Genome Editing PDF

Methods for vaccine production before the COVID-19 pandemic Producing whole virus-, proteinand vector-based vaccines requires large-scale cell culture Resourceintensive process limits rapid vaccine production in response to outbreaks and pandemics https://www.nobelprize.org/prizes/medicine/2023/pre...

Methods for vaccine production before the COVID-19 pandemic Producing whole virus-, proteinand vector-based vaccines requires large-scale cell culture Resourceintensive process limits rapid vaccine production in response to outbreaks and pandemics https://www.nobelprize.org/prizes/medicine/2023/press-release/ Observation: bases in RNA from mammalian cells, but not in vitro transcribed mRNA, frequently chemically modified • produced different variants of mRNA, with unique chemical alterations in their bases • inflammatory response abolished if with base modifications! Further studies: delivery of mRNA generated with base modifications markedly increased protein production compared to unmodified mRNA mRNA vaccines: barriers Issues with in vitro transcribed mRNA: • considered unstable • challenging to deliver: requires sophisticated carrier lipid systems • triggers inflammatory reactions The Nobel Laureates discovered that base-modified mRNA can be used to block activation of inflammatory reactions (secretion of signaling molecules) and increase protein production when mRNA is delivered to cells. mRNA vaccines: delivery via lipid nanoparticles Lipid nanoparticles protect RNA from being broken down in the body, help to ferry it through cell membranes Moderna, Pfizer RNA vaccines carried by lipid nanoparticles with PEG ➢PEG addition to surface of nanoparticles makes them last in the body much longer mRNA vaccines currently in clinical trials mRNA therapeutics currently in clinical trials Genome editing Knockout mice used to study genetic diseases Knockout mice good model systems for ❑investigating nature of genetic diseases ❑efficacy of different types of treatment ❑for developing effective gene therapies Many inbred mice strains exist e.g, for obesity, body size, muscularity Site-directed mutagenesis • Technique wherein changes in DNA are made at a desired position • Usually done to determine effect of changing DNA sequence on gene or DNA function • Usually done using PCR Site – directed mutagenesis: steps ❑ Clone DNA of interest into plasmid vector ❑ Denature plasmid DNA to produce single strands ❑ Anneal synthetic oligont with desired mutation to target region ❑ Extend mutant oligonucleotide using a plasmid DNA strand as the template. ❑ Transformation in E. coli Figure 8-63 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008) Original plasmid digested by Dpn1, which recognizes methylated DNA Figure 8-63 (part 2 of 2) Molecular Biology of the Cell (© Garland Science 2008) Directed changes in DNA Substitutions: oligonucleotide-directed mutagenesis ❑e.g., for changing amino acid specified ❑Use oligo primer that is complementary to region of the gene except for target base ❑mismatch of 1 of 15 bp’s tolerable if annealing is carried out at appropriate temp Site-Directed Mutagenesis of Subtilisin • residues of the catalytic triad mutated to alanine • activity of mutated enzyme was measured • mutations in any component of the catalytic triad cause a dramatic loss of enzyme activity Cassette mutagenesis A variety of mutations, e.g., insertions, deletions, multiple point mutations, can be introduced into gene of interest Random vs. targeted gene insertion • early vectors used for gene insertion place GOI anywhere in the genome • However, possible to design vectors that replace GOI ➢Can restore function in a mutant animal ➢Can knock out function of a particular gene Leptin receptor knockout mice db/db DB/DB Analysis of gene function: gene silencing • RNAi = post transcriptional gene silencing ❑mRNA is made, but then degraded ❑Blocks gene function • RNAi pathway produces small interfering RNAs (siRNAs) that silence complementary target genes • Elucidated by Fire and Mello ❑2006 Nobel Prize for Physiology or Medicine • Function in organisms: immune response ❑Defensive mechanism against molecular parasites (transposons, viral genetic elements) RNAi • initially discovered, characterized in C. elegans • dsRNA 10x more effective in silencing target gene expression than antisense or sense RNA alone • RNAi can be used on many organisms where genetic analysis has been unavailable RNA interference Synthetic siRNAs • Synthetic siRNAs that target any sequence can be prepared by chemical synthesis • In mammalian cells, siRNAs range in effectiveness at knocking down target gene expression (50-95%) • effectiveness of an siRNA is dependent upon target sequence sense 5´-NNNNNNNNNNNNNNNNNNNUU-3´ ||||||||||||||||||| 3´-UUNNNNNNNNNNNNNNNNNNN-5´ antisense Example of siRNA knockout • siRNA targeting rev mRNA sequence encoding rev-EGFP fusion protein • Sense (S) or antisense (AS) strand of siRNA alone does not effect knockdown of rev-EGFP expression • An irrelevant siRNA sequence (IR) does not effect knockdown of rev-EGFP expression Gene disruption/gene knockout provides clues to gene function • A mutated version of the target gene is introduced into an embryonic stem cell • recombination takes place at regions of homology • Normal copy “knocked out” by foreign gene • Cell is inserted into embryos, producing knockout mice Gene knock-in A knock-in replaces an endogenous gene with an alternative sequence Gene inserted into a specific locus; "targeted" insertion Genome editing What’s the difference from genetic engineering?!? Altering the sequence of DNA “in situ” ❑Knock-outs ❑Point mutations ❑Gene insertions DNA is inserted, deleted or replaced in the genome using engineered nucleases, or "molecular scissors“ Recall DSB DNA repair mechanisms! non-homologous end joining (NHEJ) produces random mutations (gene knockout) • NHEJ while HDR. homology-directed repair (HDR) uses additional DNA to create a desired sequence within the genome (gene knock-in) Genome editing mechanisms • Restriction enzymes! • Zinc finger nucleases (ZFNs) • Transcription Activator-Like Effector-based Nucleases (TALENs) • CRISPR-Cas9 system Zinc finger nucleases (ZFNs) 2 domains: 1. zinc finger DBD: recognizes 3bp site on DNA; can be combined to recognize longer sequences 2. engineered nuclease (Fokl): makes nonspecific ds break Applications: ✓used to disable CCR5 on human T-cells, a major receptor for HIV ✓used to edit tumor-infiltrating lymphocytes; treatment for metastatic melanoma TALENS gene editing: single nucleotide resolution • Transcription activator-like effector nucleases (TALENs) • Also 2 components: 1. Fokl nuclease to cut DNA 2. DBD: transcription activator-like effectors (TALEs), tandem arrays of 33-35 amino acid repeats aa repeats have single nt recognition → increased targeting, specificity compared to ZFNs Issues with 1st gen genome editing tools ZFNs: G-rich target sites more efficient at editing versus nonG-rich sites ❑Also, interaction with DNA is modular, so editing efficiency low TALENS: Delivery into cells challenging; TALENs much larger than ZFNs (~6kb vs. ~2kb) CRISPR-Cas9: RNA-guided genome editing CRISPR/Cas = clustered, regularly interspaced short palindromic repeats - CRISPR-associated proteins Known mechanism in bacteria used to fight off invading viruses direct repeats tracrRNA Cas9 Cas1 Cas2 Csn2 Streptococcus pyogenes CRISPR array spacers CRISPR sequences transcribed to RNA CRISPR repeats and spacer sequences derived from viral pathogens; transcribed to RNA upon viral entry Natural CRISPR Pathway 1. transcription of pre-crRNA and tracrRNA 2. binding of tracrRNA to pre-crRNA 3. cleavage of guide RNA from pre-crRNA 4. binding of inactive Cas9 nuclease to the guide RNA to produce the active Cas9 nuclease Engineered CRISPR: transcription of Guide RNA as a single sequence 1. transcription and translation of Cas9 nuclease 2. binding of Guide RNA to Cas9 and Activation of Cas9 https://sites.tufts.edu/crispr/genome-editing/ Homework! Due 10/6/23 Research on newer genome editing approaches that came after CRISPR-Cas9 • Identify at least 3 • Compare methodologies and specific example of where applied thus far • Advantages/disadvantages compared to CRISPRCas9 Class activity for 10-10-23 Assemble into your groups and discuss the following about the guest lecture of Dr. Villacastin ✓Three things you learned about plant tissue culture ✓Two things that are the most practical/useful about the techniques discussed ✓1 question you have about any part of the lecture You have 10 minutes! Discussion outputs will be uploaded to Canvas Genome editing via CRISPR CAS9 CRISPR/Cas9 expression plasmids used to edit the genome of human cells have codons optimized for human cells Variations of CRISPR-Cas9 applications If nuclease active sites of Cas9 are mutationally inactivated, nucleasedeficient complex still binds tightly to its target ❑ can block movement of RNA polymerase ❑ Gene silencing! If inactivated Cas9 protein fused to gene activator protein, can trigger enhanced transcription CRISPR variants Published by AAAS E Pennisi Science 2013;341:833-836 How CRISPR lets you edit DNA Andrea M. Henle CRISPR genome editing CRISPR gene therapy for sickle cell anemia Harvest bone marrow stem cells Before chemotherapy After chemotherapy CRISPR/Cas 9 genome editing Cas Guide 9 RNA Implant edited stem cells in patient Who’s doing genome editing, and for what?!? Medical applications ❑engineering human cells to treat a disease or controlling a human pathogen ❑upstream medical research tools: ➢ edited human cell lines ➢ animal models for human diseases ➢ animal sources for xenotransplantation ❑100 diseases, covering most categories of the international classification of diseases ➢ Cancer: 131 patent families; 31 describe immunotherapy ➢ Treatment of viral infections : 112 patents ❑Gene therapy, eg, gene replacement in somatic cells: ➢ Alzheimer’s and other nervous system disorders ➢ blood diseases (eg, βthalassemia and anemia) ➢ musculoskeletal diseases (eg, bone diseases and rheumatoid arthritis) ➢ muscular dystrophies (eg, Duchenne’s disease) ➢ nucleotide repeat disorders ➢ retinal or other ocular diseases (e.g. glaucoma) ❑Induced pluripotent stem cell (iPSC) modifications for ex vivo therapy Medical applications Gene knockout to: ❑treat allergic, endocrine, nutritional and metabolic diseases (diabetes, cystic fibrosis, hypercholesterolemia, hyperlipidemia, obesity, etc) ❑prevent coronary atherosclerotic, heart and other cardiovascular disease -destroy senescent cells ❑target metastasis-related genes Microorganisms (fungi or bacteria) ❑identification of serotypes ❑growth of microorganisms ❑suppression of resistance to antibiotics ❑biofuel production ❑production of molecules of interest CRISPR: industrial applications Animal cells ❑high-throughput screens of volatile flavor and fragrance compounds ❑activation of taste receptor genes ❑manufacturing skeletal muscle for dietary consumption ❑kit for detecting pyrogen ❑production of hypoallergenic cats ❑silk production Plant breeding (130 patents) ❑rice (64 patents) ❑maize (11) ❑wheat (5) ❑tomato (4) ❑potato (3) ❑tobacco (2) Targeted traits: ❑male sterility (16 patents) ❑virus resistance or detection (9 patents) ❑herbicide tolerance (6 patents) ❑fungi, bacteria and pest resistance ❑Cotton ❑plant stature or architecture ❑nut grass ❑flowering time ❑oilseed plants ❑pollination and fertility parameters ❑sorghum ❑plant aging, fruit shelf-life Agricultural applications ❑yield ❑stress resistance Agricultural applications Private companies filed 27% of ‘plant’ patents, 5% of ‘farm animal’ patents ❑DuPont-Pioneer (USA; 12 patents) ❑KWS Saat (Germany; 5 patents) ❑Keygene (Netherlands; 4) ❑Dow Agrosciences (USA; 3 patents) ❑Beijing DBN Technology (China; 3 patents) For farm animals: Breeding (50 patents) ❑pigs (22 patents) ❑sheep (12) ❑mammals in general (5) ❑cows or rabbits (1 each) ❑fish (4) ❑birds (3) Risk considerations of genomeedited crops Risk assessment of GM plants in Europe based on comparing conventional crop, its GM counterpart Genome editing reduces the amount of uncertainties due to precise targeting ➢ Consists of: Crops with specific mutation developed via genome editing considered safer than crops with that same mutation resulting from mutation breeding 1. molecular characterization of GM plant 2. comparative analysis of compositional phenotypic, agronomic properties 3. safety assessment for humans and animals (allergenicity, nutritional value, toxicology) 4. safety assessment of the environment ➢ May many additional random mutations ➢ Effects of random mutations unknown Artificial selection These common vegetables were cultivated from forms of wild mustard. This is evolution through artificial selection. What are gene drives? Gene drives = systems of biased inheritance Ability of a genetic element to pass from parent to offspring via sexual reproduction is enhanced Key features and potential uses of gene drives Defining features: ❑Spread and persistence ❑Potential to cause irreversible ecological change Two potential uses: ❑Population suppression: Decrease numbers ❑Population replacement: Change genetic characteristic(s) Defining features of gene drives Potential to cause irreversible ecological change • If successful, genome changes become parts of genetic profile of organism ❑Population suppression: Decrease numbers ❑Population replacement: Change genetic characteristic(s) CRISPRCas9 has allowed researchers to more effectively insert a modified gene and the gene drive components Potential uses: eliminate diseases such as malaria, dengue, yellow fever, West Nile, sleeping sickness, Lyme, and others by altering insect species to no longer spread them 'Gene drive' mosquitoes engineered to fight malaria NATURE | NEWS 23 November 2015 Humans contract malaria from mosquitoes infected by Plasmodium parasite Crispr-Cas9 used to introduce 2 genes that cause resistance to Plasmodium resulting mosquitoes passed on modified genes to >99% of offspring Gene drive: questions about science, ethics, and governance • Could gene drives have unintended consequences for public health and the environment? • Do we know enough to consider releasing gene-drive modified organisms into the environment? • Should a gene drive be used to suppress or eliminate a pest species? • How do we decide where gene-drive modified organisms could be released? What should be governments’ role? Gene editing can now change an entire species - forever Jennifer Kahn • TED2016 Human genetic engineering: two potential applications 1. "Somatic" genetic engineering • targets the genes in specific organs and tissues of the body of a single existing person without affecting genes in their eggs or sperm. • Basis of gene therapy 2. "Germline" genetic engineering • targets the genes in eggs, sperm, or very early embryos. • alterations affect every cell in the body of the resulting individual, and are passed on to all future generations. • banned in many countries (but not in the US) ..\..\youtube downloads\NS50\We Can Now Edit Our DNA. But Let's Do it Wisely _ Jennifer Doudna _ TED Talks.mp4 • Past efforts to manipulate the genetic code of life have been slower, more cumbersome and more unpredictable • CRISPR technology is vastly more efficient; possibility of it being practiced widely is that much more real • Many countries ban human germline editing outright • The US forbids the use of federal funds for such research Some potential reasons for genome editing human cells, including those of the germ line and early embryos • Basic understanding of human biology: the role of specific genes and processes • To create and study models of human genetic disease in vitro • To treat disease (somatic cells) • Germline changes to avoid/prevent genetic disease • Germline alterations to give “genetic enhancement”