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CRISPR-Cas9 Mechanism Reference Cheat Sheet
A printable reference covering CRISPR repeats, guide RNA, PAM recognition, Cas9 cutting, DNA repair, and gene editing outcomes for grades 11-12.
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CRISPR-Cas9 is a gene-editing system adapted from a bacterial defense mechanism against viruses. This cheat sheet covers how guide RNA directs Cas9 to a target DNA sequence, how Cas9 recognizes a PAM site, and how DNA repair creates edits. Students need this reference to connect molecular biology vocabulary with the actual sequence of events in genome editing. It also helps organize the difference between cutting DNA and successfully changing a gene. The core mechanism is guide RNA base-pairing with a complementary DNA target next to a PAM sequence, usually NGG for commonly used SpCas9. Cas9 makes a double-strand break about 3 base pairs upstream of the PAM. The cell repairs the break by non-homologous end joining, which often creates insertions or deletions, or by homology-directed repair if a repair template is provided. Editing success depends on target sequence choice, PAM presence, delivery method, repair pathway, and off-target risk.
Key Facts
- CRISPR stands for clustered regularly interspaced short palindromic repeats, which are DNA sequences used by bacteria to remember viral infections.
- Cas9 is an enzyme called a nuclease because it cuts DNA at a specific location directed by guide RNA.
- The guide RNA must be complementary to the target DNA sequence, using base-pairing rules A pairs with U in RNA-DNA pairing and C pairs with G.
- SpCas9 usually requires a PAM sequence of NGG, where N can be A, T, C, or G, immediately next to the target DNA.
- Cas9 cuts both DNA strands about 3 base pairs upstream of the PAM, producing a double-strand break.
- Non-homologous end joining, or NHEJ, repairs broken DNA quickly but often creates small insertions or deletions called indels.
- Homology-directed repair, or HDR, can copy a supplied repair template into the genome to make a precise sequence change.
- Off-target editing happens when guide RNA binds and directs Cas9 to a similar but unintended DNA sequence.
Vocabulary
- CRISPR
- A bacterial DNA system made of repeated sequences and spacer sequences that helps recognize genetic material from viruses.
- Cas9
- A CRISPR-associated nuclease that cuts DNA when guided to a matching target sequence.
- Guide RNA
- An RNA molecule designed to match a target DNA sequence and bring Cas9 to that location.
- PAM
- A short DNA sequence next to the target site that Cas9 must recognize before it can cut the DNA.
- Double-strand break
- A break in both strands of a DNA molecule that activates cellular DNA repair pathways.
- Indel
- A small insertion or deletion of bases that can occur when DNA is repaired after a cut.
Common Mistakes to Avoid
- Thinking guide RNA cuts the DNA is wrong because guide RNA only directs Cas9, while Cas9 is the enzyme that makes the cut.
- Ignoring the PAM sequence is wrong because Cas9 usually cannot bind and cut a target unless the correct PAM is next to it.
- Assuming every CRISPR edit is precise is wrong because NHEJ often creates random indels instead of a planned sequence change.
- Confusing NHEJ with HDR is wrong because NHEJ joins broken ends without a template, while HDR uses a matching repair template.
- Forgetting off-target effects is wrong because a guide RNA can sometimes bind similar sequences elsewhere in the genome.
Practice Questions
- 1 A target DNA site uses SpCas9 and has the sequence 5'-ACCTGATCGTACGTTAGCAA-NGG-3'. How many bases long is the target sequence before the PAM if the target is the 20 bases shown?
- 2 If Cas9 cuts about 3 base pairs upstream of the PAM, how far from the PAM is the double-strand break in base pairs?
- 3 A guide RNA is designed against a 20-base target, and 4 of the bases do not match the DNA target. How many bases match, and why might this increase or decrease editing accuracy?
- 4 A scientist wants to knock out a gene rather than replace one exact base. Which repair pathway, NHEJ or HDR, is more likely to be useful, and why?