B08_Attachment_1__SOW_Zebrafish_Knockout_Line.docx

United States Department of the Interior

U. S. GEOLOGICAL SURVEY

STATEMENT OF WORK

GENERATION OF A THIAMINASE-KNOCKOUT ZEBRAFISH LINE

I. GENERAL INFORMATION

a. INTRODUCTION. The Department of the Interior (DOI), U.S. Geological Survey (USGS), Columbia Environmental Research Center (CERC) has a requirement for a zebrafish knockout line bearing a deletion of the tenA-like thiaminase I tandem duplication on chromosome 4 of the zebrafish genome.

b. BACKGROUND. The generation of the zebrafish knockout line bearing a deletion of the tenA-like thiaminase I genes will support lab capabilities to investigate thiaminase production in fish, and will provide a key component for research funded by a 5-year reimbursable grant from DoD, Strategic Environmental Research and Development Program (SERDP) to characterize the roles of multiple stressors in thiaminase production by prey fish, and to determine the contribution of the microbiome to thiaminase activity in prey fish.

c. DEFINITION/APPLICABLE DOCUMENTS.

i. N/A.

d. SCOPE. The contractor shall provide all materials, labor, equipment, travel, supervision, technical expertise, bioinformatic design & reagent development, zebrafish reagent preparation & validation, CRISPR founder screening & identification, zebrafish husbandry, and zebrafish CRISPR line confirmation necessary for generation of a sequence-confirmed zebrafish USGS01 line (F2 animals) carrying a CRISPR KO-deletion of tenA-like thiaminase I tandem duplication on chromosome 4 [si:ch1073-67j19.1 + si:ch1073-67j19.2].

II. WORK REQUIREMENTS

a. TECHNICAL REQUIREMENTS

i. Zebrafish TenA-like Thiaminase I tandem duplication knockout

1. All work shall be coordinated with the USGS Technical Liaison.

2. PHASE I - CRISPR DESIGN will include bioinformatic identification of candidate sgRNAs targeting locus of interest.

a. A variety of databases and algorithms will be used to identify the best sgRNAs with high likelihood of on-target specificity and cutting activity.

b. Selection of sites as close to the targeted region as possible will be preferred.

3. PHASE I - sgRNA SELECTION & SYNTHESIS

a. Four sgRNAs will be selected from bioinformatic analysis for subsequent testing.

b. Individual sgRNAs will be complexed with Cas9 protein prior to microinjection.

4. PHASE I - VERIFICATION OF sgRNA CUTTING

a. sgRNAs will be tested in vivo to determine cutting efficiency.

b. Tests will be conducted in Zebrafish strain: ABC wild-type (WT)

c. Injection mixes will consist of 4 individual sgRNAs (i.e. cutting efficiency test) and 4 combinations of one 5’ sgRNA + one 3’ sgRNA (i.e. deletion detection test), with each sgRNA complexed with Cas9 protein prior to microinjection.

d. Injected embryos will be screened for mutagenesis using standard methods.

e. If no mutagenesis is observed a repeat injection will be performed free of charge.

f. Poor cutting may result. If so, the client will be contacted to discuss further options, which may include testing alternative sgRNAs.

5. PHASE II - F0 INJECTIONS & SOMATIC MUTAGENESIS ASSESSMENT

a. Final CRISPR KO deletion mix will be assembled and injected to assess somatic editing.

b. Work will be conducted in Zebrafish strain: ABC WT

c. Injection mix will consist of two sgRNAs, Cas9 protein, and donor homology template (optional).

d. 400-500 embryos will be injected; a subset of injected embryos will be screened to confirm detection of somatic editing in F0 mosaic embryos.

e. Remaining F0 injected embryos will be reared to adulthood using zebrafish husbandry best practices. This will include daily monitoring to ensure the animals’ welfare consistent with NIH/OLAW guidelines.

f. At least 50% of F0 embryos submitted to the facility must survive to adulthood in order to progress with the project.

6. PHASE III - IDENTIFY GERMLINE TRANSMISSION OF DESIRED EDIT

a. F0 mosaic animals will be screened for detection of desired edit in F1 progeny.

b. Adult animals will be screened for germline transmission using a combination of screening techniques to identify germline founders. Because germline transmission is mosaic, clutches from identified founders will be raised to adulthood to isolate heterozygous F1 candidates.

c. Fin clips will be harvested from 2-3 month old F1 fish to confirm the edit by sequencing. Only animals bearing the desired edit will be retained.

d. The sequence-confirmed line carrying the edit will be propagated to ensure a sufficient number of animals will be available.

7. PHASE III - TRANSGENIC LINE DELIVERY

a. Sequence-confirmed mutants will be prepared for shipment.

b. Shipment will include either sequence-confirmed F1 fin-clipped adults, or F2 embryos from identified F1 heterozygous animals incrossed and/or outcrossed to ABC WT based on discussions with with the USGS Technical Liaison.

c. Transgenic animals will be sequence-confirmed and shipped, along with the following:

i. PCR reagents necessary to track the modified locus

ii. Donor homology repair template (if used)

iii. Authentication report describing line generation, validation & reagents

III. SUPPORTING INFORMATION.

a. GOVERNMENT FURNISHED PROPERTY (GFP): None.

b. SECURITY. N/A

c. PLACE OF PERFORMANCE. Contractor facility

d. PERIOD OF PERFORMANCE.

i. Ten to twelve months of bioinformatic and wet laboratory work is expected after the start date determined by acceptance of purchase order.

ii. The F2 animals of the transgenic line will be available no later than 24 months after the start date. Comment by Schneider, Kimberly R: What is the USGS01 line? And this will be a 2-year requirement for a final deliverable? Comment by Richter, Cathy A: Yes, 2 years

e. USGS TECHNICAL LIAISON

i. To be provided upon award.

f. SPECIAL CONSIDERATIONS

i. Any changes affecting the scope of work shall be originated from and approved by the Contracting Officer.

ii. All materials and workmanship shall be subject to the inspection and approval of the USGS Technical Liaison. All work unsatisfactorily performed shall be promptly corrected and made acceptable to the Government at no additional Cost.

iii. Final inspection and acceptance shall be performed with the contractor, on site by the USGS Technical Liaison upon completion of job performance.

iv. MATERIALS. The contractor shall furnish all supplies, materials, and equipment necessary for performance of the work included in this contract unless otherwise specified. Materials and supplies to be used shall be commercially available products of reputable manufacturers or suppliers and shall be of a quality to conform to applicable Federal Specifications.

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