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miniPCR bio TM Chopped! Using CRISPR/Cas9 to cut DNA - Student's Guide Version: 1.0 - Release: May 2022 - © 2022 by miniPCR bio™ Student's Guide P./14 Figure 1. Cas9 cuts DNA as instructed by a guide RNA (gRNA). (Left) The scaffold region of the gRNA (yellow) binds to the Cas9 nuclease (red) to form a gRNA/Cas9 complex. The spacer region of the gRNA is made up of 20 bases that are complementary to the target DNA sequence. (Right) The Cas9 enzyme unwinds a small section of DNA. If the spacer region of the gRNA encounters a complementary DNA sequence, the Cas9 nuclease will cut both strands of the DNA. If the gRNA is not complementary to the DNA, the Cas9 nuclease does not cut and will continue to a new region of the DNA. In this way, the gRNA allows the CRISPR/Cas9 system to be both programmable and specific. The CRISPR/Cas9 system is programmable because the gRNA can be designed and synthesized in the lab to complement almost any DNA sequence a scientist would like to target. And it is specific because Cas9 is only expected to cut the DNA when the match between the 20 base gRNA and the DNA is exact. The chance of any specific 20 base sequence matching a random 20 base stretch is less than one in a trillion. This means that even in a genome that is billions of base pairs long, it is likely that the only place the Cas9 enzyme will target is the specific place in the genome for which the gRNA is designed. Complementary RNA-DNA duplex gRNA scaffold region gRNA spacer region Cas9 nuclease Cas9 nuclease DNA Guide RNA Guide RNA DNA cut What's in a name: CRISPR CRISPR stands for clustered regularly interspaced short palindromic repeats—what a mouthful! It refers to an area of the bacterial genome involved in the immune defense against viruses. This bacterial defense mechanism relies on two main components, the DNA region we call CRISPR and the Cas9 nuclease. In bacteria, the CRISPR DNA region codes for many different RNAs that will each recognize and target a unique viral DNA sequence. Bacteria use CRISPR/Cas9 to specifically recognize viral DNA sequences and then destroy the virus by cutting that recognized DNA. The CRISPR/Cas9 genome editing technique that scientists use relies heavily on Cas9 nuclease, but in the lab, scientists don't actually use the CRISPR region. Instead, they design their own gRNAs. So why is the term CRISPR more famous than Cas9? Probably just because saying CRISPR sounds a lot catchier than calling it Cas9 genome editing.