Cas9
The endonuclease that makes a double-stranded break next to a PAM site
What is Cas9?
The 3 base pairs upstream from the PAM sequence
What is the location where Cas9 cuts DNA in relation to the PAM sequence?
The big orange blob
Where is the Cas9 enzyme?
The full non-abbreviated name of CRISPR
What is Clustered Regularly Interspaced Palindromic Repeats?
Identify the PAM sites (the NGG sequences) on the Target Gene
What is the first step of designing an sgRNA sequence?
Defends against attacks from viruses
What is the purpose of Cas9 in bacteria?
Cell death due to DNA degradation
What happens if a cell is cut but not repaired?
The small red box section
Where is the PAM site?
The 5'-NGG-3' sequence that Cas9 binds to
What is Protospacer Adjacent Motif (PAM)?
Including the PAM site, count 23 base pairs upstream to put into NCBI Blast
What is the second step to designing an sgRNA sequence?
It causes the DNA strands to separate so sgRNA can bind to its complementary strand
What is the purpose of a PAM site in Cas9's function?
When enzymes patch a break with a donor template, NOT through self-repair (which can cause more disruptions)
How is Homologous-Directed Repair (HDR) different from Non-Homologous End Joining (NHEJ)?
The grey strip
Where is the sgRNA sequence?
An engineered 100-nucleotide strand of RNA that forms a complex with the Cas9 enzyme?
What is a Single Guide RNA (sgRNA)?
After finding a PAM site with a sequence that has the least off-target cut possibilities and the least chance of impact, make the sgRNA
What is the third step to designing an sgRNA sequence?
The repair mechanism that uses a donor template to repair the cell's DNA
What is Homology Directed Repair (HDR)?
The amount of matching bases on the target sequence and genes that are essential to life or could cause fatal diseases when cut
What is something to take into consideration when designing an sgRNA sequence?
The natural cellular repair mechanism that binds two sticky ends of DNA together
What is Non-Homologous End Joining (NHEJ)?