Ward's World Activity Guides

miniPCR bio TM Chopped! Using CRISPR/Cas9 to cut DNA - Instructor's and Student's Guide Version: 1.0 - Release: May 2022 - © 2022 by miniPCR bio™ P./48 Instructor's Guide Notes on lab design This lab is designed to introduce students to the CRISPR/Cas system using an in vitro experiment. We believe this approach provides the right balance between intellectual engagement, inquiry, and accessibility. Certain experimental design choices were made with this approach in mind. Some of these choices include: • In this lab activity, students performed an in vitro assay and analyzed the results of their CRISPR/Cas9 reaction by running the cut DNA samples on a gel. This approach provides access to CRISPR/Cas9 technology using tools that are already commonly found in most teaching laboratories. It also allows for protocols that can be completed easily in typical 45- minute class periods. • The majority of CRISPR/Cas9 applications aim to modify DNA sequences in live organisms. Modifying DNA in vivo requires a mechanism to repair double strand breaks induced by the Cas9/gRNA complex. While repair mechanisms are critical to gene editing systems in live organisms, they function separately from the Cas9 enzyme, and are typically a function mediated by the cell. This lab chooses to focus explicitly on Cas9 function, and therefore does not deal with DNA repair mechanisms directly. • A critical limiting factor in designing gRNAs is the PAM sequence. The PAM sequence is a three-nucleotide sequence (5' NGG 3', where N may be any nucleotide) that must be directly adjacent to the target sequence on the strand of DNA opposite to that which the gRNA binds. The PAM sequence is integral to Cas9 binding to the DNA and, importantly, the PAM sequence is recognized by the Cas9 enzyme and not by the gRNA. For the Cas9/gRNA complex to recognize a target sequence, the Cas9 enzyme must first recognize a PAM sequence. To focus on the importance of gRNAs in directing the Cas9 enzyme, discussion of PAM sequences has been omitted from this lab. We encourage you to introduce the concept of PAM sequences in classes where this is appropriate. ° As the paper model in the Pre-lab activity contains the actual DNA sequence used in this experiment, PAM sequences are present, though students are not asked to locate them. ° PAM sequences will further reduce the likelihood of the gRNA binding to off-target sequences. In the section Using mathematical thinking, to obtain correct likelihood of matches including the PAM sequence, answers should be multiplied by 1/16, the likelihood of a target sequence being adjacent to the sequence 5' NGG 3'.

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