Download CRISPR-Cas Genome Manipulation

Risk Assessment Considerations:
CRISPR-Cas Mediated Genome Manipulation
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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
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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
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Revision date: 9/20/2016 JBishop