Regents of the University of California Since 1954 the Regional Oral History Office has been interviewing leading participants in or well-placed witnesses to major events in the development of Northern California, the West, and the Nation. Oral history is a method of collecting historical information through tape-recorded interviews between a narrator with firsthand knowledge of historically significant events and a well- informed interviewer, with the goal of preserving substantive additions to the historical record. The tape recording is transcribed, lightly edited for continuity and clarity, and reviewed by the interviewee. The corrected manuscript is indexed, bound with photographs and illustrative materials, and placed in The Bancroft Library at the University of California, Berkeley, and in other research collections for scholarly use. Because it is primary material, oral history is not intended to present the final, verified, or complete narrative of events. It is a spoken account, offered by the interviewee in response to questioning, and as such it is reflective, partisan, deeply involved, and irreplaceable. ************************************ All uses of this manuscript are covered by a legal agreement between The Regents of the University of California and Arthur Kornberg, M.D., dated June 18, 1997. The manuscript is thereby made available for research purposes. All literary rights in the manuscript, including the right to publish, are reserved to The Bancroft Library of the University of California, Berkeley.
    [Show full text]
  • Pooled CRISPR-Activation Screening Coupled with Single-Cell RNA-Seq in Mouse Embryonic Stem Cells
    ll OPEN ACCESS Protocol Pooled CRISPR-activation screening coupled with single-cell RNA-seq in mouse embryonic stem cells Celia Alda-Catalinas, Melanie A. Eckersley-Maslin, Wolf Reik celia.x.aldacatalinas@gsk. com (C.A.-C.) [email protected]. uk (W.R.) Highlights Protocol for CRISPRa screens with single- cell readout to interrogate gene function Detailed description of CRISPRa screening procedures in mouse embryonic stem cells Detailed steps on how to construct derived single-cell sgRNA amplicon libraries CRISPR/Cas9 screens are a powerful approach to identify key regulators of biological processes. By combining pooled CRISPR/Cas9 screening with a single-cell RNA-sequencing readout, individual perturbations can be assessed in parallel both comprehensively and at scale. Importantly, this allows gene function and regulation to be interrogated at a cellular level in an unbiased manner. Here, we present a protocol to perform pooled CRISPR-activation screens in mouse embryonic stem cells using 103 Genomics scRNA-seq as a readout. Alda-Catalinas et al., STAR Protocols 2, 100426 June 18, 2021 ª 2021 The Authors. https://doi.org/10.1016/ j.xpro.2021.100426 ll OPEN ACCESS Protocol Pooled CRISPR-activation screening coupled with single-cell RNA-seq in mouse embryonic stem cells Celia Alda-Catalinas,1,4,7,* Melanie A. Eckersley-Maslin,1,5,6 and Wolf Reik1,2,3,8,* 1Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, UK 2Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK 3Centre for Trophoblast Research, University of
    [Show full text]
  • A CRISPR Activation and Interference Toolkit for Industrial Saccharomyces Cerevisiae Strain KE6‑12 Elena Cámara, Ibai Lenitz & Yvonne Nygård*
    www.nature.com/scientificreports OPEN A CRISPR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6‑12 Elena Cámara, Ibai Lenitz & Yvonne Nygård* Recent advances in CRISPR/Cas9 based genome editing have considerably advanced genetic engineering of industrial yeast strains. In this study, we report the construction and characterization of a toolkit for CRISPR activation and interference (CRISPRa/i) for a polyploid industrial yeast strain. In the CRISPRa/i plasmids that are available in high and low copy variants, dCas9 is expressed alone, or as a fusion with an activation or repression domain; VP64, VPR or Mxi1. The sgRNA is introduced to the CRISPRa/i plasmids from a double stranded oligonucleotide by in vivo homology‑directed repair, allowing rapid transcriptional modulation of new target genes without cloning. The CRISPRa/i toolkit was characterized by alteration of expression of fuorescent protein‑encoding genes under two diferent promoters allowing expression alterations up to ~ 2.5‑fold. Furthermore, we demonstrated the usability of the CRISPRa/i toolkit by improving the tolerance towards wheat straw hydrolysate of our industrial production strain. We anticipate that our CRISPRa/i toolkit can be widely used to assess novel targets for strain improvement and thus accelerate the design‑build‑test cycle for developing various industrial production strains. Te yeast Saccharomyces cerevisiae is one of the most commonly used microorganisms for industrial applications ranging from wine and beer fermentations to the production of biofuels and high-value metabolites1,2. How- ever, some of the current production processes are compromised by low yields and productivities, thus further optimization is required3.
    [Show full text]
  • Advances in Genomics for Drug Development
    G C A T T A C G G C A T genes Review Advances in Genomics for Drug Development Roberto Spreafico , Leah B. Soriaga, Johannes Grosse, Herbert W. Virgin and Amalio Telenti * Vir Biotechnology, Inc., San Francisco, CA 94158, USA; Rspreafi[email protected] (R.S.); [email protected] (L.B.S.); [email protected] (J.G.); [email protected] (H.W.V.) * Correspondence: [email protected] Received: 24 July 2020; Accepted: 13 August 2020; Published: 15 August 2020 Abstract: Drug development (target identification, advancing drug leads to candidates for preclinical and clinical studies) can be facilitated by genetic and genomic knowledge. Here, we review the contribution of population genomics to target identification, the value of bulk and single cell gene expression analysis for understanding the biological relevance of a drug target, and genome-wide CRISPR editing for the prioritization of drug targets. In genomics, we discuss the different scope of genome-wide association studies using genotyping arrays, versus exome and whole genome sequencing. In transcriptomics, we discuss the information from drug perturbation and the selection of biomarkers. For CRISPR screens, we discuss target discovery, mechanism of action and the concept of gene to drug mapping. Harnessing genetic support increases the probability of drug developability and approval. Keywords: druggability; loss-of-function; CRISPR 1. Introduction For over 20 years, genomics has been used as a tool for accelerating drug development. Various conceptual approaches and techniques assist target identification, target prioritization and tractability, as well as the prediction of outcomes from pharmacological perturbations. These basic premises are now supported by a rapid expansion of population genomics initiatives (sequencing or genotyping of hundreds of thousands of individuals), in-depth understanding of disease and drug perturbation at the tissue and single-cell level as measured by transcriptome analysis, and by the capacity to screen for loss of function or activation of genes, genome-wide, using CRISPR technologies.
    [Show full text]
  • Gene Therapy Glossary of Terms
    GENE THERAPY GLOSSARY OF TERMS A • Phase 3: A phase of research to describe clinical trials • Allele: one of two or more alternative forms of a gene that that gather more information about a drug’s safety and arise by mutation and are found at the same place on a effectiveness by studying different populations and chromosome. different dosages and by using the drug in combination • Adeno-Associated Virus: A single stranded DNA virus that has with other drugs. These studies typically involve more not been found to cause disease in humans. This type of virus participants.7 is the most frequently used in gene therapy.1 • Phase 4: A phase of research to describe clinical trials • Adenovirus: A member of a family of viruses that can cause occurring after FDA has approved a drug for marketing. infections in the respiratory tract, eye, and gastrointestinal They include post market requirement and commitment tract. studies that are required of or agreed to by the study • Adeno-Associated Virus Vector: Adeno viruses used as sponsor. These trials gather additional information about a vehicles for genes, whose core genetic material has been drug’s safety, efficacy, or optimal use.8 removed and replaced by the FVIII- or FIX-gene • Codon: a sequence of three nucleotides in DNA or RNA • Amino Acids: building block of a protein that gives instructions to add a specific amino acid to an • Antibody: a protein produced by immune cells called B-cells elongating protein in response to a foreign molecule; acts by binding to the • CRISPR: a family of DNA sequences that can be cleaved by molecule and often making it inactive or targeting it for specific enzymes, and therefore serve as a guide to cut out destruction and insert genes.
    [Show full text]
  • CRISPR RNA-Guided Integrases for High-Efficiency and Multiplexed
    bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209452; this version posted July 18, 2020. 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. CRISPR RNA-guided integrases for high-efficiency and multiplexed bacterial genome engineering Phuc Leo H. Vo1, Carlotta Ronda2, Sanne E. Klompe3, Ethan E. Chen4, Christopher Acree3, Harris H. Wang2,5, Samuel H. Sternberg3 1Department of Pharmacology, Columbia University Irving Medical Center, New York, NY, USA. 2Department of Systems Biology, Columbia University Irving Medical Center, New York, NY, USA. 3Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA. 4Department of Biological Sciences, Columbia University, New York, NY, USA. 5Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY, USA. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.17.209452; this version posted July 18, 2020. 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. Tn7-like transposons are pervasive mobile genetic elements in bacteria that mobilize using heteromeric transposase complexes comprising distinct targeting modules. We recently described a Tn7-like transposon from Vibrio cholerae that employs a Type I-F CRISPR–Cas system for RNA-guided transposition, in which Cascade directly recruits transposition proteins to integrate donor DNA downstream of genomic target sites complementary to CRISPR RNA.
    [Show full text]
  • Prime Editing €“ an Update on the Field
    Gene Therapy (2021) 28:396–401 https://doi.org/10.1038/s41434-021-00263-9 PERSPECTIVE Prime editing – an update on the field 1,2 3 Janine Scholefield ● Patrick T. Harrison Received: 12 January 2021 / Revised: 15 April 2021 / Accepted: 5 May 2021 / Published online: 24 May 2021 © The Author(s) 2021. This article is published with open access At Gene Therapy, we are continually monitoring the land- containing RNA template as a contiguous extension of the scape of research, noting those technologies which advance guide RNA (known as the pegRNA), and M-MLV reverse the goal of clinical translation of the field. In one of the transcriptase (RT) fused to the C terminus of Cas9 (H840A) most exciting ‘needle-shifting’ gene editing publications nickase. Use of the Cas9 nickase avoids the formation of a published in 2019, David Liu et al., (who pioneered base DSB, and simply cuts the non-complementary strand of editing; BE [1]), developed ‘Prime editing (PE) [2]’, erasing the DNA three bases upstream of the PAM site. This several limits of CRISPR that have caused bottlenecks in its exposes a DNA flap with a 3’ OH group which binds to the therapeutic and biotechnological applicability. Traditional primer binding site (PBS) of the RNA template, serving as a gene editing strategies directing specific changes to the primer for RT, which extends the 3’ flap by copying the edit 1234567890();,: 1234567890();,: genome sequence itself largely reply on a common step – sequence of the pegRNA (Fig. 1). Despite this extended 3’ creating a double-strand break (DSB) to attract and exploit flap being thermodynamically less likely to hybridise to the the DNA repair pathway machinery in executing the desired unedited complementary strand compared to the unedited 5’ repair.
    [Show full text]
  • Long-Read Cdna Sequencing Identifies Functional Pseudogenes in the Human Transcriptome Robin-Lee Troskie1, Yohaann Jafrani1, Tim R
    Troskie et al. Genome Biology (2021) 22:146 https://doi.org/10.1186/s13059-021-02369-0 SHORT REPORT Open Access Long-read cDNA sequencing identifies functional pseudogenes in the human transcriptome Robin-Lee Troskie1, Yohaann Jafrani1, Tim R. Mercer2, Adam D. Ewing1*, Geoffrey J. Faulkner1,3* and Seth W. Cheetham1* * Correspondence: adam.ewing@ mater.uq.edu.au; faulknergj@gmail. Abstract com; [email protected]. au Pseudogenes are gene copies presumed to mainly be functionless relics of evolution 1Mater Research Institute-University due to acquired deleterious mutations or transcriptional silencing. Using deep full- of Queensland, TRI Building, QLD length PacBio cDNA sequencing of normal human tissues and cancer cell lines, we 4102 Woolloongabba, Australia Full list of author information is identify here hundreds of novel transcribed pseudogenes expressed in tissue-specific available at the end of the article patterns. Some pseudogene transcripts have intact open reading frames and are translated in cultured cells, representing unannotated protein-coding genes. To assess the biological impact of noncoding pseudogenes, we CRISPR-Cas9 delete the nucleus-enriched pseudogene PDCL3P4 and observe hundreds of perturbed genes. This study highlights pseudogenes as a complex and dynamic component of the human transcriptional landscape. Keywords: Pseudogene, PacBio, Long-read, lncRNA, CRISPR Background Pseudogenes are gene copies which are thought to be defective due to frame- disrupting mutations or transcriptional silencing [1, 2]. Most human pseudogenes (72%) are derived from retrotransposition of processed mRNAs, mediated by proteins encoded by the LINE-1 retrotransposon [3, 4]. Due to the loss of parental cis-regula- tory elements, processed pseudogenes were initially presumed to be transcriptionally silent [1] and were excluded from genome-wide functional screens and most transcrip- tome analyses [2].
    [Show full text]
  • Improving Treatment of Genetic Diseases with Crispr-Cas9 Rna-Guided Genome Editing
    Sanchez 3:00 Team R06 Disclaimer: This paper partially fulfills a writing requirement for first-year (freshmen) engineering students at the University of Pittsburgh Swanson School of Engineering. This paper is a student paper, not a professional paper. This paper is not intended for publication or public circulation. This paper is based on publicly available information, and while this paper might contain the names of actual companies, products, and people, it cannot and does not contain all relevant information/data or analyses related to companies, products, and people named. All conclusions drawn by the authors are the opinions of the authors, first- year (freshmen) students completing this paper to fulfill a university writing requirement. If this paper or the information therein is used for any purpose other than the authors' partial fulfillment of a writing requirement for first-year (freshmen) engineering students at the University of Pittsburgh Swanson School of Engineering, the users are doing so at their own--not at the students', at the Swanson School's, or at the University of Pittsburgh's--risk. IMPROVING TREATMENT OF GENETIC DISEASES WITH CRISPR-CAS9 RNA-GUIDED GENOME EDITING Arijit Dutta [email protected] , Benjamin Ahlmark [email protected], Nate Majer [email protected] Abstract—Genetic illnesses are among the most difficult to treat as it is challenging to safely and effectively alter DNA. INTRODUCTION: THE WHAT, WHY, AND DNA is the basic code for all hereditary traits, so any HOW OF CRISPR-CAS9 alteration to DNA risks fundamentally altering the way someone’s genes are expressed. This change could lead to What Is CRISPR-Cas9? unintended consequences for both the individual whose DNA was altered and any offspring they may have in the future, CRISPR-Cas9 is an acronym that stands for “Clustered compounding the risk.
    [Show full text]
  • Spatiotemporal Control of CRISPR/Cas9 Gene Editing
    Signal Transduction and Targeted Therapy www.nature.com/sigtrans REVIEW ARTICLE OPEN Spatiotemporal control of CRISPR/Cas9 gene editing Chenya Zhuo1, Jiabin Zhang1, Jung-Hwan Lee2, Ju Jiao3, Du Cheng4, Li Liu5, Hae-Won Kim2,YuTao1 and Mingqiang Li 1,6 The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) gene editing technology, as a revolutionary breakthrough in genetic engineering, offers a promising platform to improve the treatment of various genetic and infectious diseases because of its simple design and powerful ability to edit different loci simultaneously. However, failure to conduct precise gene editing in specific tissues or cells within a certain time may result in undesirable consequences, such as serious off-target effects, representing a critical challenge for the clinical translation of the technology. Recently, some emerging strategies using genetic regulation, chemical and physical strategies to regulate the activity of CRISPR/Cas9 have shown promising results in the improvement of spatiotemporal controllability. Herein, in this review, we first summarize the latest progress of these advanced strategies involving cell-specific promoters, small-molecule activation and inhibition, bioresponsive delivery carriers, and optical/thermal/ultrasonic/magnetic activation. Next, we highlight the advantages and disadvantages of various strategies and discuss their obstacles and limitations in clinical translation. Finally, we propose viewpoints on directions that can be explored to
    [Show full text]