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Risk Assessment Considerations: CRISPR-Cas Mediated Genome Manipulation [email protected] (805) 893 - 8894 Risk Assessment for Researcher Exposure Practices and precautions will mainly depend on the host cell type(s) and the delivery method. 1. Which organism(s) and cell type(s) are being targeted? 2. If the guide RNA (gRNA) sequences are targeted to an animal, have potential on- and offtarget sites been identified in the human genome? http://crispr.mit.edu/ (Zhang lab, MIT); http://www.e-crisp.org/E-CRISP/aboutpage.html (German Cancer Research Center); http://www.rgenome.net/cas-offinder/ (Center for Genome Engineering, Institute for Basic Science, Korea) 3. What’s the function of the gene(s) being targeted? 4. What’s the objective in using CRISPR? a. Gene drive b. Gene knockout/indel c. Genome-wide screening d. Activate, enhance or repress expression e. Imaging or purification of gene loci f. Fuse gene with a reporter g. Generate a point mutation 5. How will the CRISPR components be delivered, and how will they be expressed? CRISPR components can be delivered via transfection/transformation, electroporation, transduction, or injection as protein, RNA, or DNA. Expression system Lentiviral vector Delivery method Transduction; single or separate transfer vectors Application Stable expression; common for genome-wide screens Adeno-associated viral vector Transduction Transient or stable expression Cas9 protein or mRNA and gRNA Microinjection, cationic lipid transfection or electroporation Transfection Transient expression; used to generate transgenic embryos or create a stable cell line Transient or stable expression in a mammalian cell line Mammalian expression vector 6. If viral vectors will be used to express and deliver CRISPR components, consider Page 1 of 2 Revision date: 9/20/2016 JBishop i. the potential for replication competent viruses ii. vector tropism iii. insertional activation of oncogenes/tumor suppressor gene inactivation 7. CRISPR/Cas9 mediated gene drive determinants: a. Cas9 gene and guide RNA are physically linked on the same construct, b. Cas9 and gRNA flanked by homologous arms that target the insertion site, and c. Introduced into embryos or germ line cells Risk Assessment for Environmental Release Generally of concern with experiments targeting a quickly developing, sexually reproducing organism, e.g., fruit flies or mosquitoes. Omar S. Akbari et al. Science 2015; 349:927-929 Page 2 of 2 Revision date: 9/20/2016 JBishop