CRISPR-CAS9 Flashcards
what does CRISPR stand for?
clustered regular interspaced short palindromic repeats
What is the difference between nucleases and endonucleases?
endonucleases work inside the nucleus
What is CRISPR-Cas9?
found naturally in all bacteria cells
a complex formed between gRNA and Cas9 which can cut a target sequence of DNA. Bacteria use this complex for protection from viruses and scientists have modified it to edit genomes
What do humans use CRISPR-Cas9 for?
many applications
- knock out genes (make ineffective)
- knock in genes
- insert/delete nucleotides
- labelling genes with markers
What is Cas9?
A protein (endonuclease) that cuts DNA at sugar phosphate bonds
It is not specific and must be guided by RNA
How do humans guide Cas9?
they can construct any RNA sequence to guide Cas9 to any target DNA
What is sgRNA?
single guide RNA, this is made in a lab and has a single strand
What is gRNA?
guide RNA, has a specific sequence determined by CRISPR to guide Cas9 to a specific site
What is ligase?
an enzyme that joins molecules, including DNA or RNA, together by catalysing the formation of phosphodiester bonds
What are spacers?
viral DNA “mugshots”, they are DNA captured from bacteriophages
Viral DNA 1 is complementary to spacer 1
Viral DNA 2=spacer 2 ect.
Why do viruses infect other cells?
viruses don’t have any organelles but want proteins, therefore infects bacteria which has ribosomes and energy to make proteins
What is the function of Cas9?
first looks for a PAM site before unwinding the DNA and checking for a match with the gRNA
How does bacteria not destroy its own DNA?
there are no PAM sites in bacterial DNA
What is a PAM site?
Protospacer adjacent motif
A few nucleotides found in bacteriophages which signal to Cas1 and Cas2 to remove a sequence of DNA
NGG will be recognised as a PAM
What are the components that make up a monomer of sgRNA?
ribose sugar, phosphate and nitrogenous bases
- will be the same for all RNA
What are the components that make up a monomer of gRNA?
ribose sugar, phosphate and nitrogenous bases
- will be the same for all RNA
What is a protospacer?
a short sequence of DNA extracted from a bacteriophage by Cas1 and Cas2, which has yet to be incorporated into the CRISPR gene
What happens when Cas9 finds a PAM region?
What is CRISPR?
