(This is only relevant for the mouse experiment portion of the simulation) The first guide sequence you design must be complementary to the top strand of mouse DNA, and it should bind somewhere to the left (upstream)of the sequence that you want to remove. Remember, you are designing RNA, not DNA. Furthermore, remember that two strands of nucleic acid base pair with antiparallel polarity.
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Prerequisite skills needed:
Transcription
Translation
Relevant book section: pages 246-248
By completing this simulation, you will:
Apply the process of CRISPR-mediated interference to defend a bacterium against bacteriophage infection
Develop a CRISPR system to excise genes from a eukaryotic cell
CRISPR
InstructionsIn this simulation, we’ll explore how a bacterium uses its CRISPR-Cas defense system to protect against invading bacteriophage. When a bacteriophage attacks a bacterium, the cell stores a portion of the invading genetic material within its own DNA. Known as adaptive immunity, this mechanism enables the bacterium to defend itself against future attacks from the same type of bacteriophage. To begin, attack the bacterium with the bacteriophage.
This bacteriophage stores DNA in the topmost part of its structure. If you look carefully, you can see the red DNA through the translucent blue "head."
The CRISPR-Cas locus of the bacterial genome is shown looped out from the rest of the chromosome. Hover over the different regions to see what they are. Spacer sequences are fragments of foreign DNA from different strains of bacteriophage that have previously infected this cell. In the first step towards establishing immunity, nucleases (cutting enzymes) in the cell cleave the viral DNA into fragments. Drag the cutting enzyme (symbolized here by the scissors) onto the viral DNA.
The next step is to insert the viral DNA into the CRISPR locus. Drag the cut piece of viral DNA onto the CRISPR repeat located immediately adjacent to the green leader portion of the bacterial CRISPR locus.
Drag the RNA Polymerase onto the loop of bacterial DNA in order to initiate transcription.
Drag the ribosome onto the cas mRNA transcript.
The long CRISPR RNA transcript needs to be cut into smaller fragments. Drag the cutting enzyme (symbolized here by the scissors) onto the RNA.
Drag the Cas9 protein/tracrRNA onto any one of the gRNAs.
Drag the appropriate CRISPR-Cas9 complex onto the incoming viral DNA.
Amino Acid (ex. ALA)
Anti-Codon (ex. GAA)
5' 3'
gRNA sequence (4 bases) to associate with the top strand, left side of the deletion sequence
5' 3'
gRNA sequence (4 bases) to associate with the bottom strand, right side of the deletion sequence
This is the wild type mouse. It is a black mouse and will remain black for the remainder of the experiment.
Your two engineered guide RNAs, along with the mRNA for Cas9, are injected into fertilized mouse eggs. The fertilized eggs are then returned to a wild type mother mouse for gestation. If you designed your guide RNAs correctly and the segment of the Atp7a gene was successfully deleted in at least some of the fertilized eggs, then some of the pups will be mottled. You can be sure that any mottled pups are heterozygous for the Atp7a mutation, because homozygous Atp7a mutants do not survive to birth.
Instructions Now that you have zoomed into the mRNA strand, please assemble the initiation complex. Drag and drop the appropriate molecules to the correct area to assemple the complex.
Congratulations Now that you have assembled the initiation complex, use the tRNA builder and the drag/drop menu to the left to elongate the amino acid chain. When appropriate, terminate the translation process and recycle the ribosome.
Congratulations You have successfully completed this activity!
Amino Acid (ex. ALA)
Anti-Codon (ex. GAA)
5'
5'
5'
3'
3'
5'
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RNA Processing - Simulation Results
Your Answer:
Correct Answer:
NOTE: This is not the actual sequence of the BRCA-II gene, but rather just a random sequence created for this exercise.
Results:
Congratulations! You figured out the sequence based on the chromatogram. If you would like to try the exercise again with a new dna sequence, please click the button below.
Results:
Sorry, you selected the wrong nucleotides. If you would like to try the exercise again with a new dna sequence, please click the button below.
