CRISPR_Cas Flashcards
What are the three steps in the CRISPR-Cas defence mechanism?
Spacer acquisition
Expression: crRNA biogenesis and processing
Interference: target DNA degradation
What type of Cas proteins form the Class 1 systems and how many proteins are in the complex?
Types,1, 3 and 4 and multiple proteins are there
What types of Cas proteins are in the Class 2 systems and how many make up the complex?
Types 2, 5 and 6, only one multidomain Cas protein make up the complex
Describe the spacer acquisition step.
The protospacer (small frag of DNA to be integrates) contain a PAM which is the recognition motif. Cas1/2 and other host factors integrate protospacer into CRISPR locus between direct repeats.
Describe the expression step
transcription of the pcrRNAs and the specific endoribonucleases cut pcrRNA into small crRNAs. In the Type 2 system a trans encoding small crRNA is transcribed and forms a duplex with the other crRNA, this then binds with cas to form the complex
Describe the Interference step
crRNA in complex recognizes the incoming foreign DNA or RNA and Cas protein cuts target DNA close to its PAM site
How long are the repeats and spacer lengths in the CRISPR locus
repeat: 21-48bp, spacer: 26-72
What is the Cas9 protein
signature protein to type 2 CRISPR-Cas, 2 nuclease domains HNH and RuvC, two REC domains, 1-binds crRNA and 2- likely regulates function od HNH domain, PI domain, for interacting with PAM
What are the PAM
it’s near the 3’-end of target DNA site, recognition site for Cas9-crRNA-tracrRNA
What are the single RNA guide
crRNA and tracrRNA joined together by a linker to make sgRNA
How is CRISPR-Cas9 used in Gene Editing
Cas9 forms complex with sgRNA which recognizes and binds target sequence, Cas9 cuts target DNA near PAM site and creates DSB, results in non-homologous end joinind or homology directed repair
What does each repair system do?
NHEJ: add or remove nucleotides
HDR: repair using outside template provided
What are the major issues of CRISPR-Cas gene editing?
DSBs are normally joined by NHJ which isnt precise so there are strategies to increase repair by HDR
some of the targets are not specific enough
What are some other applications of the CRISPR technology?
Fuse proteins-reduce gene expression, activate gene expression, epigenetic changes, chromatin imaging or deaminase fusion
How are they able to not cut DNA?
dCas9 is deactivated nucleases and when bound to sg-RNA it can still recognize and bind sequences
