Location: Cell Wall Biology and Utilization Research
Project Number: 5090-31000-028-048-A
Project Type: Cooperative Agreement
Start Date: Jul 1, 2023
End Date: Jun 30, 2024
Objective:
1. Objective 1: Develop methods for targeted, cattle transcript depletion to enrich tissue-embedded microbial total RNA (TEMTR) in RNA samples extracted from cattle liver.
2. Objective 2: Develop methods for targeted, cattle transcript depletion to enrich TEMTR in RNA samples extracted from cattle blood sample.
Approach:
First, ARS will provide a list of highly expressed cattle genes for the liver, rumen, and blood. Cooperator will perform in silico analysis to identify CRISPR-Cas9 guiding sequences that will direct precise cleavage of targeted transcripts. Depletion performance will be estimated in silico against representative RNA-Seq data. This component will be carried out in the first 6 months of the collaboration.
Second, Cooperator will perform synthesis of all the designed products and package them into kits to be easily applied to RNA sequencing library preparation workflows. Due to the nature of transcript expression changes at different developmental stages, or disease and health conditions, liver and blood samples will be collected from various developmental stages and disease conditions. This component will be carried out in the 7th to 12th month of the first year.
Third, once the kit is designed, sequencing libraries will be prepared by ARS using the selective enrichment method. Liver and blood samples will be processed for sequencing libraries with or without the designed product. Multiple wet-lab procedures might be tested, specifically concerning when to add the cDNA digestion by Cas9.
Fourth, we will perform comprehensive bioinformatic analyses of all the samples we have sequenced to assess the efficacy of the targeted removal kit and to examine if there are any regions or transcripts from the host that are not efficiently removed, such as highly repetitive regions. Once such transcripts are identified, we will be continuing work with the Cooperator to optimize the targeted removal by designing and providing a booster pool of guides. Sequencing library preparation, sequencing, and bioinformatics analyses will follow to confirm the efficacy of the revised targeted removal kit. This component will be carried out in the remainder of the second year.
