Unit 4. Gene Editing. CRISPR

Unit 4. Gene editing. CRISPR. SECTION II: CELL STRUCTURE AND FUNCTION. GENE EDITING Previous technology: Recombinant DNA Genetic editing: Recombinant Gene editing DNA Modifications...

Unit 4. Gene editing. CRISPR. SECTION II: CELL STRUCTURE AND FUNCTION. GENE EDITING Previous technology: Recombinant DNA Genetic editing: Recombinant Gene editing DNA Modifications directed at the genome, their contexts (epigenetic marks) or their results (transcripts), using endonucleases * *Endonuclease: enzyme that cuts DNA Gene editing tools ZFNs (Zinc Finger Nucleases) High cost and difficulty, operating problems TALENs (Transcription Activator-Like Effector Nucleases) More difficult and expensive than CRISPR CRISPR-Cas What is CRISPR-Cas? Natural defense system against viruses in bacteria and archaea CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats Cas: CRISPR-associated The natural system acts in 3 phases: Acquisition, expression and interference CRISPR as a gene editing tool Francisco Martínez Mojica 1993. Describes repeated DNA sequences in archaea Haloferax mediterranei 2000. Same sequences in 20 different microorganisms 2005. The biological function of CRISPR is intuited  2012. Emmanuelle Charpentier 2013. Feng Zhang uses and Jennifer Doudna elucidated CRISPR-Cas9 on the genome the mechanism of CRISPR/Cas 9 of a living mammalian cell and demonstrated that it had potential use for gene editing Historical distribution of CRISPR documents available in Pubmed How does the technique work? DNA repair mechanisms ▪ nonhomologous end-joining ▪ homology-directed repair APPLICATIONS  Medical applications ❑GERMINAL GENETIC MODIFICATION In reproductive cells (sperm and eggs), which are modified by introducing functional genes into their genomes or by disrupting the wrong genes In early embryonic development Germ gene editing leads to permanent changes that are passed on to the next generations  2015 China 3PN EMBRYOS H U M A N GERM LINE MODIFICATION 2016 China CLONED VIABLE EMBRYOS 2016 China 3PN EMBRYOS The first experiment to modify the genome of human embryos is authorized in Spain https://www.observatoriobioetica.org/2020/02/se-autoriza-el-primer-experimento- de-modificacion-del-genoma-de-embriones-humanos-en-espana/32621 November 2018  Two twin girls were born, Lulu and Nana, whose DNA was modified with CRISPR/Cas to give them 'protection' against HIV The research has not been published in any scientific journal, so its data could not be verified "It's irresponsible," say scientists He Jiankui photographed in his lab Jiankui case specifications Falsified ethical approval documents Totally unfavorable benefit risk balance. He chose a target disease that can be prevented and treated. Chose a genetic target, the CCR5 gene, with important functions (risks) His experiment cannot be considered therapeutic, it is an “enhancement" Embryonic destruction Are the risks acceptable? Off-target effects Repair by NHEJ route: random insertions or deletions (mutations on target, low efficiency HDR route) Mosaicism  “the gene-edited embryos were mosaic. For example, embryo No. 16 contained many different kinds of alleles” (Liang P, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. 2015) “Because the edited embryos are genetically mosaic, it would be impossible to predict gene editing outcomes through pre-implantation genetic diagnosis (PGD)” Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human embryos. Alanis-Lobato et al. bioRxiv 2020.06.05.135913; doi: https://doi.org/10.1101/2020.06.05.135913 Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos. Zuccaro et al. Cell. 2020 Dec 10;183(6):1650-1664.e15. doi: 10.1016/j.cell.2020.10.025. Epub 2020 Oct 29. PMID: 33125898. Frequent gene conversion in human embryos induced by double strand breaks. Liang et al. bioRxiv 2020.06.19.162214; doi: https://doi.org/10.1101/2020.06.19.162214 "If human embryo editing for reproductive purposes or germline editing were space flight, the new data are the equivalent of having the rocket explode at the launch pad before take-off" Fyodor Urnov, estudia edición del genoma en la Universidad de California, Berkeley, https://www.nature.com/articles/d41586- 020-01906-4 Resource ‘‘What other valuable research is not being done as a result of this allocation investment? (...) what other medical In which cases is there no alternative for parents? needs are being underfunded?” 4-8 x 10-8 % 4-8 per ten billion couples for any gene Cases of severe monogenic diseases in which all children would We could also ask, more concretely, inherit the genotype of the disease. whether research efforts, talents, and funds should not be directed to the Autosomal dominant disease Autosomal recessive disease X-linked recessive diseases studies investigating somatic GE, If one parent carries two disease-causing alleles If both parents carry two disease-causing alleles If the expectant mother carries two disease- which does not involve so many (homozygous affected), all children will inherit in the same gene (homozygous affected), causing alleles (homozygous affected) contentious ethical aspects as GGE, all children will inherit and the male father the disease-causing genotype. the disease-causing carries one disease- yet, is a promising approach to treat genotype. causing allele on his only X chromosome (hemizygous affected), or even cure a number of genetic all the offspring would be affected. diseases.  What is the current position of the scientific community? The second international summit on human genome editing (27-29 November 2018) recommends charting the path towards germline gene editing: “the scientific understanding and technical requirements for clinical practice remain too uncertain and the risks too great to permit clinical trials of germline editing at this time. Progress over the last three years and the discussions at the current summit, however, suggest that it is time to define a rigorous, responsible translational pathway toward such trials” Statement by the Organizing Committee of the Second International Summit on Human Genome Editing November 28, 2018 https://www.nationalacademies.org/news/2018/11/statement-by-the-organizing-committee-of-the-second- international-summit-on-human-genome-editing ❑SOMATIC GENETIC MODIFICATION Clinical trials in this field are few but have In 2015, Layla Richards, a one-year-old girl who at 14 weeks of age already started, had been diagnosed with aggressive leukemia, received an especially ex vivo experimental treatment based on TALENs: the so-called UCART19 cells. trials CAR-T cell therapy (Chimeric Antigen Receptor T-Cell or chimeric antigen receptor of T cells) consists of extracting T lymphocytes from the patient (cells of the immune system). These are later modified by gene editing to recognize and attack tumor cells, and are transferred back to the patient's body so that, after being reprogrammed, they can recognize, attack and destroy cancer cells. A single 1 ml dose of these cells was administered and within 2 months the cancer cells disappeared. She is currently cancer-free and has no problems associated with the technique. More patients are receiving this treatment: https://clinicaltrials.gov/ct2/show/NCT02808442?intr=UCART19&rank=1 (EDIT-101) is an experimental medicine The trial will assess the safety, delivered via sub-retinal injection under tolerability, and efficacy of EDIT-101 development for the treatment of Leber in approximately 18 patients with this congenital amaurosis 10 (LCA10), an disorder. inherited form of blindness caused by mutations in the CEP290 gene.  24/01/2024 https://www.npr.org/sections/health- shots/2024/01/24/1226595039/gene-therapy-shows- promise-for-an-inherited-form-of- deafness#:~:text=For%20the%20first%20time%2C%2 0gene%20therapy%20is%20showing,at%20least%20 some%20hearing%20for%20five%20of%20them. Defect in the otoferlin gene. Protein necessary for the transmission of sound signals from the ear to the brain. First experimental programs in the United States, Europe and China. 5 clinical trials. First known case: Aissam Dam, 11 years old. He traveled to the United States to be treated at the Children's Hospital of Philadelphia. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8713252/ https://bioethicsobservatory.org/2023/03/wom an-with-sickle-cell-disease-cured-with-crispr- gene- editing/45101/#iLightbox[gallery45101]/0 Biotechnological applications “We should be aware that, if CRISPR technology is going to influence our lives in any way, it will probably do so sooner through what we are going to eat and not through new innovative treatments, which will still be late in coming and will not do so until the required levels of safety and efficacy are achieved”. Lluís Montoliu ❑LIVESTOCK ✓ The piglets subjected to editing lived with seven other normal conspecifics in the same pen, and then all of them were inoculated with the PRRS (porcine reproductive and respiratory syndrome) virus. ✓ Five days later the ordinary pigs contracted a fever and fell ill, but the genetically modified pigs remained healthy Nat Biotechnol. 2016 Jan;34(1):20-2. Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus. Whitworth KM, et al. CRISPR/Cas9- mediated generation of animal models and application in human health ❑AGRICULTURE ✓ CRISPR permite obtener cultivos de arroz resistentes a la contaminación radiactiva, inactivando el transporte de cesio radiactivo depositado en el suelo Plant J. 2017 Oct;92(1):43-56. Production of low-Cs+ rice plants by inactivation of the K+ transporter OsHAK1 with the CRISPR-Cas system. Nieves- Cordones M, et al.  Number of genes modified using CRISPR/Cas system with the aim of crops improvement Anna KorotkovaS. V. GerasimovaS. V. Current achievements in modifying crop genes using CRISPR/Cas system February 2019Vavilov Journal of Genetics and Breeding 23(1):29-37 DOI: 10.18699/VJ19.458 CRISPR-edited plants Soybean (Glycine max) with drought and salt tolerance; achieved by disrupting the Drb2a and Drb2b genes (double-stranded RNA-binding protein2 genes) Camelina with increased oil content; target genes not disclosed Setaria viridis, or green bristlegrass, with delayed flowering time; achieved by deactivating the S. viridis homolog of the Zea mays ID1 gene Waxy corn with starch composed exclusively of amylopectin; achieved by inactivating the endogenous waxy gene Wx1 that encodes a granule-bound start synthase catalyzing production of amylose White button mushroom (Agaricus bisporus) with antibrowning properties; achieved by knocking out a gene coding for polyphenol oxidase (PPO) Nat Biotechnol. 2018 Jan 10;36(1):6-7. With a free pass, CRISPR-edited plants reach market in record time. Waltz E. Crops that are made resistant to diseases via CRISPR/Cas9 Crop Disease/symptoms Targeted gene Triticum aestivum Powdery mildew disease TaMLO-A1 (wheat mildew resistance locus1) Oryza sativa Bacterial blight of rice OsSWEET11, OsSWEET14 (rice bacterial blight susceptibility genes) Rice blast disease OsERF922 (ethylene responsive factor transcription factor) Arabidopsis Turnip mosaic virus elF(iso)4E (elF transcription factor) thaliana disease Gossypium Cotton leaf curl disease CLCuD IR and Rep regions hirsutum Cucumis sativus L Ring spot disease, vein elF4E (eukaryotic translation initiation factor yellowing disease 4E) Nicotiana Leaf thickening, BeYDV (short intergenic region, trans acting benthamiana chlorosis, curling replication initiation protein) Gene Editing and Crop Improvement Using CRISPR-Cas9 System. Leena Arora, Alka Narula. Front Plant Sci. 2017; 8: 1932. TRANSGENIC ORGANISMS VS. GENE EDITING GMO Gene editing They contain foreign They contain small genes randomly alterations in existing introduced into the genes that give the genome that produce organism a beneficial new proteins in the trait by tweaking the organisms, giving it a levels of a protein beneficial trait that it that was already in did not have before the body  https://www.nature.com/articles/d 41586-018-05814-6 In the European Union, the Court of Justice (CJEU) ruled on July 25, 2018 that gene-edited crops must be subject to the same strict regulations as conventional genetically modified organisms. https://ec.europa.eu/info/sites/info/files/201 8_11_gcsa_statement_gene_editing_1.pdf Safety: What is important, the product or the method? The changes introduced by random mutagenesis are often more dramatic than those that result from gene editing techniques, and include not only numerous point mutations, but also significant deletions and rearrangements of genome fragments. The resulting mutant organisms require a lengthy analysis of the characteristics of the organisms to identify the few mutants that possess a novel desirable characteristic and do not exhibit unwanted characteristics. Despite this lengthy selection process, the end products ultimately selected are likely to have additional mutations beyond those that result in the desired trait.  Unwanted effects will occur less frequently in gene-edited products - potentially safer than traditional random mutagenesis products. The characteristics of the final product must be examined regardless of the underlying technique used. The safety of an organism is determined by multiple factors such as the specific characteristics of the organism, the environment in which it is grown, the agricultural practices used, and exposure to humans and animals rather than the technique used to produce it. The current approach does not adequately respect the motivation behind the precautionary principle of ensuring product safety. Situations may arise where two products are identical, but due to the different methods used in their production, they would have to meet completely different regulatory requirements. Europe has long been a bastion of skepticism about genetically engineered organisms, but today the European Parliament voted to lessen regulatory oversight of crops created through one type of DNA manipulation: gene editing. a “significant step forward” that would increase innovation and agricultural sustainability. Gene-edited plants can produce higher yields and better resist pests and pathogens, reducing the need for pesticides, for example.. The legislation passed by a relatively narrow margin of 307 to 263, with 41 members abstaining. https://www.science.org/content/article/european- parliament-votes-ease-regulation-gene-edited-crops ❑GENE DRIVE Later called gene drive Gene drive Ethical issues Unpredictable consequences for the ecosystem Appearance of other pests Imbalance in the food chain... Possible spread of mutated guide RNAs Dual-use technology