CRISPR - CAS 9 Flashcards
definitions
What is a virus?
A non-cellular, infectious agent composed of genetic material enclosed in a protein coat that requires a host cell to multiply.
What is a bacteriophage?
A virus that infects prokaryotic organisms.
What is CRISPR-Cas9?
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 is CRISPR-associated protein 9 (Cas9)?
An endonuclease that creates a blunt end cut at a site specified by guide RNA (gRNA).
is an enzyme with two active sites which cuts both strands of DNA to create a
blunt end cut.
What is CRISPR?
Short, clustered repeats of DNA found in prokaryotes which protect them against viral invasion.
- is naturally found in prokaryotes
What is a spacer?
Short sequences of DNA obtained from invading bacteriophages that are added into the CRISPR sequence.
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 is a protospacer adjacent motif (PAM)?
A sequence of two-six nucleotides that is found immediately next to the DNA targeted by Cas9.
What is guide RNA (gRNA)?
RNA which has a specific sequence determined by CRISPR to guide Cas9 to a specific site.
What is genetic modification?
The manipulation of an organism’s genetic material using biotechnology.
What is a deleterious mutation?
A change in DNA that negatively affects an individual.
What is gene therapy?
Repairing genetic mutations by replacing a defective gene with a healthy one.
What is single guide RNA (sgRNA)?
Guide RNA utilised by scientists to instruct Cas9 to cut a specific site when using CRISPR-Cas9 in gene editing.
What is gene knockout?
A technique in gene editing where scientists prevent the expression of a target gene to understand its function in an organism.
What is a zygote?
The diploid cell formed by the combination of two haploid gamete cells.
What is gene knock-in?
A technique in gene editing where scientists substitute or add nucleotides in a gene.
What is an embryo?
An early stage of development in an organism. In humans, used to refer to the organism during the first eight weeks of development.
What is differentiation?
The process in which cells develop specialised characteristics, typically transforming them from one cell type to another more specialised cell type.
process of CRISPR- CAS9 defense system
1- exposure - bateriaphage injects its dna into the bacterium - which it identifies the viral DNA as a forgien substance - CAS 1 and 2 are in charge of cutting out a short section of the DNA which becomes a protospacer then it is introduced into the bacterium’s CRISPR gene and become a spacer.
2- expression- CRISPR spacer is transcibed with half palindrome from the repeat either side of it, - converts into gRNA. - gRna bind to CAS9 making CRISPR-CAS9 complex - directed to any viral DNA inside the cell that is complementary to the gRNA. gRNA forms a hairpin loop-like structure from the transcribed palindromic repeats either side of the spacer.
3- extermination - CRISPR-CAS9 complex scans for invading viral DNA that is complementary to the ‘mugshot’ on the gRNA. when it does s, Cas9 cleaves the phosphate-sugar backbone to inactivate the virus. Cas9 contains two active sites to cut both strands of DNA and create blunt ends
WHAT IS PAM
protospacer adjacent motif- is a short (2–6 nucleotides) sequence of nucleotides that Cas1 and Cas2 can consistently recognise
Cas1 or Cas2 encounter PAM,- signalled to extract a protospacer from invading DNA- the enzymes cut the viral DNA before the PAM, so PAM does not get included in final protospacer.
To speed up time the Cas9 only looks for PAMs
PAMs are on the target sequence DNA
COMPONENTS OF THE gRNA
gRNA molecule is formed by two smaller RNA molecules creating a hybrid of CRISPR RNA (crRNA) and trans-acting CRISPR RNA (tracrRNA)
crRNA = of a spacer and a repeat, which are transcribed and cleaved to produce the ‘mugshot’ for Cas9 to cut DNA.
tracrRNA = complementary sequence to the crRNA repeat which enables the two molecules to bond and establish the final gRNA structure
-tracrRNA is responsible for binding tightly with Cas9 to establish the CRISPR-Cas9 complex
