Lecture 4: basic techniques - CRISPR-Cas Flashcards
What is CRISPR-Cas9 (in nature)?
CRISPR-Cas9 is an adaptive immune system against viruses that has been found in bacteria and archaea.
Where does CRISPR stand for?
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas9 gene consists of short repeating sequences, interspaced by unique sequences called spacers and accompanied by a number of Cas genes. What seems to be the function of the Cas genes and the spacer sequences?
The Cas genes encode for DNA cutting enzymes. The spacer sequences appeared to have similar sequences compared to viral sequences, this may be a clue for a defense mechanism against viruses.
Shortly describe how CRISPR-Cas was discovered as a defense mechanism in bacteria (don’t learn by heart).
A milkfactory depended on bacteria to convert milk into yoghurt. But sometimes they would lose entire cultures to bacteriophages (bacteria-killing viruses). The company tested what would happen when these bacteriophages infected bacteria and saw that sometimes few bacteria survived and had become resistant. And when these bacteria multiplied, their progeny would also be resistant. This indicated that something changed at a genetic level. They found that bacteria had included some of the bacteriophage’s DNA into their genome (then called spacers) and when they would remove this new DNA, the bacteria would lose their resistance.
Describe what genes the entire CRISPR-Cas locus is composed of and what proteins or RNA result from these genes.
- Cas genes (Cas9, Cas1, Cas2) encode for Cas proteins/enzymes
- Spacers, containing viral DNA, encode for crisprRNA
- CRISPR repeats surrounding the spacers that encode for tracrRNA (trans-activating crispr RNA)
A bacteria with a CRISPR-Cas defense system encounters a virus (i.e. bacteriophage) which RNA is already integrated into the bacteria its CRISPR locus. How does the CRISPR-Cas defense system of the bacteria work upon second attempt of infection?
Upon second infection:
- translation of CRISPR-Cas genes which results in the production of crisprRNA, tracrRNA and Cas proteins.
- crisprRNA forms a double stranded hybrid with tracrRNA
- crisprRNA-tracrRNA forms a RNA-protein complex with Cas9 protein.
This way Ca9 can use the copy from the DNA archive to look for the virus (foreign) invading RNA. When a complete match is found, the protein cuts out the viral RNA to make it useless.
How many unique spacers are in the double stranded hybrid crisprRNA-tracrRNA?
Every hybrid crRNA-tracrRNA contains 1 single spacer (and thus 1 single viral sequence).
How does Cas9 cut out the foreign viral invading DNA?
By making a double stranded break
How can the hybrid crRNA-tracrRNA also be called and why?
guideRNA (gRNA), because it this complex guides Cas9 to where it needs to cut DNA. The gRNA is complementary to the DNA that needs to be cut.
You can use CRISPR-Cas9 for genome engineering in mice, cells or even gene therapy for humans. You can knock out a gene or insert a new gene. How can a new gene by incorperated into a genome (what proteins are needed for example)?
You need the Cas9 protein, gRNA and a piece of DNA you want to incorparate (host or donorDNA). Here, the gRNA guides the complex with Cas9 to where DNA needs to be cut. Here, helicase splits the double stranded DNA so that gRNA can bind to its complementary DNA strand. Cas9 can then cut out a piece of DNA by making a double stranded break. Then the hostDNA is incorparated into the genome by the DNA-repair system.
What happens after Cas9 has made a double stranded break?
The cells DNA-repair system tries to repair the break. It does this by non-homologous end joining (NHE) since there is no template for the break. NHE can be very error-prone. This causes random insertions or deletions (indels) that will result in a frame shift of the open reading frame.
You can use CRISPR-Cas9 for genome engineering in mice, cells or even gene therapy for humans. You can knock out a gene or insert a new gene. How is it also called when you make use of donorDNA to knock in/insert this DNA into the genome?
Homology-directed repair
