MBB 267 Week 6: Corrigan 5 Flashcards
What are the defence strategies in bacteria against pathogens?
Bacterial defence strategies:
a. Surface receptor modification
b. Restriction digestion of foreign DNA
c. CRISPR-Cas systems
What is CRISPR-Cas?
Clustered Regularly Inter-Spaced Short Palindromic Repeats. Small interfering RNA system in which Bacteria and Archaea that survive a bacteriophage attack, capture a piece of the attacker’s DNA to counteract future attacks
What is the general CRISPR-Cas structure?
Structure;
a. CRISPR loci: short (~30 bp) direct repeat sequences separated by spacers (20-72 bp depending on the species)
b. Repeats and spacers are not protein coding. Direct repeats flank the spacers
c. Number of repeat-spacer units vary between species A conserved leader sequence is located on one side of the cluster
d. Adjacent to these sequence clusters are protein encoding genes (cas). CRISPR associated genes (cas) surround the CRISPR locus
What are the key features of CRISPR systems?
Features;
a. Adaptation: insertion of new spacers into the CRISPR locus
b. Expression: transcription of the CRISPR locus and processing of CRISPR/guide RNA
c. Interference: detection and degradation of mobile genetic elements by CRISPR RNA and Cas protein(s).
What is a Type II-A CRISPR-Cas system?
System that cuts specific parts of DNA.
What is the structure of a type-II CRISPR-Cas?
Contains;
a. A tracrRNA
b. Cas operson, which consists of;
i. Cas9, cas1, cas2 and csn2
c. A CRISPR repeat-spacer array
How does a type-II CRISPR-Cas system work?
Step:
a. cas1, cas2 and csn2 (csn2 is a nuclease) are used to cut the phage DNA. DNA fragment is inserted in repeat spacer array.
b. When the phage attacks again, the repeat spacer sequence, tracrRNA and cas9 are transcribed. RNAse 3 comes and cuts the spacer array to find the needed part. tracrRNA binds to the PAM (or repeater?) sites
c. The cas9 complex (which contains; spacer-repat+crRNA+tracrRNA) binds to the incoming phage DNA. Phage DNA and spacers would bind together, PAM and domain sites are used to activate the cutting of the phage DNA and then degrading.
How is a cas9 system activated?
Using a PAM site
How did scientists simplify the cas9 mechanism?
By linking the tracrRNA and the crRNA together using a linker loop which simplifies the process.
How are blunt double-strands repaired?
Can be fixed by one of these methods;
a. Nonhomologous end joining
b. Homology-directed repair
How does Nonhomologous end joining works?
The DNA would be digested back until, then filled with an insertion.
a. This is not good of a repair method as the gene is disrupted and most likely create mutations
How does homology-directed repair work?
A homologous DNA sequence is used, and the correct DNA is used to repair the faulty strand as they are homologous.
How can cas9 be used as a nickase?
When engineered to contain 1 domain active site instead of 2, so it will introduce only single stranded breaks
What is the use of a double nickase cas9?
Since cas9 target sites are not always accurate, 2 cas9 can be used to improve the accuracy. As each cas9 nickase cuts a strand, making a staggered double strand break which is more accurate.
How can cas9 have other uses?
So the domains active sites are deactivated, and This catalytically inactive or dead Cas9 (dCas9) can mediate transcriptional down-regulation or activation.
a. Also, the dcas9 can fuse to domains to have more specilised functions.
