An Undergraduate Laboratory on Recombineering and CRISPR/Cas9-Assisted Gene Editing in Escherichia coli

Laboratory experience is vital to undergraduate science education. It allows students to observe and conduct engaging experiments to enhance their skills and literacy, helps them retain knowledge, and deepens their understanding of related content covered in lectures. This paper reports a 4-week undergraduate laboratory exercise on Escherichia coli gene editing by recombineering, recombination-mediated genetic engineering, with or without clustered regularly interspaced short palindromic repeats and their associated protein 9 (CRISPR/Cas9). Gene editing makes precise modifications to the DNA of living organisms that influence their development and functions. As technology evolves, recombineering and CRISPR/Cas9 have replaced methods that use restriction enzymes and DNA ligase and are applied to a wide variety of research and applications. It is necessary to introduce undergraduates to these two rapidly growing technologies. Student results obtained from the lab indicate that antisense single-stranded oligodeoxynucleotide (ssODN) has a 15-20 times higher recombineering efficiency than the sense strand. Treatment with a plasmid containing the crRNA target of CRISPR/Cas9 increased recombineering efficiency. Instructional assessments, based on student feedback, revealed that the lab had clear objectives, instructions, and explicit protocols, with sufficient time to complete them, and was found to be interesting and worthwhile. Student learning outcomes, assessed by comparing pre-lab questions and post-lab tests, suggested that they learned the underlying principles and detailed molecular mechanisms. Besides learning the technologies and acquiring basic laboratory skills, students practiced key components of scientific research, such as data collection, analysis, and scientific communication.