CRISPR: A Bacterial Defence Turned into a Tool
Some bacteria acquire short foreign DNA sequences as CRISPR spacers. In Cas9 systems, RNA guides and a nearby DNA motif help select targets for cutting. Researchers can redirect Cas9 to compatible sites; cellular repair then shapes the editing outcome.
What a learner can do afterwards
- Explains how foreign DNA sequences become spacers and how spacer transcripts supply guide information.
- Explains how guide pairing and a compatible PAM select Cas9 targets and help spare the native array.
- Links a Cas9 break to possible repair outcomes without assuming that cutting guarantees the intended edit.
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You have seen plasmids move DNA between cells. Some bacteria can store a short sequence from foreign DNA, such as phage or plasmid DNA, as a spacer between CRISPR repeats. The array is transcribed, and RNA processing produces guides carrying spacer information. These guides work with Cas proteins in defence. Not every bacterium has CRISPR, and encountering foreign DNA does not always add a spacer.
Here we focus on Cas9, a CRISPR protein that cuts DNA. Its guide pairs with complementary target DNA. Cas9 also needs a compatible nearby DNA motif called a PAM. Guide pairing alone is not enough. The native array is normally spared because its stored spacer lacks the required adjacent PAM. Other CRISPR systems can have different targeting and self-recognition rules.
After a Cas9 break, repair determines the resulting DNA sequence. End joining reconnects the ends without copying a donor. It can restore the original sequence or leave insertions or deletions. These changes may disrupt a gene, but need not do so. Homology-directed repair can copy a designed change from a supplied donor with matching sequence. Making a cut or supplying a donor does not guarantee the intended edit.
To target a different compatible DNA site, researchers can keep the same Cas9 protein and change the guide. The new site must meet that Cas9 system's PAM requirement. Changing the guide redirects targeting; it does not specify the repair outcome. A chosen replacement also needs suitable sequence information, such as a matching donor that the cell can copy.
A guide may also recognise similar DNA elsewhere beside a compatible PAM. Cutting at an unintended site is called off-target cutting. A predicted unique match does not prove that no other cuts occur. Researchers assess plausible lookalikes and test the resulting DNA. Sequence similarity identifies a possible target; it does not establish that an edit occurred.
Spacers supply guide information. Guide pairing and a PAM help select Cas9 targets. Repair shapes the sequence, and testing checks the outcome.
Lesson, questions, process diagrams and flashcards written for this dataset.
Readings and flashcards written for this dataset. Linked videos: How CRISPR lets you edit DNA, by TED-Ed; Applications of DNA technologies, and DNA cloning and recombinant DNA, by Khan Academy.
More to read
From foreign sequence to immune memory
Phages and plasmids can introduce DNA into bacteria. Some bacteria acquire short sequences from foreign DNA and store them as spacers between CRISPR repeats. This is one way an encounter can leave an inherited record. Acquisition does not happen after every encounter, and not every bacterium has this defence.
The stored record and the active guide are different molecules. Transcription produces RNA from the array. RNA processing supplies guides whose sequences come from the spacers. Cas proteins use this information during defence. The spacer therefore stores targeting information; it is not itself an enzyme that cuts an invader.
Pairing and the nearby motif
Cas9 recognises DNA using an RNA guide together with a nearby DNA motif called a PAM. Different Cas9 proteins can have different PAM requirements. A sequence complementary to the guide is not enough if the required PAM is absent.
The PAM requirement also helps distinguish a foreign target from its stored record. In a native Cas9 system, the stored spacer normally lacks the required adjacent PAM. A similar sequence elsewhere with a compatible PAM may instead be recognised. This account concerns Cas9; other CRISPR systems can recognise targets and distinguish self from foreign material in different ways.
What follows the break?
A DNA break can be followed by different repair outcomes. End joining reconnects broken ends without copying a donor. The original sequence may return, or repair may leave insertions or deletions. A sequence change does not necessarily disable a gene.
A matching donor can carry a chosen replacement. If repair copies that information into the target, the repaired DNA can contain the intended change. Delivering the donor does not force the cell to copy it. This is why an editing claim needs evidence about the resulting DNA, rather than evidence of cutting or delivery alone.
Retargeting and checking
Researchers can redirect a Cas9 system by changing its guide while keeping the cutting protein. The new DNA site must still meet that protein's targeting requirements, including its PAM requirement. Retargeting changes where the system may cut; it does not dictate how the cell repairs the break.
A guide may recognise similar DNA at an unintended site. Predictions help identify such sites for testing. They do not establish that a predicted site was edited or that every other site remained unchanged. Checking the intended allele and assessing possible off-target sites answer different questions about the experiment.
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Where it sits
8 questions wait behind this lesson, each with its answer explained. Every answer feeds the sky: stars light as they are learned, and dim when it is time to come back.