Final Report of the Emerging Technologies Subcommittee Biotechnology
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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. -
CRISPR/Cas9: Tools and Applications for Eukaryotic Genome Editing
CRISPR/Cas9: Tools and Applications for Eukaryotic Genome Editing Fei Ann Ran Broad Institute Cambridge, Massachusetts [email protected] I will provide some background on the CRISPR/Cas9 technology, some of the rationale for how we came to develop and use this tool, and I will address immediate questions concerning the specificity of the technology. I will also discuss some of the more interest- ing applications. Figure 1 reflects how the cost of DNA sequencing has decreased dramatically over the past two decades due to technological progress. As a result, there has been an explo- sion of data, not only in the sequences of different species, but in sequence differences between individuals within species, between cell types and between diseased and healthy cells. It suffices to say that this is an exciting time to be working in the field of genome engineering. Genome Engineering Typically, genome engineering is achieved by leveraging the cell’s own repair machinery. This can come from the error-prone NHEJ pathway that leads to insertion/deletion (in- del) mutations, which can be used to knock out genes, or, alternatively, we can supply a repair template to overwrite the site of a double-stranded break (DSB) for more-precise genome engineering via the HDR pathway (Figure 2). Figure 1. Advances in DNA-sequencing technologies. (Stratton MR et al., 2009) When we started working on CRISPR/Cas technology1, several well developed tools were already being used—and still are being used—to achieve impressive results in bio- technology, medicine, agriculture, and other fields. At the outset, we were interested in developing an alternative technology to make cloning easier at lower cost with greater scalability. -
Gene Drives for Malaria Control and Elimination in Africa
GENE DRIVES FOR MALARIA CONTROL AND ELIMINATION IN AFRICA HIGH LEVEL APET REPORT • GENE DRIVES FOR MALARIA CONTROL AND ELIMINATION IN AFRICA About the AU and NEPAD The African Union (AU) The African Union (AU) is a continental union consisting of all 55 countries on the African continent. It was established on 26 May 2001 in Addis Ababa, Ethiopia, and launched on 9 July 2002 in South Africa, with the aim of replacing the Organisation of African Unity (OAU). The most important decisions of the AU are made by the Assembly of the African Union, a semi-annual meeting of the heads of state and government of its member states. The AU’s secretariat, the African Union Commission, is based in Addis Ababa. The AU was established following the 9th September 1999 Sirte Declaration of the Heads of State and Governments of the Organisation of the African Unity (OAU). The AU is based on a common vision of a united and strong Africa and on the need to build a partnership between governments and all segments of civil society, in particular, women, the youth and the private sector, in order to strengthen solidarity and cohesion amongst the peoples of Africa. As a continental organization, it focuses on the promotion of peace, security and stability. The development work of the AU is guided by the AU Agenda 2063, which is a 50-year plan to harness Africa’s comparative advantage to deliver on the vision of “The Africa We Want”. The New Partnership for Africa’s Development (NEPAD) Created by the African Union, the New Partnership for Africa’s Development (NEPAD) is a strategic framework for Pan-African socio- economic development. -
Development and Applications of CRISPR-Cas9 for Genome Engineering
Leading Edge Review Development and Applications of CRISPR-Cas9 for Genome Engineering Patrick D. Hsu,1,2,3 Eric S. Lander,1 and Feng Zhang1,2,* 1Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02141, USA 2McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 3Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cell.2014.05.010 Recent advances in genome engineering technologies based on the CRISPR-associated RNA- guided endonuclease Cas9 are enabling the systematic interrogation of mammalian genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. In this Review, we describe the development and applications of Cas9 for a variety of research or translational applications while highlighting challenges as well as future directions. Derived from a remarkable microbial defense system, Cas9 is driving innovative applications from basic biology to biotechnology and medicine. Introduction of the genome and its functions. In biotechnology, precise The development of recombinant DNA technology in the 1970s manipulation of genetic building blocks and regulatory machin- marked the beginning of a new era for biology. -
Recombinant S. Pyogenes CRISPR-Cas9 Catalog Number: 9957-C9
Recombinant S. pyogenes CRISPR-Cas9 Catalog Number: 9957-C9 DESCRIPTION Source E. coli-derived s. pyogenes CRISPR-Cas9 protein S. pyogenes CRISPR-Cas9 KRPAATKKAGQAKK- APKKKRKVGIHGVPAA (Asp2-Asp1368) HHHHHH KKGYGRKKRRQRRRG Accession # Q99ZW2 N-terminus C-terminus N-terminal Sequence Ala Analysis Predicted Molecular 164 kDa Mass SPECIFICATIONS SDS-PAGE 133 kDa, reducing conditions Activity Measured by its ability to cleave a targeted DNA substrate. S. pyogenes CRISPR-Cas9 achieves >80% substrate cleavage, as measured under the described conditions. Endotoxin Level <0.10 EU per 1 μg of the protein by the LAL method. Purity >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining. Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, EDTA, Glycerol and TCEP. See Certificate of Analysis for details. Activity Assay Protocol Materials Assay Buffer: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 µg/mL BSA, pH 7.9 Recombinant Streptococcus pyogenes CRISPR-Cas9 (rS. pyogenes Cas9) (Catalog # 9957-C9) PBR322 vector (NEB, Catalog # N3033S) digested with EcoRI-HF (NEB, Catalog # R3101S)* Dharmacon synthetic sgRNA, targeting sequence: GAGGCAGACAAGGTATAGGG Ethidium Bromide, 10 mg/mL (Amresco, Catalog # X328) Ultrapure DNase/RNase-Free Distilled Water (Invitrogen, Catalog # 10977015), to prepare Assay Buffer DNA gel *Digest was gel purified using gel purification kit and eluted in EB buffer (10 mM Tris-HCl, pH 8.5). Assay 1. Prepare RNP Complex: a. 600 nM sgRNA (6 µL addition from 3 µM stock prepared in Assay Buffer) b. 0.25 μg rS. pyogenes Cas9 c. Add Assay Buffer for a final RNP Complex volume of 26.5 µL d. -
What Is Gene Drive?
The Science: What is gene drive? What is gene drive? Gene drive is a genetic phenomenon that occurs in nature and causes a selected trait to spread rapidly through a species via sexual reproduction over several generations. Gene drive works by increasing the likelihood that a modified gene will be inherited by its offspring. Normally, genes have a 50/50 chance of being inherited, but gene drive How is Target Malaria using systems could increase that chance to upwards gene drive technology? of 99 percent. This means that over the course of several generations, a selected trait could become We aim to tackle malaria at the source. Target increasingly common within a specific species. Malaria is using gene drive approaches to insert a modification in malaria mosquitoes that would Researchers have been studying how to harness affect the mosquito’s ability to reproduce. By gene drives to solve some of society’s most reducing the population of malaria mosquitoes, intractable problems for a long time. Public health we aim to reduce the transmission of the disease. and ecosystem conservation are two of the main areas where research has focused, although other Worldwide there are more than 3,500 species of uses are also possible. mosquito, with 837 of them in Africa. Of these, a single cluster of three very closely related What are gene drive applications species are responsible for most of the malaria transmission – Anopheles gambiae, Anopheles in public health? coluzzii and Anopheles arabiensis. In public health, several proposals have been The project is investigating the use of genes that made which would use gene drive to limit the produce enzymes (called nucleases) that cut spread of diseases, particularly those spread specific sequences of DNA. -
CRISPR/Cas9 System and Gene Editing Tools – on Patent Rights, Recent Disputes and Its Potential Commercial Applicability in Biotechnology and Medicine
ISSN 2003-2382 CRISPR/Cas9 system and gene editing tools – On patent rights, recent disputes and its potential commercial applicability in biotechnology and medicine By Thomas Hedner and Jean Lycke ABSTRACT may also find a future use in “de-extinction” of various animals such as the woolly mammoth The CRISPR/Cas9 discovery has emerged as a and passenger pigeon. powerful technology tool to edit genomes, which The recent discoveries and developments have allows researchers, innovators and life science led to extensive patenting efforts, resulting in some entrepreneurs to alter DNA sequences and modify major patent disputes. The extensive patenting may gene function in a range of species. The simplicity, risk creating a scenario, which could hamper the high efficiency and seemingly broad use of the further development of this technology and ultima- CRISPR/Cas9 system has led to hopes that this tely limit full value creation of this technology for disruptive technology may have the potential to major societal and industrial stakeholders. transform important sectors of biotechnology and medicine. The technology will enable users to make changes in the sequence or expression of virtually 1. INTRODUCTION any gene, cell type or organism. The rapid progress The CRISPR technology, which allows researchers to easily in the development of CRISPR/Cas9-based techno- alter DNA sequences and modify gene function has over logies over the past years has been extraordinary. the past decade emerged a simple and powerful tool for In spite of that, many outstanding questions remain editing genomes1 The CRISPR/Cas9 is a system initially to be addressed, and potentially interesting applica- found in bacteria as a mechanism involved in immune tions as well as potential risks yet need to be explored. -
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. -
Engineering of Primary Human B Cells with CRISPR/Cas9 Targeted Nuclease Received: 26 January 2018 Matthew J
www.nature.com/scientificreports OPEN Engineering of Primary Human B cells with CRISPR/Cas9 Targeted Nuclease Received: 26 January 2018 Matthew J. Johnson1,2,3, Kanut Laoharawee1,2,3, Walker S. Lahr1,2,3, Beau R. Webber1,2,3 & Accepted: 23 July 2018 Branden S. Moriarity1,2,3 Published: xx xx xxxx B cells ofer unique opportunities for gene therapy because of their ability to secrete large amounts of protein in the form of antibody and persist for the life of the organism as plasma cells. Here, we report optimized CRISPR/Cas9 based genome engineering of primary human B cells. Our procedure involves enrichment of CD19+ B cells from PBMCs followed by activation, expansion, and electroporation of CRISPR/Cas9 reagents. We are able expand total B cells in culture 10-fold and outgrow the IgD+ IgM+ CD27− naïve subset from 35% to over 80% of the culture. B cells are receptive to nucleic acid delivery via electroporation 3 days after stimulation, peaking at Day 7 post stimulation. We tested chemically modifed sgRNAs and Alt-R gRNAs targeting CD19 with Cas9 mRNA or Cas9 protein. Using this system, we achieved genetic and protein knockout of CD19 at rates over 70%. Finally, we tested sgRNAs targeting the AAVS1 safe harbor site using Cas9 protein in combination with AAV6 to deliver donor template encoding a splice acceptor-EGFP cassette, which yielded site-specifc integration frequencies up to 25%. The development of methods for genetically engineered B cells opens the door to a myriad of applications in basic research, antibody production, and cellular therapeutics. -
A CRISPR Endonuclease Gene Drive Reveals Two Distinct Mechanisms of Inheritance Bias
bioRxiv preprint doi: https://doi.org/10.1101/2020.12.15.421271; this version posted December 15, 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 4.0 International license. A CRISPR endonuclease gene drive reveals two distinct mechanisms of inheritance bias Sebald A. N. Verkuijl1,2,+, Estela Gonzalez1,+, Ming Li3, Joshua Ang1, Nikolay P. Kandul3, Michelle A. E. Anderson1, Omar S. Akbari3, Michael B. Bonsall2, and Luke Alphey1,* 1Arthropod Genetics, The Pirbright Institute, Ash Road, Pirbright GU24 0NF, U.K 2Mathematical Ecology Research Group, Department of Zoology, University of Oxford, 11a Mansfield Road, Oxford OX1 3SZ, U.K 3Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, La Jolla, San Diego, United States, 92093 +these authors contributed equally to this work *[email protected] ABSTRACT RNA guided CRISPR gene drives have shown the capability of biasing transgene inheritance in multiple species. Among these, homing endonuclease drives are the most developed. In this study, we report the functioning of sds3, bgcn, and nup50 expressed Cas9 in an Aedes aegypti homing split drive system targeting the white gene. We report their inheritance biasing capability, propensity for maternal deposition, and zygotic/somatic expression. Additionally, by making use of the tight linkage of white to the sex-determining locus, we were able to elucidate mechanisms of inheritance bias. We find inheritance bias through homing in double heterozygous males, but find that a previous report of the same drive occurred through meiotic drive. -
Resistance to Gene Drive
Resistance to gene drive Many existing methods of malaria control are facing difficulties in maintaining effective performance, from insecticide and drug resistance to increasing costs of interventions. Controlling the malaria vector, the mosquito, remains the frontline for disease elimination. Target Malaria aims to develop new genetic technologies in a strategy known as gene drive to reduce the malaria mosquito population and thus reduce malaria transmission. This means that both the CRISPR/Cas transgene and female infertility spread through mosquito Gene drive involves a genetic mechanism population at far greater rates than would occur designed to spread through a population by normal inheritance mechanisms, ultimately while at the same time reducing the leading to reductions in the overall numbers of mosquito population and decreasing malaria vectors. malaria transmission. Gene drive is an especially appealing strategy as it could be deployed in remote locations, can Resistance potential work in parallel with the health care system while not being dependent on it, Just as antibiotic use to treat bacterial infection is potentially cost effective, can be used can lead to drug-resistant bacteria, any method in complementarity with other vector of mosquito population control - such as the control methods and has the potential for use of insecticides - can lead to mosquitoes that long term and sustainable impact. are resistant to that method. Likewise, the risk exists that some form of resistance to the gene drive could emerge. Resistance arises through a The Target Malaria gene drive approach exploits common cause of natural selection, whatever the a transgene encoding the nuclease CRISPR/ resistance is against. -
User Method: Alt-R CRISPR-Cas9 RNP Complexes for Microinjection Into B
user method genome editing Microinjection of Bactrocera tryoni (Queensland fruit fly) embryos How to prepare Alt-R® CRISPR-Cas9 ribonucleoprotein complexes for microinjection Contributed by Amanda Choo, University of Adelaide, Adelaide, South Australia The method presented here is provided by customers who have used the Alt-R CRISPR-Cas9 System. This can serve as a starting point for using the Alt-R CRISPR-Cas9 System in similar biological systems, but may not be fully optimized for your gene or application. IDT does not guarantee these methods, and application specialists at IDT can only provide general guidance with limited troubleshooting and support. Materials Kits and reagents Ordering information Enzyme dilution reagents: Option 1: HEPES and KCl General laboratory supplier Option 2: 1X Phosphate buffered saline (PBS) General laboratory supplier Option 3: Opti-MEM® media Thermo Fisher (cat # 51985091) IDT predesigned and custom crRNA: Alt-R CRISPR-Cas9 crRNA www.idtdna.com/CRISPR-Cas9 Alt-R CRISPR-Cas9 tracrRNA IDT (cat # 1072532, 1072533, 1072534) or Alt-R CRISPR-Cas9 tracrRNA – ATTO™ 550 IDT (cat # 1075927, 1075928) Alt-R S.p. Cas9 Nuclease 3NLS IDT (cat # 1074181, 1074182) Nuclease-Free Duplex Buffer IDT (cat # 11-01-03-01) Reagents for 10X Injection buffer: Sodium phosphate General laboratory supplier KCl General laboratory supplier Nuclease-Free Water IDT (cat # 11-05-01-14) HDR template: Ultramer® DNA Oligonucleotides IDT (www.idtdna.com/Ultramer) See what more we can do for you at www.idtdna.com. user method genome editing Methods A. Prepare buffers and enzyme 1. Prepare the Cas9 enzyme working buffer as described in the following table: Component Amount Final concentration 1 M HEPES, pH 7.5 200 µL 20 mM 1 M KCl 1.5 mL 150 mM Nuclease-Free Water ~6.5 mL* — Final volume 10 mL — * Add 6.5 mL of water, verify pH 7.5, and add more water to reach final volume.