Discovery of Escherichia coli CRISPR sequences in an undergraduate laboratory

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPRs) represent a novel type of adaptive immune system found in eubacteria and archaebacteria. CRISPRs have recently generated a lot of attention due to their unique ability to catalog foreign nucleic acids, their ability to destroy foreign nucleic acids in a mechanism that shares some similarity to RNA interference, and the ability to utilize reconstituted CRISPR systems for genome editing in numerous organisms. In order to introduce CRISPR biology into an undergraduate upper-level laboratory, a five-week set of exercises was designed to allow students to examine the CRISPR status of uncharacterized Escherichia coli strains and to allow the discovery of new repeats and spacers. Students started the project by isolating genomic DNA from E. coli and amplifying the iap CRISPR locus using the polymerase chain reaction (PCR). The PCR products were analyzed by Sanger DNA sequencing, and the sequences were examined for the presence of CRISPR repeat sequences. The regions between the repeats, the spacers, were extracted and analyzed with BLASTN searches. Overall, CRISPR loci were sequenced from several previously uncharacterized E. coli strains and one E. coli K-12 strain. Sanger DNA sequencing resulted in the discovery of 36 spacer sequences and their corresponding surrounding repeat sequences. Five of the spacers were homologous to foreign (non-E. coli) DNA. Assessment of the laboratory indicates that improvements were made in the ability of students to answer questions relating to the structure and function of CRISPRs. Future directions of the laboratory are presented and discussed. V C 2016 by The

Genome-editing applications of CRISPR–Cas9 to promote in vitro studies of Alzheimer’s disease

“…While sequencing an isozyme of the alkaline phosphatase gene, they uncovered a unique sequence nearby with a set of repeat sequences and spacer regions. 20 , 21 Later, homologous DNA sequences were identified in several bacterial species, including different Mycobacteria , E. coli , Streptococcus , and Campylobacter species. The CRISPR structure in bacteria contained viral (bacteriophage) sequences and could acquire additional repeats inside the sequence.…”

CRISPR-Cas Technology In and Out of the Classroom

“…In some cases, these experiences can be used to generate both laboratory reagents and data as well as data pertaining to student outcomes resulting from the implementation of CUREs. [21][22][23][24][25][26] Given student interest and the increased need to communicate cutting-edge research to undergraduate students, colleges and universities have begun instituting CRISPR-Casrelated CUREs with great success. Part of the reason why CUREs and other forms of course-based research that utilize CRISPR-Cas technologies are successful is due to the current curricula in molecular biology-based laboratory courses at diverse academic institutions, particularly fouryear colleges and universities.…”

“…Although data collection on the outcomes of CRISPR-Cas-based CUREs is ongoing, instructors report increased student learning and engagement after implementing CRISPR-Cas-based CUREs using various metrics. [21][22][23][24][25][26] Academic institutions have only recently started using CRISPR-Cas-based CUREs in their coursework; with increased interest in CRISPR-Cas-based CUREs, more rigorous studies on student achievement will undoubtedly help inform future activities at more institutions.…”

“…Students were asked to isolate genomic DNA from various E. coli strains, amplify the CRISPR locus, and then run DNA sequence analyses in order to identify previously uncharacterized CRISPR loci that can be studied in greater detail later in the future. 25 The Université Laval (Quebec City, Canada) has also used CRISPR-Cas-based CUREs to further examine the complexities of the CRISPR-Cas system. Dr. Sylvain Moineau's CRISPR-Cas-based CURE investigates natural CRISPR-Cas immunization in Streptococcus thermophilus and introduces students to how bacteria can expand their CRISPR array in response to challenge with virulent bacteriophage.…”

An experimental protocol for teaching CRISPR/Cas9 in a post‐graduate plant laboratory course: An analysis of mutant‐edited plants without sequencing

“…4 Several undergraduate laboratory courses using CRISPR/Cas systems with activities oriented to work with a number of model organisms such as Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster have been succefully carried out. [5][6][7][8][9] Here we present a laboratory activity using Arabidopsis thaliana as our model system that was designed to update a postgraduate course on plants, which helped students to learn theoretical and practical skills associated with CRISPR-Cas9. Moreover, we wanted to introduce postgraduate students to the main challenges in plant gene editing by CRISPR-Cas9 and how these can be overcome.…”