Interference 1 Flashcards
How does cascade-crRNA deliver the immune response?
Interference reactions
What are the two classes of CRISPR groups?
Class I which has effector interference complexes with multiple proteins. Class II made up of a single polypeptide.
Is class I or II the focus of editing technology?
Class II as easier to manipulate 1 protein than multiple.
How does Cas9 work?
Two RNA molecules combine (base-pair) to form an RNA scaffold around which cas9 folds. Cas9 binds to an RNA called tracRNA; ‘tra’ because it is a trans activating factor. The tracrRNA guides Cas9 to transcribed pre-crRNA. Cas interacts with RNaseIII, which cuts the pre-crRNA into crRNA.
What do biotech CRISPR systems contain?
Contains crRNA, artificial linker and tracRNA. All in one complex. Forms a single guide crRNA. Used to have three separate plasmids with each in.
What are Cas 9’s two nuclease active sites?
RuvC-like based on Asp residues and RNase-like based on a His residue. They each cut one DNA strand in opposite positions. Leads to a blunt end ds break. Acid base catalysis.
What is a protospacer?
A precisely measured piece of DNA from invader virus/plasmid that is captures by CRISPR-cas adaption and inserted into a CRISPR site as newly acquired immunity.
What is PAM?
Typically 3-4 ntds marks the site of a subsequent target sequence for the interference complex. But isnt integrated into CRISPR.
In natural CRISPR systems how does PAM link adaptation with interference?
The virus constantly tries to escape being destroyed by constantly mutating. The immune system updates so the virus can’t escape.
What are the potentials for CRISPR?
Plant biotech, livestock improvement, correction of genetic and metabolic diseases, control of bacterial and viral diseases, drug development, biofuel production.
What are the steps to CRISPR-cas9 systems?
Cas9 searches the DNA for PAM binding sites. If PAM is found, Cas9 binds and forms an R-loop with the DNA. Cas9 generates a double strand DNA break. The DNA is left for repair by other enzymes.
What enzymes do gene editing after CRISPR has cut it?
DNA deletion via non homologous end joining or insertion via homologous dependent recombination.
What are negatives of homologous dependent recombination?
Difficult to do and contributes to genome instability.
What is nickase engineered Cas9 (nCas9)?
nCas9 is created by inactivating either NHN-active site histidine residue or RuvC active site aspartic acid. Both change to alanine to prevent acid base catalysis.
What is prime editing?
The prime editor complex includes a Cas9 enzyme, modified to only nick one strand of DNA and a reverse transcriptase enzyme which can generate new DNA by copying an RNA template. An engineered peg RNA (prime editing guide) sends the editor to its target where Cas9 nicks the DNA. DNA mismatching occurs. This frees CRISPR editing from reliance on HDR.
What is base editing?
Removing individual base mutations in genes. Spontaneous cellular deamination of C to U. U is then read as A. A is then paired with T so C-G from A-T mutation occurs. Reverses the nucleotide that causes the change in disease. Additionally, A-T to G-C mutation where A is changes to Inosine which is read as G.
What is nCas9-cytosine base editor (CBE) fusions?
Naturally occurring cytosine deaminase enzymes (C to U), which therefore can reverse mutant C-G base pairs to wild type T-A and therefore reverse genetic disease. Tool compromises Nickase Cas9 (nCas9) to avoid dsDNA breaks, cytidine deaminase and uracil DNA glycosylase inhibitor UGI to prevent the removal of U into AP site.
What is nCas9-adenine base editor ABE fusions?
Evolved E. coli TadA protein, a tRNA adenine deaminase to become active on DNA. To reverse A-T to G-C.
What is SPACE?
A dual nCas9-adenine and cytosine base editor ABE-CBEs. SPACE= synchronous programmable adenine and cytosine editor.
