CRISPR Flashcards
What is origin story of CRISPR?
It is bacteria’s adaptive immune responses to viruses.
CRISPR
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a powerful gene editing technology that allows scientists to selectively modify DNA sequences in living organisms. It was first discovered in bacteria as a natural defense mechanism against invading viruses, and has since been adapted for use in many different organisms.
The CRISPR system consists of two components: a guide RNA molecule, which directs the CRISPR-associated (Cas) protein to a specific target sequence in the DNA, and the Cas protein itself, which cuts the DNA at that site. By modifying the guide RNA, scientists can target virtually any DNA sequence of interest and make precise changes to the genetic code.
PAM
Protospacer Adjacent Motif (NGG)
CRISPR array
Two components: spacers and repeats
Pre-crRNA
Pre CRISPR RNA molecule transcribed from the CRISPR array
Three components:
1. Spacers
2. Repeats
3. Unprocessed tracrRNA (complementing the repeats)
Cr:tracrRNA
Three components:
1. Spacer
2. Repeats
3. TracrRNA (acting as an anchor / skyfold)
CAS9 protein
See pic
Guide RNA (gRNA): cas9 complex
See pic
Describe bacteria “remember” past viral infection?
See pic for detail:
- Step 1: Cas 1 — Cas2 complex scans the viral DNA for PAM
- Step 2: Once the complex finds PAM, they will cut a piece of the viral DNA (coding strand) upstream of the PAM sequence, this piece of DNA is called the protospacer (~20 nucleotides)
- Step 3: the complex will then insert the Protospacer to the CRISPR array
- Step 4: Build the repeat flanking the Spacer
Describe how bacteria “defend” themselves against for future viral infections?
See pic for detail:
- Step 1: Transcription of the CRISPR array to pre-crRNA
- Step 2: pre-crRNA then cut by RNAase to cr:tracrRNA
- Step 3: cr:tracrRNA / gRNA form a complex with cas9
- Step 4: cas9 recognizes the PAM due to its PAM domain
- Step 5: Opens up the DNA and see if there is sequence upstream the PAM sequence complementing its gRNA spacer sequence
- Step 6: if complementing sequence are found, cas9 will make ds break a few bases upstream the PAM
Step 4 to 6 are the essential steps of the modern CRISPR technology
SgRNA:cas9 complex
Single guide RNA (sgRNA): basically just cr:tracrRNA but linked and synthesized in the lab
How does bacteria know not to cut its own DNA?
Because of lack of PAM sequence in their own DNA sequence and the plenty of Chi sites that cas9 wont cut
How does modern CRISPR technology work to cut DNA?
- Step 1: cas9 recognizes the PAM due to its PAM domain
- Step 2: Opens up the DNA and see if there is sequence upstream the PAM sequence complementing its sgRNA (spacer) sequence
- Step 3: if complementing sequence are found, cas9 will make ds break a few bases upstream the PAM
How does CRISPR perform gene knockouts?
Through NHEJ (non-homologous end joining)
What is NHEJ / non-homologous end joining
See pic
- DNA repair w/o donor DNA template;
- Most common form of DNA repair mechanism in eukaryotic cells
- Very error prone and cause a gene mutation that knocks out a gene
How we use HDR for gene editing?
Homology arms: homologous arms to the DNA where the cas9 made cuts
How we use HDR for gene editing?
Homology arms: homologous arms to the DNA where the cas9 made cuts
How we use HDR for gene editing?
Homology arms: homologous arms to the DNA where the cas9 made cuts
