Bio-ARROW - SmartForm - Recombinant Synthetic DNA/RNA Materials
- Drug Resistance
- Drug Resistance Trait-Compromise
- Genes and DNA/RNA Fragments
- Construct Risk Attenuation
- Genome Editing and Gene Drives
- Research Involving Human Subjects
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.
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.
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
Genes and DNA/RNA Fragments
Examples of how to enter genes fragments used to knock-down or silence genes.
|
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 |
|
|
Gene name |
Cas9 |
|---|---|
|
Gene source |
Streptococcus pyogenes |
|
Nature of insert or protein |
Nuclease |
|
Gene use |
Cut nucleic acid |
|
Gene use – other details |
|
|
Gene name |
Estrogen receptors: alpha, beta, GPER |
|---|---|
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Gene source |
Human, Non-human primate |
|
Nature of insert or protein |
DNA-binding transcription factor |
|
Gene use |
Upregulating |
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Gene use – other details |
|
|
Gene name |
Fragments of gene X |
|---|---|
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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:
|
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 |
|
|
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 |
|
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 name |
Hsp |
|---|---|
|
Gene source |
E. coli, yeast, Drosophila |
|
Nature of insert or protein |
Chaperones |
|
Gene use |
Point mutation Gene expression |
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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.
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 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 |
|
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|
|
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|
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 |
|
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|
|
|
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|
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 |
|
|
|
|
|
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|
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 |
|
|
|
|
|
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|
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. |
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Example table: Sexually reproducing organism, single construct designed to allow integration and nuclease bred out of subsequent generations to avoid gene drive |
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|
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 |
|
|
|
|
|
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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 |
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.
Still have questions? Call the Office of Biological Safety (OBS) at 608-263-2037. We are happy to help.