NINDS 25-006671

STATEMENT OF WORK (SERVICES)
(SOW)

GENERAL INFORMATION
Title of Project:
Crispr Genomic Editing Service, Knock-in Service: PC12/VAMP2-SNAP-tag and Syx1A-Halo-tag.

Statement of Need and Purpose:
The National Institute of Neurological Disorders and Stroke (NINDS), Synaptic Transmission Section (STS), requires the use of CRISPR-Cas9 genome editing to insert tags into the endogenous loci of VAMP2 and Syntaxin in neuronal cells. This modification will enable precise tracking and characterization of these synaptic proteins, supporting ongoing research into mechanisms underlying synaptic function and their relevance to neurodevelopmental and neurodegenerative disorders.
Background Information and Objective:
The National Institutes of Health (NIH) is the nation’s leading medical research agency and the primary federal agency whose mission is to seek fundamental knowledge about the nature and behavior of living systems and to apply that knowledge to enhance health, lengthen life, and reduce illness and disability. NIH conducts and supports biomedical discoveries that improve public health and save lives.
The National Institute of Neurological Disorders and Stroke (NINDS) is one of 27 Institutes and Centers at the NIH. Its mission is to seek fundamental knowledge about the brain and nervous system and to use that knowledge to reduce the burden of neurological disease for all people.
The Synaptic Transmission Section (STS) at NINDS employs a combination of physiological, molecular, and imaging techniques to understand the mechanisms and dynamics of membrane trafficking during synaptic vesicle exocytosis and endocytosis. These tightly regulated processes are mediated by specialized proteins such as VAMP2 and Syntaxin, which orchestrate vesicle docking, fusion, and retrieval at the synaptic terminal. However, the precise molecular organization and interaction of these proteins in their native context remain poorly resolved.
To overcome current limitations in protein visualization at the nanoscale, STS utilizes advanced super-resolution microscopy, such as single-molecule localization microscopy (SMLM). However, conventional antibody-based labeling methods introduce spatial inaccuracies due to the large size of antibody complexes (up to ~16 nm when using primary and secondary antibodies), which is incompatible with the high resolution (1–3 nm) of modern imaging systems. These bulky probes also contribute to molecular crowding in the synaptic environment, potentially distorting the true architecture of the synapse.
To address these challenges and enable high-precision visualization, the current project will use CRISPR-Cas9 genome editing to insert small tags into the endogenous loci of VAMP2 and Syntaxin in neuronal cells. Tagging endogenous proteins at their native expression levels will preserve their physiological localization and function while allowing accurate super-resolution imaging.
This project will advance the spatial and functional characterization of synaptic proteins and provide critical insights into the nanoscale organization of the vesicle trafficking machinery. Ultimately, these efforts aim to deepen our understanding of synaptic transmission and its dysregulation in neurodevelopmental and neurodegenerative disorders.

Period of Performance:
The period of performance shall be seven (7) months from award.
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 specific requirements of this Statement of Work.
Specific Requirements:
The Contractor shall perform the services in five (5) different phases as follows:
Phase 1. Feasibility and Cell Line Preparation
a) The Contractor shall perform host cell quarantine, expansion, and mycoplasma testing.
b) Contractor shall confirm target gene sequences in the client-provided PC12 cell line using PCR and Sanger sequencing.
c) A report shall be provided summarizing results of sequence verification and any recommendations or limitations for proceeding with gene editing.

Phase 2. CRISPR Design and Donor Synthesis
a) The Contractor shall design and synthesize CRISPR guide RNAs targeting VAMP2 and Syntaxin1A based on reference genomic sequences or those verified in Phase 1.
b) The Contractor shall prepare Cas9 protein, gRNAs, and HDR donor templates (with SNAP-tag or other specified tags).
c) Donor sequences may include silent mutations to reduce off-target cleavage by gRNA.
d) QC (e.g., sequencing confirmation) will be documented and provided.

Phase 3. Genome Editing and Cell Pool Generation
a) Transfection of host cells with RNP complexes and donor constructs will be conducted.
b) The edited pools will be expanded, and editing efficiency will be evaluated using PCR and Sanger sequencing.
c) A brief report summarizing knock-in rates and editing outcomes for both targets will be provided.

Phase 4. Single Cell Clone Generation
a) The Contractor shall isolate single cells from edited pools using limiting dilution.
b) Up to 100 clones per target will be screened via PCR and Sanger sequencing to confirm successful integration.
c) A summary report will be provided with the genotype of verified clones, zygosity status, and editing success rates.

Phase 5. Clone Cryopreservation and Quality Control, Final Deliverables, and Documentation
a) Positive clones will be expanded, cryopreserved, and tested for viability and mycoplasma.
b) Two vials per clone will be prepared and stored for up to 6 months.
c) Final deliverables include a complete report with cell identity, QC results, sequence maps, and Sanger data and will be delivered to NINDS.

GOVERNMENT RESPONSIBILITIES
The Government will not furnish property, facilities, workspace, computers, or other equipment. Performance of the services shall be on-site at the Contractor’s facility. The Government will have the responsibility to review, approve, and accept the reports for each phase described in the SOW.

DELIVERY OR DELIVERABLES
The Contractor shall provide interim reports at the conclusion of each phase for review and approval by NINDS before moving to the next phase. The interim reports shall be sent electronically to the Contracting Officer Representative (COR). 
The Contractor shall provide one (1) final report encompassing the results of all phases of the specific requirements of this Statement of Work as well as information all genome-edited cell lines, including documentation of their properties, quality control, and validation. The final report shall be sent electronically to the COR.
Delivery of the genome-edited PC12 cell lines is required within seven (7) months after award. Delivery shall be Freight on Board (FOB) Destination and shall be delivered to Building 35, Room 2B-1006, 35 Convent Drive
Bethesda, MD 20892-3706.

REPORTING REQUIREMENTS 
The Contractor shall provide by-weekly updates on the progress of the project, or more often as appropriate. Updates shall be provided electronically to the COR. The Contractor shall update about the progress after each stage through email and/or other forms of electronic communication accepted by NINDS.

OTHER CONSIDERATIONS
Data Rights:
FAR Clause 52.227-14, Rights in Data-General (May 2014) is included in the solicitation and resultant award. Under 52.227-14(c)(1), the Contractor must obtain approval from the contracting officer to assert copyright in any data first produced in performance of this contract and published in academic, technical or professional journals, symposia proceedings, or similar works. Under 52.227-14(d)(2), the Contractor must obtain approval from the contracting officer to use, release to others, reproduce, distribute, or publish any data first produced or specifically used by the Contractor in the performance of this contract.

In addition, the Contractor shall keep the results of the acquisition confidential.