Dong, Jixin, authorYang, Hua, advisorBelk, Keith E., committee memberMetcalf, Jessica L., committee memberWeir, Tiffany L., committee member2021-09-062021-09-062021https://hdl.handle.net/10217/233750The CRISPR-Cas9 system has emerged as a programmable and versatile tool for precise gene editing purposes. In addition to gene editing, the CRISPR-Cas9 system can be developed to kill targeted bacteria. In our previous studies, we developed a CRISPR-Cas9 targeted killing system with a guide RNA designed to specifically recognize the Shiga-toxic genes (stx1 and stx2). Delivery of this system into E. coli cells could effectively kill Shiga-toxin producing E. coli (STEC) cells. This current study was conducted to estimate potential biosafety risk associated with our CRISPR-Cas9-based targeted killing system when applied to kill STEC cells in a bovine cell line model system using next generation sequencing (NGS) analysis. A bovine cell line CPA 47 (ATCC® CRL-1733™) was cultured to reach 90% confluence. Then the bovine cells were subjected to one of four treatments: 1) bovine cell control: without any CRISPR treatment; 2) CRISPR/gRNA: treated with phages that carry the CRISPR system with the guide RNA targeting stx genes (106 PFU/flask); 3) CRISPR+O157: treated with phages that carry the CRISPR system but without the guide RNA targeting stx genes (106 PFU/flask) and E. coli O157:H7 strain Sakai cells (105 CFU/flask); and 4) CRISPR/gRNA+O157: treated with phages that carry the CRISPR system with the guide RNA targeting stx genes (106 PFU/flask) and E. coli O157:H7 strain Sakai cells (105 CFU/flask). Each treatment was conducted in four replicates for a total of 16 samples. After application of treatments, bovine cells from each sample were collected and divided into two portions: half for whole genome sequencing (WGS) and half for protein analysis. Whole genome DNA from each sample was extracted, purified, and sent to Novogene Bioinformatics Technology (Beijing, China) for library construction and WGS. Raw reads were subjected to quality control (QC) procedures to remove unusable reads. Clean reads after QC were aligned to the bovine reference genome (NCBI access number: ARS-UCD 1.2 USDA ARS) using Burrows-Wheeler Aligner (BWA) with default parameter. Based on the mapping results, SAMtools was used to detect individual SNP/InDel variants, and ANNOVAR was used for functional annotation of the detected variants. A total of 1078 Gb of output with 3593.12 million paired end reads (150 bp) were obtained for all 16 samples after NGS. After QC, a total of 1073 Gb of clean data output were obtained for all 16 samples. The average output for the four replicates within the control, CRISPR/gRNA, CRISPR+O157, and CRISPR/gRNA+O157 treatments was 59.1, 72.3, 72.3, and 65.9 Gb, respectively. The mapping rate of each sample ranged from 99.48% to 99.75%, and the 4X coverage ranged from 89.45% to 98.23%. For SNP detection, a total of 94,796,157 SNPs were identified in all 16 samples when compared with the reference genome. The number of SNPs with each sample ranged from 5,225,269 to 6,192,930. Of the total number of SNPs, 60.01% were located in intergenic regions (regions between genes), 36.40% in intronic regions (non-coding sequences of genes), and 0.79% in exonic regions (coding sequences of genes). Further analysis of exonic regions showed that the average SNPs for each treatment of control, CRISPR/gRNA, CRISPR+O157, and CRISPR/gRNA+O157 was 0.1964%, 0.2002%, 0.2002%, and 0.1948%, respectively. SNPs within each functional class, for example, stop loss, stop gain, synonymous, non-synonymous, at slicing sites, and upstream or downstream from transcription termination sites, the number of SNPs all showed no significant differences (P > 0.05) among the control and the three CRISPR treatments. For InDel detection, a total of 11,949,421 InDels were identified in all 16 samples, with each sample having between 604,387 and 817,716 InDels. Of the total number of InDels, 62.78% were located in intergenic regions, 38.54% in intronic regions, and 0.15% in exonic regions. The average exonic InDels in the control, and CRISPR/gRNA, CRISPR+O157, and CRISPR/gRNA+O157 treatments was 0.0371%, 0.0372%, 0.0375%, and 0.0372%, respectively. InDels within each functional class, for example, stop loss, stop gain, frameshift insertion/deletion, non-frameshift insertion/deletion, at slicing sites, and upstream or downstream from transcription termination sites, the number of InDels all showed no significant differences (P > 0.05) among the control and the three CRISPR treatments. Neither the SNP nor InDel data showed significant differences (P > 0.05) in the number of SNPs/InDels between the control and the CRISPR treatments when their WGS data were compared with the bovine reference genome. These results go along with our initial prediction that the biosafety concern of our CRISPR-Cas9 system should be low because our CRISPR system was designed to make a cleavage on target bacterial genomes but not on cattle genomes. In addition to coding regions, we are continuing with our analysis of the variants in non-coding regions. Results from this study will provide insights on how to further improve approaches or develop criteria on biosafety evaluation of the CRISPR-Cas9 system. Completion of this project will provide the beef industry with biosafety information regarding application of CRISPR as an alternative to antibiotics in cattle production.born digitalmasters thesesengCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.beef cattle productionCRISPR-Cas9 systemShiga toxin-producing E.colibiosafety riskantimicrobialomic-based analysisText