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From 2014.igem.org

Contents 1 Ethics of CRISPR-Cas Mediated Phage Therapy 2 Safety 2.1 Organism and Biological Part Safety 2.2 Risks Associated with Synthetic and Recombinant Biological Parts 2.3 Institutional Safety 2.4 Safety Concerns when using Antibacterials

Ethics of CRISPR-Cas Mediated Phage Therapy

An ethical question encountered during the experiment design concerns the manipulation of genetic material in pathogenic organisms. Concerns were raised regarding the possibility that genome cleavage by CRISPR-Cas9 could result in accelerated mutation rates in pathogenic organisms. Through limited experimentation, it appears that cells that survive CRISPR-Cas9 cleavage received a non-targetable copy of the plasmid, rather than acquiring new mutations that disrupts the target sequence in the bacterial genome. Therefore, it does not appear that CRISPR-Cas9 selects for mutations.

Safety

Organism and Biological Part Safety

Escherichia coli K12 variants

K12 strains of E. coli are commonly used, biosafety level 1 organisms. They pose little risk to lab members or the environment. They can be potentially harmful if ingested so basic lab safety such as gloves and hand washing were implemented to ensure lab member safety.

M13 phage

M13 phage are biosafety level 1 viruses. Phage only infect bacteria so the risk to humans is negligible. The helper phagemid have a weakened packaging signal which reduces their replication efficiency, however, these phage could infect E. coli and other bacterial species containing the F’ episome if the phage escaped laboratory containment. M13 phage are not synthetic organisms; their environmental impact if they escaped laboratory containment is small due to their disrupted packaging signal so they would be quickly outcompeted by wild-type phage. Precautions such as hand washing and use of gloves were implemented to reduce risk to lab members. Proper disposal of samples was implemented to avoid environmental contamination.

Endogenous CRISPR-Cas(BBa_K1218011)

Part BBa_K1218011 was apart of the 2014 iGEM distribution. The encoded CRISPR-Cas9 system is derived from Streptococcus pyogenes, a biosafety level 1 organism, and is widely used in gene editing. Malicious use of the CRISPR-Cas9 system is theoretically possible as it is a system that targets specific genetic sequences and could be used to target certain alleles. However, this technology has been used for several years for the purpose of gene editing and no health risks have been reported.

Risks Associated with Synthetic and Recombinant Biological Parts

No organism used in these experiments pose any significant risk to health or the environment. The Cas9 can only target DNA sequences that are adjacent to a PAM site and are complementary to its guide RNA. The CRISPR-Cas9 plasmid contains the M13 packaging signal but no other phage genes; therefore, this part can be packaged into, but cannot make, phage. A separate helper phagemid is needed to assemble the phage coat but the packaging signal on this part is disrupted; thus, it cannot be efficiently packaged into a phage. This reduces the chances of uncontrolled phage replication. Some phage may still contain the helper phagemid but they can only infect bacteria with an F’ episome.

Institutional Safety

The University of Colorado has an Institutional Biosafety Committee(IBC) that oversees the safety and ethics of projects at the university. The IBC has not raised any concerns regarding the safety protocols or synthesized organisms used in this experiment. All reagents and organisms used in this experiment were disposed of by the University Environmental Health and Safety services in accordance with institutional, national and international safety standards.

Safety Concerns when using Antibacterials

The overuse of antibiotics is a major cause of emerging antibiotic resistance. This project is aimed at developing a system that enables physicians to counter bacterial resistance to therapeutics at a rate that is more competitive to that of evolution. The health and safety of humanity is threatened by gene flow through bacterial species by conjugation and other methods. Through bioengineering, strain-specific CRISPR-Cas therapeutics can be designed within several days to target pathogenic strains with new mutations, while simultaneously leaving the symbiotic species in the microbiome unaffected. The end result is safer, more adaptive, and more versatile treatment methods.

Online safety form found here.