CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats
Short Palindromic Repeats
segments of DNA containing short, repetitive base sequences
In a palindromic repeat, the sequence of nucleotides is the same in both directions
Nucleobases of DNA
Nucleotide = Nitrogenous base + Pentose + Phosphate
Pentose = Sugar (monosaccaride)
Nucleoside = Nitrogenous base + Pentose
Nucleobase = Nitrogenous base
Clusterly Regularly Interspaced
(In between spacers) (Short Palindromic Repeats are the same)
by so called "Spacers" = Spacer DNA
(Spacers are unique)
Spacer DNA = intergenic spacer (IGS) is a region of non-coding DNA between genes
snippets of DNA from viruses that have attacked the bacterium.
CRISPR/Cas = Cas + CRISPR
Cas = CRISPR Associated Genes
Gene = A sequence of DNA/RNA
(codes a specific function - e.g. color of eyes, etc.)
encode proteins that mediate the defense process ~ They create proteins:
Nuclease Proteins (Cut DNA)
Helicase Proteins (Unwind DNA)
Bacteriophage
A virus that infects and replicates within a bacteria.
E. Coli
A prokaryotic bacteria in which the CRISPR/Cas9 immune system was observed
Viral Reproduction Cycles
Lytic cycle
Lysogenic cycle
CRISPR/Cas system
= prokaryotic bacteria immune system against foreign genetic elements
(1) Virus invades bacterial cell
(2) Spacer acquisition
Process of acquiring a 'spacer DNA' - from virus
Cas1
Cas2
proteins & dimeres
Dimere =  oligomer consisting of two structurally similar monomers joined by bonds that can be either strong or weak, covalent or intermolecular
Nuclease & Integrase
Endoribonuclease
Nuclease - can cut DNA
Integrase - can integrate DNA into an existing DNA
can cut RNA
(because some viruses can have/inject RNA genome instead of DNA)
create Cas1-Cas2 complex
X-ray crystal structure of E. coli Cas1-Cas2 (blue/green and yellow) in complex with double-stranded DNA (red) – image copied directly from Nuñez et al., 2015
(3) CRISPR RNA = crRNA processing
(3.1) Transcription of CRISPR DNA single strand to mRNA
(mRNA = "messenger RNA" is transcription - basically a copy of one DNA strand)
(3.2a) Type I crRNA processing
(3.2a.1) CRISPR repeats are looped - in mRNA
(3.2a.2) mRNA is cut to each spacer with looped repeat (1+1)
using either Cas6e or Cas6f proteins
(Cas5 can also be part of the process)
(pre-crRNA = mRNA)
(3.2a.3) Cut spacer + repeat is called CRISPR RNA = crRNA
(3.2b) Type II crRNA processing
(3.2b.1) tracrRNA = trans-activating CRISPR RNA
(Trans-acting RNA/gene codes for a protein that will be used in the regulation of another target gene.)
(3.2b.2) tracrRNA bounds to non-looped repeats of mRNA
(3.2b.3) mRNA is cut to each space with non-looped repeat and bounded tracrRNA
using Cas9 & RNase III proteins
(3.2c) Type III crRNA processing
(3.2c.1) Cas6 Homolog cuts mRNA directly into crRNA (spacer + repeat)
(4) Interference (We are close to the most significant part)
Merge of (crRNAs + Cas Protein/Enzyme)
Protospacer Adjacent Motif = PAM
= A 'flag' in injected genome of virus (bacteriophage in our case)
Guide RNA = gRNA (artificial - made by humans)
recognizable by
Cas Protein/Enzyme
(4a) Type I
(4a.1) Recognition of PAM by Cas Protein
(4a.2) Recognition of viral genome/DNA by crRNA Spacer
(4a.3) Creation of Cas Cascade (= Complex of multiple Cas proteins)
(Cas Cascade)
(Cas Cascade)
(Noone very well knows how that happens yet)
(4a.4) Cas Cascade will attract Cas3
(4a.5) Cas3 cuts viral genome ■
("Target degradation" - Virus Attacked Bounced Off!)
(4b) Type II
CRISPR/Cas9 System
(4b.1) Recognition of PAM sequence by Cas9 Protein
(4b.2) Recognition of viral genome/DNA with crRNA Spacer (on image above = pre-crRNA) = with RNA sequence by Cas9 Protein
(See https://kurzgesagt.com for more explanatory videos!)
(4b.3) "Double strand break" (DSB) by Cas9 enzyme ■
Cuts both strands!
At the same place!
Cas9 uses HNH and RuvC to DSB
Cas9
has 6 domains (Rec I, Rec II, Bridge Helix, RuvC, HNH, and PAM)
(4c) Type III
there is no PAM
(4c.1) Recognition of viral genome/DNA by crRNA Spacer
(4c.2) Creation of Cas Cascade (= Complex of multiple Cas proteins)
(4c.3) Cut of viral genome ■
Guide RNA = gRNA = tracrRNA + crRNA
Artificially created by
Emmanuelle Charpentier
Jennifer Doudna
With CRISPR/Cas9 system we are now able to cut DNA programatically where we want to.
And on the place of cut we can insert host DNA of our choice.
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