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Drug Resistance

Antibiotics, herbicides, etc. that are used for selection purposes, even if they are not used to treat infections, should be included in this section.

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Drug Resistance Trait-Compromise

The deliberate transfer of a drug resistance trait to a microorganism when such resistance could compromise the ability to control the disease agent in humans, veterinary medicine, or agriculture requires NIH Director approval. It  is considered a "Major Action" under the NIH Guidelines: https://osp.od.nih.gov/biotechnology/faqs-about-major-actions-under-section-iii-a-of-the-nih-guidelines-for-research-involving-recombinant-or-synthetic-nucleic-acid/ .

To assist the IBC in determining the NIH Guideline III-A applicability, please comment in the Research Description of the protocol if the antibiotic resistance trait being introduced can be naturally acquired by the pathogen.
 
A therapeutically useful drug must be recognized in the scientific literature as a useful drug in vivo. The drug does not have to be the "first line" agent. A drug can be useful to treatment even if its use is limited to the treatment of a specific patient population, such as children or pregnant women, or primarily used outside of the U.S. where alternative drugs are not available.

To determine if a particular drug is considered a therapeutically useful drug, consult the following references:

Up to Date Online:  http://www.uptodateonline.com/utd/index.do
Johns Hopkins ABX Guide: http://www.hopkins-abxguide.org/
Red Book: Report of the Committee on Infectious Diseases: http://redbook.solutions.aap.org.ezproxy.library.wisc.edu/
Sanford Guide to Antimicrobial Therapy: http://search.library.wisc.edu/catalog/ocm37280724
Principles and Practice of Infectious Diseases:  http://search.library.wisc.edu/catalog/ocn370605770
Principles and Practice of Pediatric Infectious Disease: https://search.library.wisc.edu/catalog/9910053220302121
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Genes and DNA/RNA Fragments

Examples of how to enter genes fragments used to knock-down or silence genes.

Example A: sgRNA used for CRISPR modifications:

Gene name

sgRNAs

Gene source

Synthetic

Nature of insert or protein

Describe the function of the sgRNA you are using for CRISPR modifications

Gene use

Select all that apply (e.g., Knockout, Silencing, Up-regulations, etc.)

Gene use – other details

 

 

Example B:  Cas9 gene used for CRISPR modifications:

Gene name

Cas9

Gene source

Streptococcus pyogenes

Nature of insert or protein

Nuclease

Gene use

Cut nucleic acid

Gene use – other details

 

Example C: DNA carried on a vector that is introduced to upregulate a gene:

Gene name

Estrogen receptors: alpha, beta, GPER

Gene source

Human, Non-human primate

Nature of insert or protein

DNA-binding transcription factor

Gene use

Upregulating

Gene use – other details

 

Example D: siRNA

Gene name

Fragments of gene X

Gene source

Synthetic

Nature of insert or protein

Describe the function of the gene you are knocking down or silencing

Gene use

Knockout

Silencing

Gene use – other details

 

Examples on how to broadly describe a category of genes:

Example E

Gene name

Genes involved in energy metabolism (e.g., ldh, ackA, pyk, pta)

Gene source

E. coli, yeast

Nature of insert or protein

Metabolic genes

Gene use

Gene expression

Complement knockout (restore function in trans)

Gene use – other details

 

 

Example F

Gene name

Virulence factors (e.g., SpA, Protein A)

Gene source

S. aureus

Nature of insert or protein

Virulence factors

Gene use

Downregulating

Knockout

Complement knockout (restore function in trans)

Gene use – other details

Do not anticipate causing increased pathogenicity

Example of how to describe the use of a genome library:

Gene name

cDNA library

Gene source

Aspergillus

Nature of insert or protein

cDNA library

Gene use

Gene expression

Complement knockout (restore function in trans)

Gene use – other details

Not targeting toxin genes

Gene expression for protein production:

Gene name

Hsp

Gene source

E. coli, yeast, Drosophila

Nature of insert or protein

Chaperones

Gene use

Point mutation

Gene expression

Gene use – other details

 




 
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Construct: Construct Risk Attenuation

Plasmids that are non-conjugative or non-mobilizable lack the nic/bom elements required for mobility. Plasmids that lack F factors are considered nonconjugative (see http://en.wikipedia.org/wiki/F-plasmid for more information).

Agrobacterium plasmids that are disarmed lack the tumor inducing genes needed for pathogenicity.

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Genome Editing and Gene Drives

This section of the biosafety protocol is for information on the use of gene editing tools such as CRISPR/Cas9, TALENs, or ZFNs. It is also used to enter information on organisms or cells you are working with been created using genome editing tools. Even if these genome editing tools are not used directly for editing purposes, they should be added to this section.

If you are using microbes, animals, or plants that were generated by a collaborator or company using CRISPR-based genome editing tools, this should be clearly stated. Any additional information related to the potential risk of gene drive and how it would be mitigated that is not already included elsewhere in the protocol should be described here. Please clarify if the organism being modified would be at an advantage if inadvertently released into the environment. Describe how risk of release will be mitigated. If you are using CRISPR systems for purposes other than genome editing (e.g., CRISPRi, CRISPRa, CETChseq), the components of the editing system and their intended purpose should be clearly described. Many non-traditional uses of CRISPR are based on mutants that lack nuclease activity. Without this activity, there may be no risk of gene drive. Other risks (e.g., oncogenic potential) may still exist, however, and should be addressed.

This section uses branching logic; only questions relevant to the organism or cells being modified will be displayed. 

Example Tables

Example table: Gene drive is not relevant; only in somatic cells where oncogenic potential exists

Organism or Cells Being Modified

Human Cells or Cell Culture (Somatic)  

Targeted Sequence(s)

Genes involved in chromatin remodeling

Nuclease Type

CAS9 gene (cutting intact)  

Nuclease Type - Other

No Value Entered  

Delivery Vehicle(s)

pQCXIN , pCDNA

Delivery Vehicle(s) - Other

 No Value Entered  

Delivery Method(s)

Transfection; Other  

Delivery Method(s) - Other

liposome  

Integration of Genome Editing Components

No Value Entered  

Integration of Genome Editing Components - Other

No Value Entered  

Gene Drive

No Value Entered  

Gene Drive Additional Information

No Value Entered  

 

 

 

 

Example table: Gene drive is not relevant; only non-sexually reproducing bacteria.

Organism or Cells Being Modified

Microbe  

Targeted Sequence(s)

Genes involved in biofilm production  

Nuclease Type

Zinc finger nuclease (ZFN)  

Nuclease Type - Other

No Value Entered  

Delivery Vehicle(s)

PZFN-1  

Delivery Vehicle(s) - Other

No Value Entered  

Delivery Method(s)

Bacterial transformation  

Delivery Method(s) - Other

No Value Entered  

Integration of Genome Editing Components

No Value Entered  

Integration of Genome Editing Components - Other

No Value Entered  

Gene Drive

No Value Entered  

Gene Drive Additional Information

No Value Entered  

 

 

 

 

Example table: Sexually reproducing organism, gene drive could not occur because purified nuclease protein used

Organism or Cells Being Modified

Animal  

Targeted Sequence(s)

Genes involved in insulin production

Nuclease Type

Cas9 protein (no DNA, protein only)  

Nuclease Type - Other

No Value Entered  

Delivery Vehicle(s)

No value Entered

Delivery Vehicle(s) - Other

 Purified gRNA  

Delivery Method(s)

Direct injection  

Delivery Method(s) - Other

No Value Entered  

Integration of Genome Editing Components

Purified nuclease protein, will not integrate  

Integration of Genome Editing Components - Other

N/A  

Gene Drive

No  

Gene Drive Additional Information

No Value Entered  

 

 

 

 

Example table: Sexually reproducing organism, separate constructs designed to prevent integration and potential for gene drive

Organism or Cells Being Modified

Animal  

Targeted Sequence(s)

Genes involved in superoxide dismutase  

Nuclease Type

Cas9 nickase gene (single stranded cut)  

Nuclease Type - Other

No Value Entered  

Delivery Vehicle(s)

sgRNA, pSuperOx

Delivery Vehicle(s) - Other

No Value Entered 

Delivery Method(s)

Viral transduction  

Delivery Method(s) - Other

No Value Entered  

Integration of Genome Editing Components

Separate constructs, will not integrate at the target site (i.e., flanking sequences not homologous to sequences near the target site)  

Integration of Genome Editing Components - Other

N/A  

Gene Drive

No  

Gene Drive Additional Information

No Value Entered  

 

 

 

 

Example table: Sexually reproducing organism, single construct designed to allow integration for purpose of creating gene drive

Organism or Cells Being Modified

Animal  

Targeted Sequence(s)

Genes involved in fertility

Nuclease Type

CAS9 gene (cutting intact)  

Nuclease Type - Other

No Value Entered  

Delivery Vehicle(s)

pCRISPR  

Delivery Vehicle(s) - Other

No Value Entered  

Delivery Method(s)

Direct injection  

Delivery Method(s) - Other

No Value Entered  

Integration of Genome Editing Components

Single construct, could integrate at the target site (i.e., flanking sequences are homologous to sequences near the target site and could have the potential to be used as template for homology-based DNA repair)  

Integration of Genome Editing Components - Other

Integration required for experimental design.  

Gene Drive

Yes  

Gene Drive Additional Information

The purpose of the study is to enact gene drive to test approach for changing population dynamics of wild type mosquito. To prevent accidental release, work will be performed at ACL2 using double containment housing and traps; see Research Description for additional details.  

 

 

 

 

Example table: Sexually reproducing organism, single construct designed to allow integration and nuclease bred out of subsequent generations to avoid gene drive

Organism or Cells Being Modified

Plant  

Targeted Sequence(s)

Genes encoding human antibodies

Nuclease Type

Transcription activator-like effector nuclease (TALEN)  

Nuclease Type - Other

No Value Entered  

Delivery Vehicle(s)

pTALEN 5.0  

Delivery Vehicle(s) - Other

No Value Entered  

Delivery Method(s)

Agrobacterium-mediated gene transfer  

Delivery Method(s) - Other

No Value Entered  

Integration of Genome Editing Components

Single construct, could integrate at the target site (i.e., flanking sequences are homologous to sequences near the target site and could have the potential to be used as template for homology-based DNA repair)  

Integration of Genome Editing Components - Other

The goal is to integrate the genes (stability) as goal of experimental design.  

Gene Drive

No  

Gene Drive Additional Information

No Value Entered  

 

 

 

 

Example table: Sexually reproducing organism, gene drive could not occur because using catalytically inactive nuclease for purposes other than genome editing

Organism or Cells Being Modified

Animal  

Targeted Sequence(s)

Targeting the promoter of liver enzymes

Nuclease Type

dCas9 gene (cannot cut)  

Nuclease Type - Other

No Value Entered  

Delivery Vehicle(s)

pLENTI 2.0  

Delivery Vehicle(s) - Other

No Value Entered  

Delivery Method(s)

Viral transduction  

Delivery Method(s) - Other

No Value Entered  

Integration of Genome Editing Components

Dead nuclease, no gene drive potential (e.g., dCas9)  

Integration of Genome Editing Components - Other

N/A  

Gene Drive

No  

Gene Drive Additional Information

No Value Entered  

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Research Involving Human Subjects 

Due to revisions to the NIH Guidelines in 2019, the IBC is no longer required to consider requirements for RAC review and reporting of adverse events. Additionally, since 2016 new studies no longer need to submit responses to Appendix M to NIH though if your study has this document, please provide.

 

If you need assistance in completing the biosafety protocol for a human clinical trial, please contact the Office of Biological Safety.

 

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Still have questions? Call the Office of Biological Safety (OBS) at 608-263-2037. We are happy to help.

 


Keywords:
Drug resistance, genes, DNA, RNA, risk attenuation, RAC review status, human subjects, genome editing, recombinant, gene drive, construct 
Doc ID:
43075
Owned by:
Tara S. in ARROW - Institutional Biosafety Committee
Created:
2014-08-21
Updated:
2023-07-10
Sites:
ARROW - Institutional Biosafety Committee