CRISPR Biosafety Guidance
If non-human sgRNA, what is the % homology to human guide sequences? More homology = more risk.
More targets = more risk.
Experimental risk is higher if using amphotropic or pantropic retrovirus, lentivirus, especially to express human viral/cellular oncogenes or knock-down human suppressor genes. Watch for generation of replication competent retro/lentiviruses, insertional mutagenesis, expression of oncogenes and exposure of lung epithelium to aerosolized viral particles, and needlestick accidents.
The altered gene is always inherited in offspring; without containment the offspring rapidly out-compete natural population. If so, please refer to items below.
- Delivering a DNA cassette encoding the Cas9 gene and a single sgRNA flanked with sequences homologous to the target sequences can create a gene drive by continued expression and copying of the cassette converting heterozygotes to homozygotes
- Separate constructs provide molecular control; deletion/mutation/insertion does not occur until both constructs combined
- Use of standard plasmid constructs recommended over replication competent virus (see above)
- What are the possible effects on the host population due to changes in the organism?
- What happens if the organism/ materials/altered animal are released in the environment?
Use at least 2 of the following strategies to ensure required control measures are met:
- Molecular: split drive or 2-step editing: sgRNA and Cas9 placed on separate loci e.g. generate stable Cas9 cell line and deliver sgRNA into it (deletion/mutation/insertion occurs only in the transgenic Cas9-expressing organisms) or target synthetic sequences (deletion/mutation/insertion occurs only in organisms engineered with the target sequence). Also, use of reversal drives to overwrite the original.
- Ecological: Use the gene drive system where escaped organisms cannot find mates or persist in the outside environment.
- Reproductive: Use laboratory hosts/organisms that cannot reproduce with wild hosts/organisms
- Barrier/Containment: Prevent organisms from escaping from the lab, e.g. triply-nested containers, >3 doors, etc.
Consider and evaluate the following questions:
- How will you determine the unknown off-target effects/mutations?
- How much genotype change is needed to cause an apparent physical effect?
- What are the human/animal exposure concerns with the CRISPR-cas system, vector, cells or organisms used or due to the off-target effects?
Consider and evaluate if there are antibiotic resistance, increased lethality, or disease transmission being introduced or if these can occur accidentally.
Using proper lab procedures and following Biosafety office and/or IBC requirements:
- Modify research design to lower risk, if feasible
- Apply specific organismal, containment and/or procedural controls