CRISPR Basics
The molecule that acts as a molecular GPS, using a custom 20-nucleotide sequence to direct the cutting enzymes to the target?
What is sgRNA
Short sequence that is found downstream of the protospacer target sequence and is required for Cas9 to bind?
What is the PAM site?
A small circular piece of DNA found in bacteria, used to transport new genes into a cell?
What is a plasmid.
In this lab, this color colony tells us that the original bacterial lacZ gene is completely functional?
What is blue.
This pathway is described as a "quick and dirty" repair mechanism that simply glues broken DNA ends back together?
What is NHEJ (Non-Homologous End Joining)?
This bacterial endonuclease is creates double stand breaks in the target DNA?
What is Cas9
This is the specific nucleotide sequence required for the standard Cas9 PAM site?
What is 5'-NGG
The specific DNA sequence acts as a landing site for RNA polymerase allowing a gene to be transcribed?
What is a promoter.
This is the specific enzyme produced by a functional lacZ gene that breaks down x-gal in the agar matrix?
What is B-galactosidase.
This precise repair pathway utilizes an explicit donor template to fill in the blanks or insert brand-new genes?
What are HDR (Homology Directed Repair)?
Three-Step, three word summary of how CRISPR-Cas9 system works on a genetic sequence?
What is Find, Cut, Repair
Location where the Cas9 enzyme will cut both DNA strands relative to PAM sequence?
What is 3 base pairs upstream.
The specific biological process where bacteria take up foreign DNA and express the new genes?
What is transformation.
This visual color change serves as evidence that CRISPR gene editing successfully disrupted the target gene?
What is white?
Short for "insertions and deletions", these random mutation are frequently caused by the error-prone NHEJ pathway?
What is indels?
Length of the target sequence determined by the guide RNA?
What is 20 Nucleotides
The PAM sequence performs this vital job before cutting occurs, this allows the sgRNA to attempt to bind to the DNA strand.
What is separating the DNA strands.
Vital plasmid that replicates DNA and ensures the plasmid is copied as the bacterium divides?
What is the Origin of Replication (Ori).
The chemical compound added to the agar plate that changes color when broken down by bacterial enzymes?
What is X-gal.
What is NHEJ.
If a double-strand break is made by Cas9 but the cell fails to repair itself, what is the outcome?
What is Cell death/Apoptosis
In designing an sgRNA sequence, scientists look for high on-target efficiency, the least amount of off-target effects, and ensuring the sequence will target specific early regions of a gene?
What are early consecutive exons.
This method separates and purifies components of a mixture by passing a liquid mobile phase through a column packed with a stationary resin?
What is column chromatography.
The exact biological reason why editing bacterial colonies appear white rather than blue?
What is because the lacZ gene was disrupted, this means no B-galactosidase was made to process the X-gal.
Unlike NHEJ, this is the vital extra component scientists must artificially provide to the cell for the HDR pathway to work properly.
What is a DNA template (or donor template).