Bio-ARROW - SmartForm - Recombinant Synthetic DNA/RNA Materials

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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.

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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.

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

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

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

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:

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

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 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. 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.