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CRISPR-Cas9 is revolutionizing how we conduct scientific research, treat disease, and develop new crops. The widespread impact of this genome-editing technology makes it critical for undergraduate students to understand and engage with this new tool. In this article, we describe a multi-week lab module that teaches undergraduates how to design CRISPR-Cas9 constructs and screen for CRISPR-modified genotypes. The module is conducted through the lens of independent research; students conduct a genotype screen for novel knockout mutations. In our module, students screen Zea mays (maize) seedlings for mutations in the MAD2 gene, which assists our ongoing investigation of meiotic chromosome segregation. This module can be adapted to knockout any gene in any organism, and thus align with an instructor’s research program. Engaging in original research helps undergraduate students develop independence and initiative in the lab as well as the molecular techniques of CRISPR-Cas9.
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Knockout of a secondary alcohol dehydrogenase gene in Nocardia cholesterolicum NRRL 5767 by CRISPR-Cas9 technology
In literature, Nocardia cholesterolicum NRRL 5767 (NC NRRL5767) is well-known for its ability to transform ~95% of added oleic acid, an abundant agricultural commodity, to value-added product of 10-hydroxystearic acid (10-HSA). A small amount of unwanted 10-ketostearic acid (10-KSA) was also produced. This microbe also transforms ~80% of added linoleic acid to 10-hydroxy-12(Z)-octadecenoic acid (10-OH-12-OD) (an isomer of ricinoleic acid) with minor 10-oxo-12(Z)-octadecenoic acid (10-oxo-12-OD). The conversion of oleic acid to 10-HSA and then to 10-KSA (or linoleic acid to 10-OH-12-OD and then to 10-oxo-12-OD) is catalyzed by oleate hydratase and secondary alcohol dehydrogenase (2o-ADH), respectively. The objective of this project was to knockout the 2o-ADH gene in NC NRRL5767 so that the sole biotransformation product from oleic acid would be 10-HSA. Here, we report construction of CRISPR/Cas9/sgRNA chimeric plasmid that specifically target 5’ coding region of the 2o-ADH gene by Golden Gate Assembly. The construct was confirmed by DNA sequencing and transformed into NC NRRL 5767 via electroporation. The transformants were selected by apramycin resistance and screened for the presence of the target insert (crRNA) by PCR. The ability of the selected transformants to transform oleic acid to 10-HSA was screened by TLC and further confirmed by GC-MS. Our results showed that two of the transformants produced only 10-HSA with no detectable 10-KSA from oleic acid suggesting successful knockout of the 2o-ADH gene. Final confirmation came from the isolation of genomic DNA from these two transformants and the wild type NC NRRL5767 (used as DNA template) and using 17 primers (locate at different positions along the 2o-ADH gene and the 5’ upstream of this gene) for PCR. To our best knowledge, this is the first report to knockout the target gene in Nocardia species by CRISPR-Cas9 technology.
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- Award ID(s):
- 1827209
- PAR ID:
- 10174163
- Date Published:
- Journal Name:
- 2019 ACS Midwest Regional Meeting
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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