Ward's World Activity Guides

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./31 Using mathematical thinking The CRISPR/Cas9 system is highly specific because the gRNA targets a 20-base region. This makes it unlikely that the gRNA will recognize more than one target in a given genome. Restriction enzymes are another class of nucleases that scientists have long used to modify DNA. Restriction enzymes typically target a specific DNA sequence that is only 4 to 8 bases long, making them far less specific than the gRNA targeting possible with CRISPR/Cas9. Here we will compare mathematically the difference in specificity between the CRISPR/Cas9 system and restriction enzymes. Probability of a specific DNA sequence occurring The product rule states that you can calculate the probability of a series of independent events by multiplying the probability of each event. The product rule can also be applied to calculate the probability of any DNA sequence occurring. There are four possible DNA bases (A, T, C, and G). If we assume all four bases are equally likely to occur, the probability that any specific DNA sequence will occur is (1/4) n , where n is the length of the specific sequence. For example, say you wanted to know the probability of the two-base DNA sequence AT occurring. The likelihood of having an A in the first position is 1/4 and a T in the second position is also 1/4, so the probability of the two-base sequence AT occurring in any two-base-pair stretch would be: (1/4) x (1/4) = (1/4) 2 = 1/16 or a 1 in 16 chance 1. Let's assume we have a restriction enzyme that targets the six-base sequence CGATCG. What are the chances of this specific six-base DNA sequence matching any random six-base- pair stretch of DNA? You should use a calculator, but show your work. What are restriction enzymes? Restriction enzymes are a class of nucleases that can recognize a specific DNA sequence and cut both strands of the DNA at that site. Each unique restriction enzyme recognizes a specific DNA sequence, usually 4 to 8 bases long. Similar to Cas nucleases, restriction enzymes are found naturally in bacteria and archaea as a defense mechanism against invading viruses, where the enzymes recognize and cut up the viral DNA. There are thousands of different known restriction enzymes, many of which are commonly used in the lab to cut DNA.

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