Article notes - CRISPR Flashcards
Distinguish innate and adaptive immune responses of bacteria.
Innate immune responses have a one-size-fits-all approach while adaptive immune responses have a memory forming approach, where they use the gained information of the phage infection to create a response and attack the phage. The innate immune
responses can block certain steps of infection of cleave the phage off the genome to
completely prevent transcription and replication, which is known as RM, restriction- modification. Prokaryotes produce RM and since they have a short DNA sequence the host cell can protect itself. Adaptive immune responses are known as CRISPR-Cas as
they fend off certain phages and they can adapt easily to new phages or threats
Distinguish the life cycle of a lytic phage and a lysogenic phage
Lytic phage life cycle involves the phage to co-opt the host machinery and cause the host to replicate their genome and produce phage proteins. With these phage proteins the phage will assemble new phages and cause a cell lysis which will have the phages burst
out of the cell and spread into the new host. Lysogenic phages life cycle involves them encoding the proteins and integrating their genome into the host’s genome and eventually due to an environmental trigger it will activate replication and cause the cell lysis
Within the bacterial genome, what are the two portions of a functioning CRISPR-Cas
system? What is the general purpose/function of each part?
There are cas genes and CRISPR locus. Cas genes provide instructions for the Cas proteins, which in turn carry the functions required for defense against phage.
They are under control by just a few promoters and found within a single operon,
and they are classified as a subtype of CRIPSPR system. CRISPR locus known as
the repeat-spacer array, which is where memory is stored. It is composed of a
single DNA sequence with unique stretches of DNA in between, these stretches
are called spacer
Distinguish between the spacer and the repeat. How does each end up in the bacterial genome?
Spacer is a unique stretch of DNA in between the single DNA sequence. These
spacers are captured by an inducing phage and stored within. The repeat is the
DNA sequence that is repeated many times and the spacers are placed in between them. They end up 9n the bacterial genome by the CRISPR locus, as CRISPR loci are made up of spacers and repeats
Which part is a palindrome? What is a palindrome? How does that affect the DNA or RNA?
CRISPR is known as a palindrome, as it is in its name, but it is more like a semi-palindromic sequence. A palindrome would be a sequence that can form internal base pairs into a strand of RNA, once they have been transcribed. Although Cas9 protein are not palindrome
Is the tracrRNA translated?
No the tracrRNA is not translated, it stays transcribed as it will help turn the DNA into a guide RNA sequence and this guide RNA which will help search for the phage infection. The guide RNA is transcribed directly from the CRISPR locus so it would look like the phage DNA but PAM helps avoid that.
Are the cas genes translated?
Yes cas genes are translated. Cas genes are proteins like Cas9, but in order for it
to become a protein it must go through transcription and translation as transcription causes DNA to become RNA and translation causes RNA to become protein
Is the crRNA translated?
No the crRNA is not translated. Mature crRNAs come from having the long
repeat-spacer array that is transcribed into long RNA, this is called pre-crRNA,
and it is cut into shorter pieces and this is mature crRNAs.
What is included within the interference complex?
Interference complex is made up of CRISPR proteins and guide RNA
What does the interference complex do?
The interference complex looks within the host cell for any DNA that is invading
the host by looking for a match to the spacer sequence in the guide RNA. PAM is also involved as it helps prevent Cas9 from destroying CIRSPIR locus
What is the function of the PAM? How many PAMs would you expect to find in a
bacteriophage genome (of perhaps 5000 bp, for example)?
The function of PAM is to prevent Cas9 from finding and destroying the CRISPR
locus as Cas9 is finding matches to the spacer sequence in the guide RNA and the guide RNA is transcribed from the CRISPR locus and thus it would look just like the phage DNA if PAM was not involved. Possibly around 250 PAM in a
bacteriophage genome made up of 5000 bp would be expected.
WHY doesn’t the interference complex attack the bacterial genome at the site of each
spacer?
Due to PAM since Cas9 looks for targets that are one nucleotide away from GG
and PAM is known as NGG. PAM is not present within the CRISPR repeat so Cas9 does not bind itself to the site and thus does not attack
How might phage protect themselves from CRISPR? Provide two examples?
Phage can protect themselves from CRISPR as their genome can mutate one or more nucleotides in the PAM and thus this causes the interference complex to no longer be able to recognize the target. They have also developed an anti-CRISPR, which are small
proteins that inhibit CRISPR-Cas systems by preventing Cas proteins from binding to
their target nucleic acids which can be done by physically blocking DNA sites or cleaving the guide RNA
Provide brief descriptions of five ways this system is being used in biotechnology.
The most impactful use of biotechnology using CRISPR is genome editing. Scientists use Cas9 and guide RNAs for their own design, by using them to direct the enzyme and to cut any site in the genome. Cas9 has also been used to fusing it together with transcriptional activators or repressors which can tune gene expression either up or down. Scientists also
use Cas12 and Cas13 to find and cut certain DNA and RNA targets which then they
become active and cleave the non-complementary nucleic acids. Scientists can then use
them to find certain pathogens like HPV or SARS-CoV-2 and then add fluoresce to them which helps to signal a positive for the pathogen. Cas1 and Cas2 has been used to record information in DNA and helps researchers come up with a DNA binary code for E.coli. Cas3 is used to delete thousands of base pairs of DNA which allows for a large scale
alternation.
