SOW.doc

STATEMENT OF WORK (SERVICES)

(SOW)

GENERAL INFORMATION

Title of Project:

Development of iPS report cell lines expressing HiBiT at specified target genes.

Statement of Need and Purpose:

The Stem Cell Translation Library requires the services of Synthego in order to meet project milestones. Synthego’s services portfolio includes a large selection of gene editing services that allow for knock-in of reporter tags at specific genes in iPS cells. The services offered that are of interest to the SCTL include:

· CRISPR-Edited iPS Cells – using a robust, automated CRISPR editing process that delivers consistently high editing in virtually any cell type. With over 60,000 edits completed and characterized. This expertise is applied to iPS cell editing to achieve unprecedented editing efficiencies of the highest quality. They offer knockouts, single nucleotide variants, and tags in user-supplied iPS cell lines-available in homozygous or heterozygous clone or pool format.

These services will be utilized by the SCTL to obtain reporter cell lines that will facilitate the development of novel iPSC differentiation protocols. The data obtained from this study will be used to generate new iPSC-derived cell types.

Background Information and Objective:

The SCTL has recently developed a protocol for differentiating hPSC into various cell types such as neurons, nociceptors, astrocytes and placental cells. However, one limitation in the field is that many iPSC-derived cell types retain immature phenotypes. Therefore, development of reporter cell lines for mature gene markers would facilitate the development of cell types that more closely resemble their somatic counterparts.

Likewise, stem cell field has not been able to developed robust protocols for the differentiation of iPSCs to more difficult to generate cell types, such as pancreatic beta cells and hepatocytes. Here, the proposed reporter cell line sets that follow the developmental trajectory of stem cells to somatic cell types would facilitate the establishing novel differentiation protocols.

Period of Performance:

The period of performance will commence after receipt of order (ARO) and last 20-24 weeks from receipt of the samples intended for gene editing.

SCOPE OF WORK

General Requirements:

Independently and not as an agent of the Government, the Contractor shall furnish all the necessary services, qualified personnel, material, equipment, and facilities, not otherwise provided by the Government as needed to perform the Statement of Work below:

The SCTL will provide the parental cell line. Then, the vendor shall QC the cells upon arrival, preform gene editing at specified targets, validate tag knock in, generate single cell clones, expand, re-validate knock in and QC cells. Finally, the vendor shall return gene edited iPSCs to SCTL.

Specific Requirements:

iPS Cell Editing iPS cell gene editing must be carried out on parental cell line provided by SCTL. To knock-in HiBiT tag, vendor must design CRISPR guide RNAs with low off target potential. Based on designed gRNA, vendor shall also design the donor template for tag knock-in at specified sites. Prior to gene editing cells will be qualified, CRISPR gene editing will be via electroporation. Following electroporation, cells will be allowed to recover and undergo pool analysis for gene knock-in. Then single cell clones will be expanded, reanalyzed for knock-in at specified genes followed by re-qualification for pluripotency and karyotypic abnormalities. Once gene knock in, normal karyotype and pluripotency are established reporter cell lines will be cryopreserved and shipped to SCTL.

The SCTL will provide cryopreserved parental iPS cell line for gene editing, then the vendor will:

For CRISPR gene editing:

· Qualify cells prior to gene editing

· Design CRISPR quide RNA

· Design donor template for specified genes with HiBiT tag

· Perform electroporation

· Perform pool analysis

· Perform single cell clonal expansion

· Perform clonal analysis

· Qualify cells after gene editing for knock-in at specified sites, pluripotency and karyotype

· Provide cryopreserved reporter cells and QC data to SCTL.

Specified gene targets;

1) PAX6 NM_000280.5

2) AFP NM_001134.3

3) AGRP NM_001138.2

4) ALB NM_000477.7

5) BARHL1 NM_020064.4

6) BLBP NM_001446.5

7) CALB1 NM_004929.4

8) CAMK2 NM_001220.5

9) CTIP2 NM_022898.3

10) DARPP32 NM_001242464.2

11) DLG4 NM_001365.4

12) FOXA2 NM_021784.5

13) FOXG1 NM_005249.5

14) GAD65 NM_000818.2

15) HB9 NM_005515.4

16) HEPACAM NM_152722.5

17) HLA-G NM_001363567.1

18) HOXA2 NM_006735.4

19) INS NM_000207.3

LEVEL OF EFFORT:

As per their internal standard operating procedures, the vendor shall be responsible for carrying out CRISPR gene editing, single-cell clones and QC of gene edited iPS cells.

GOVERNMENT RESPONSIBILITIES

The SCTL will provide parental iPS cell line for gene editing.

DELIVERY OR DELIVERABLES

Deliverables:

· Cryopreserved single-cell clonal reporter cell lines for specified genes.

· QC data.

REPORTING REQUIREMENTS

All reports shall be provided by Active Motif in electronic form – PDF, MS Excel sheets, MS Word, TXT files, PNG, TIFF figures, or other format based on the type of report. Raw sequencing data will be provided in the form of FASTQ files and will be furnished using as an FTP or HTML server download.

OTHER CONSIDERATIONS

Travel:

N/A Key Personnel:

Information System Security Plan:

Data Rights:

Data generated as a result of ALL services will be the properties of NIH, NCATS, SCTL.

Section 508—Electronic and Information Technology Standards:

Publications and Publicity:

The datasets derived by the SCTL from these experiments will be part of scientific publications and they will be made available to the public on the SCTL public-facing internet website. The contractor will not publish any result of the work.

Confidentiality of Information:

The Contractor will keep the results of the contract confidential. An MTA with NCATS OSA is in progress.

Version 11-30-2016-1

FAR Part 11—Describing Agency Needs