lecture 31 Flashcards
CRISPR and Cas9
surface barriers, cellular barriers, and inflammation are examples of what?
innate immunity
lymphocytes, T-cells, and B-cells are examples of what?
acquired immunity
when is acquired immunity “acquired?”
when a cell reacts to a foreign object/infection and keeps memory of it
how does viral DNA begin to be made in an bacterial cell?
a bacteriophage made of protein-coated DNA anchors itself in the cell wall of a bacteria cell and injects its DNA into the cell through a shealth; the organelles begin making this viral DNA
how does a bacteriophage-infected cell spread the virus to other cells?
the cell becomes so full of bacteriophages that its organelles have created that it bursts open, releasing the new bacteriophages to infect other cells
what does naturally occurring CRISPR in bacterial cells do?
acts as the bacteria’s immune system that protects them from viruses, especially bacteriophages, by storing a memory of previous viral infections and using that memory to recognize and fight future infections
what does CRISPR stand for?
clustered regularly interspaced short palindromic repeats
what is meant when it is said that palindromic repeats read the same way backwards and forwards?
comparing it base-by-base, it reads the same in either direction. GAATTC is an example of a palindromic repeat because the complimentary base pairs when read in the same 5’ to 3’ direction are GAATTC even though it is CTTAAG that binds
what shape do palindromic repeats form?
hairpin structure
what is spacer DNA complementary to?
viral DNA
what are the types of proteins made from CAs genes?
endonucleases and helicases
what is the order of CRISPR RNA?
CAs genes, repeats, unique spacer, repeats, unique spacer, etc.
what is spacer DNA?
unique DNA segments between each set of palindromic repeats that are derived from viral DNA that has been previously encountered and entered into the genome. it holds the “memory” of viruses.
where are CAs genes located relative to the repeats and spacers?
CAs genes are upstream
what do CAs genes do?
produce proteins(endonucleases and helicases) that carry out the immune functions of CRISPR
what type of molecule is Cas9?
protein –> enzyme –> endonuclease
in what structure is lab-created CRISPR/cas-9?
plasmid
in a bacterial cell, once the viral DNA is injected and recognized, the cell begins to transcribe CAs genes, thereby producing CAs proteins. what also happens in terms of the CRISPR DNA?
it is transcribed into a long strand of mRNA(including palindromic repeats and spacers)
what happens to the transcribed mRNA of the bacterial cell?
it is processed into short guide RNAs called crRNAs each containing a unique spacer sequence that is complimentary to the viral DNA
what does the crRNA do once it is made?
makes a complex with cas proteins that neutralize viral DNA when encountered
how does the crRNA-cas protein complex work?
when it finds viral DNA, helicase unwinds it, then the crRNA guides the complex to the complementary viral DNA sequence where endonuclease cuts the DNA, neutralizing it
what do cas1 and cas2 proteins do the new piece of viral DNA?
incorporate it into the CRISPR sequence as a spacer
what are all the elements of the plasmid?
non-coding scrambled RNA sequence, termination signal, CBh promotor(drives Cas9 expression), nuclear localization signal, SpCas9 ribonuclease, another nuclear localization signal, 2A peptide(allows for production of Cas9 and GFP from the same CBh promotor), U6 promotor(drives expression of gRNA)
what binds to the unwound viral DNA at the 5’ NGG 3’ spot?
protospacer adjacent motif(PAM)
