Central Dogma Lecture 9 CRISPR Flashcards
(35 cards)
What is gene knock-out and what are some examples of how you could do this?
-A genetic technique in which one of an organism’s genes is made inoperative
-Introduce a premature stop codon or a frameshift
-Must make homozygous knockout in diploid eukaryotes to eliminate function
-Used to study gene function by studying the effect of gene loss
Gene knock-in
-Insertion or modification of DNA that is normally found at that locus
-Mutation to swap one amino acid for another
-Repair a mutated gene, like in cystic fibrosis
-Insert foreign DNA, ex: fluorescent protein
-Add a function to a protein
What does knock-out and knock-in typically require?
-Homologous recombination
-Can be stimulated by a DSB nearby (can be repaired by NHEJ or HR
Why was knocking in or out genes difficult before CRISPR?
-Rate of HR-mediated targeting of embryonic stem cells is 1 in 10^3
-In somatic cells, rate of HR is 1 in 10^6-10^9
-HR is stimulated by DSB, and CRISPR allows you to make a DSB at a desired location
How do bacteria protect themselves against phages/viruses?
Restriction modification systems and CRISPR
What is CRISPR?
-Clustered Regularly Interspaced Short Palindromic Repeats
-Interspaced by unique 20-50 bp DNA sequences called protospacers
What are the protospacers in CRISPR?
-Sequences identical to phage DNA
-Historical record of foreign DNAs that ancestors survived
-Latest viral sequence encountered is integrated into bacterium’s genome at 5’ end of CRISPR locus as a protospacer
What are the CRISPR RNAs?
-CRISPR locus is transcribed into a single RNA, processed into ~30 nt transcripts of protospacers known as crRNAs
-A seperate transcript from a seperate gene encodes the tracrRNA (trans-activating CRISPR RNA)
-tracrRNA helps position the crRNA when bound to Cas 9
-With crRNA bound, Cas9 assumes an active conformation that is very different conformation than its inactive state without RNA bound
How does the Cas9 RNP form and what does it do?
-tracrRNA base pairs with crRNA
-tracrRNA + crRNA bind to Cas protein
-Cas + tracrRNA + crRNA form a ribonucleoprotein complex
-crRNA guides Cas to cleave complementary segments of invading viral dsDNA
-Like human immune system, CRISPR-Cas system “learns” from encounters with foreign DNA by inserting new protospacers
What is the current model system for Cas9?
Streptococcus pyogenes
What nuclease domains does spCas9 have, and what do these domains result in?
-HNH-like domain: cleaves viral target DNA on strand complementary to crRNA (the target strand)
-RuvC-like domain: cleaves viral target DNA on opposite strand (non-target strand)
-Generate a blunt DSB in target DNA, destroying the viral DNA
What is the PAM and why is it important?
-PAM = protospacer adjacent motif
-Sp PAM is 5’-NGG-3’ (fairly common)
-Cas9 cleaves target DNA 3-4 nt from 5’ end of PAM
-DNA is only cleaved if target DNA has a PAM sequence to the 3’ side of the non-target strand
-Requirement for a PAM prevents Cas9 from destroying the protospacer from which its crRNA was transcribed (autoimmunity)
-PAM is read by the Cas protein, not the RNA
What is sgRNA?
-First step toward engineering
-Fused the 3’ end of crRNA with 5’ end of tracrRNA to form sgRNA (single guide RNA, used today in gene editing and structural analysis)
-Made because one RNA is much easier to engineer than two
What happens when crRNA is bound?
Cas9 assumes an active conformation that is very different from its inactive state without RNA bound
What are some important characteristics of Cas9?
-Cas9 is a potent nuclease
-Cas9 is sequence-specific
What are some ways that bacteria reduce the chances of cutting their own genome?
-Cas9 only assumes active conformation once it is bound to crRNA/tracrRNA with an exact match to foreign DNA
-Cas9 only cleaves DNA if there is a PAM nearby
-The PAM is a component of the invading viral DNA but is not a component of the bacterial CRISPR locus
-The PAM distinguishes bacterial self DNA from non-self DNA, preventing bacterium’s CRISPR locus from being cleaved by Cas9
How can CRISPR-Cas9 be used as a tool?
-Use CRISPR system to target a DSB to a specific gene sequence
-Just need to design a sgRNA to your genomic region of interest and express sgRNA + Cas9 on a plasmid
-Now, people synthesize the sgRNA, complex it with purified Cas9 protein, and electroporate RNP into cells
-Allowed genome engineering to take a few weeks instead of many months/years
How is CRISPR used to make a knock-out cell line or animal?
-Induce a targeted DSB and hope it gets miss-repaired by NHEJ via frameshift or premature stop codon
-Can also make two cuts so a piece of DNA drops out
How is CRISPR used to make a knock-in cell line or animal?
-Induce a targeted DSB, provide a repair template containing the modification desired, and hope it gets repaired by HR
-Repair template must have long homology arms on either side of modification you wish
What else can you make with CRISPR (other than knock-out/in)
-A point mutation
-Insert foreign DNA
If all Cas9 does is generate a DSB, how does that inactivate a virus?
-Interrupts a gene (not able to completely transcribe/translate)
-Interrupts genome replication (replication fork collapse)
If CRISPR/Cas9 is so great, why are phages still a thing?
Phage inhibit, evade, or disrupt CRISPR systems
If Cas9 generates a DSB, won’t it be repaired by the cell?
Yes, but it then can be cut again until mutation in target site
What are important characteristics of using CRISPR for engineering eukaryotes?
-You are limited to cutting eukaryotic DNA sequences with PAM nearby
-You must mutate the PAM in your homology plasmid, or else your plasmid and newly edited eukaryotic genome can be cleaved again by Cas9
-You want to temporally limit expression of Cas9 in cells to reduce chance of off-target cuts i the genome of the cells you edited
