Biosafety Guidelines for CRISPR-cas Research

All research projects involving the CRISPR-cas genome editing technology must be registered with the UF Biosafety Office and reviewed by the Institutional Biosafety Committee (IBC).  The link to the registration forms is http://www.ehs.ufl.edu/programs/bio/forms
The following information is required to conduct proper risk assessment:

Risk Assessment Questions

Risk Mitigation

Will the research be done in bacteria, cell cultures, plants, insects, or whole animals?

Using human-derived cell lines will require BSL-2 practices.  Use in animals will require IACUC approval. Use in plants or insects will require specific containment.

Will you perform gene editing in embryos or germ-line cells? 

Applications of such experiments for human use are not permitted.

How will this technology be delivered to the host: using plasmids, viral vectors, nanoparticles, other delivery methods? 

The risks of using viral vectors must be considered e.g. Retro/Lentiviruses can stably integrate into the host cell chromosome and activate proto-oncogenes or suppress tumor suppressor genes at the site of insertion. Containment levels for the mode of delivery will be assessed based on the vector and gene hazards

Are the guide RNA (gRNA) sequences specific to humans/animals or can it affect both? 

What is the homology between the human and the animal gRNA sequences?  A higher containment measures may be required if high homology

How many genes are being targeted: Single? Multiple? Libraries: hundreds/thousands/more?

Off-target effects are possible if multiple genes are targeted and tumor suppressor genes may be among those targeted.  Higher containment practices maybe required if off-target effects are anticipated.

Do the experiments involve an environmental release of the modified organisms?  What are the possible effects on the population due to the changes created in the organism? Can this lead to ‘gene drive’ i.e. a 100% chance that certain traits may spread through an entire population of sexually reproducing organisms (vs. normal gene inheritance, in which there is a 50% chance)?

Gene drive containment can be obtained as follows: (labs should use at least 2 of these strategies)

Molecular: ‘Split drive’: Separate the components required for the gene drive i.e.: add gRNA to a cell line/animal already expressing CRISPR/cas9.  Higher containment may be recommended if cas9 and gRNA are delivered on the same plasmid/vector.

Have a ‘reversal drive’ in place to alter the drive created in the population

Ecological: Perform experiments in areas lacking the wild populations in which changes can occur.

Reproductive: Use laboratory strains that cannot reproduce in the wild, and synthetic sequences that are not found in the wild

Barrier: Use strict containment measures to prevent the release/escape of the modified organisms e.g. physical barriers, temperature control, air blast fans etc.

Do you anticipate any known potential off-target effects or mutations (i.e. disruption of genes not directly targeted by the gRNA sequences)?

-How will you determine the unknown off-target effects/ mutations?

-What are the human/animal exposure concerns with the off-target effects?

-How much genotype change is needed to cause an apparent physical effect on the organism? How does the route of exposure affect the outcome?

-Using a variant form of Cas9 can increase the target specificity and reduce the off-target effects.