Applications of Genomics Flashcards
2 categories of repair pathways for double-strand breaks (DSBs) upstream of the PAM sequence
- Non-homologous end joining (NHEJ)
2. Homology-directed repair (HDR)
Non-homologous end joining (NHEJ)
- NHEJ-break ends can be ligated without a homologous template
- very efficient repair mechanism that is most active in the cell
- susceptible to frequent mutation errors due to nucleotide insertions and deletions (indels)
Homology-directed repair (HDR)
- require template to guide repair
- considered the dominant mechanism for precise DSB repair
- suffers from low efficiency
- requires higher sequence similarity between the severed and intact donor strands of DNA
tracrRNA
Holds crRNA in place in Cas9
trans-activating crRNA (tracrRNA)
— a small RNA that is trans-encoded upstream of the type II CRISPR-Cas locus in Streptococcus pyogenes
CrRNA
CRISPR RNA
Fits in CAS protein and breaks down invader DNA
- up to ~105 nt, ~20 nt match target site, provides scaffold to bind tracrRNA which binds with Cas9
Cas9
Cas9 is an endonuclease, crRNA fits in it and guides it to where to cut
Cas9 binds short PAM sequence, Cas9 cleaves DNA
Cas = “CRISPR-associated” gene
Helicases (unwind DNA)
Nucleases (cut DNA)
PAM
Protospacer adjacent motif (PAM)
- 2-6 base pair DNA sequence immediately FOLLOWING the DNA sequence targeted by the Cas9 nuclease
Cas9 binds short PAM sequence then cleaves DNA
sgRNA
Single guide RNA in Cas9
tracrRNA-crRNA chimera
“CRISPR” part of CRISPR-Cas9 system
- What does CRISPR stand for?
Clustered Regularly Interspersed (Spacer DNA) Short (10-20 nt) Palindromic (Same forward/backward) Repeats
How to cut the DNA we want to cut?
Know DNA we want to cut, make corresponding gRNA, and it will cut!
Make a guide RNA that will have a corresponding bit of RNA to what we want to cut
DNA feeds into Cas9
SNIP! Now we have an inactive gene
How to insert a new gene?
As Cas9 breaks the DNA, homologous repair template is added with sequence of interest
This allows specific mutations of single strand DNA to be added, and the complementary DNA strand will be automatically added
Need…
1 gRNA (CRISPR)
2 Cas9
3 Host RNA we want to insert
- How was the guide RNA engineered in this system and what are its 2 critical features?
crRNA is CRISPR RNA with a spacer FNA segment that will match with the target DNA
tracrRNA holds crRNA in place
Put the 2 together
- What are applications of CRISPR-Cas9 mentioned here AND what are other applications that you can think of?
large-scale screening for drug targets
potentially correct genetic mutations responsible for inherited disorders.
- What is the structure of Cas9? What does each domain do? (p. 2)
endonuclease that uses a guide sequence within an RNA
duplex, tracrRNA:crRNA, to form base pairs with DNA target sequences, enabling Cas9 to introduce a site-specific double-strand break in DNA
sequence at the 5’ side of sgRNA determines the DNA target site
duplex RNA structure at the 3” side
binds to Cas9
Cas9 cuts
- What is the tracrRNA: what does it stand for and where is it located in the genome? (p. 2)
trans-activating crRNA (tracrRNA)
— a small RNA that is trans-encoded upstream of the type II CRISPR-Cas locus in Streptococcus pyogenes
Holds crRNA in place in Cas9
- What is the structure of the guide RNA and its two critical features? (p. 3)
tracrRNA and crRNA
How to knock-out a gene using CRISPR-Cas9?
Repair ds break by adding 1 or 2 nt, causes frameshift, usually 5’end of gene targeted
NHEJ repair of ds break
How to edit a gene using CRISPR-Cas9?
Homologous repair template is added with sequence of interest, allows specific mutations
HDR repair of break
What is Phylogenomics?
Phylogenomics is the use of whole organellar genomes to determine phylogenetic relationships among species
Used to align genomes and for phylogenetic tree construction
Phylogenomics in Animals and Plants?
Animals: Mitochondrial genome – small ~16kb, 13protein coding genes, barcode & sequence many at one time
Chloroplast genomes used in plants
Used to align genomes and for phylogenetic tree construction
What are applications of high-throughput sequencing to genotyping and genetic diversity studies?
Genotyping by Sequencing
- Compares sequencing profiles among samples:
Phylogenetics
- Used to align genomes and for phylogenetic tree construction
What is Genotyping by sequencing (GBS)?
Genotyping single nucleotide polymorphisms (SNPs)
What is the reason for genotyping single nucleotide polymorphisms (SNPs)?
SNPs between individuals allows comparison of sequencing profiles among samples: SNP discovery, relatedness comparisons, mutation detection, etc.
How does Genotyping by sequencing (GBS) compare to genomic sequencing
Not whole genome sequencing; only sequences a small part of a genome
Cheaper, faster
What is the purpose of Barcoding in GBS?
To multiplex samples on high throughput
sequencing, usually on Illumina sequencer
Barcoding identifies which sequences go with which sample
How are Reduced Representation Libraries (RRLs) made?
By digesting each DNA sample with a restriction enzyme (ApeK1).
- leaves sticky ends at restriction sites
How are barcodes added to DNA in a RRL?
Custom barcoded adaptors are ligated to sticky ends of restriction sites
Each sample has its own unique barcode sequence
Steps of Genotyping by Sequencing.
- make RRLs
- Barcode each sample
- Pool digested and barcoded DNA into a single tube, perform PCR amplification, library preparation, and sequencing on Illumina platform
- Use barcodes to assign sequences to samples. Produce a file of DNA sequence data for each sample.
How does CRIPSR-Cas9 gene editing tool have the ability to edit (fix) genes?
RNA guide leads the enzyme to the cutting site of the host DNA. CAS9 comes in and snips the DNA
The host cell’s natural DNA repair mechanism fixes the cut while integrating the donor DNA – therefore altering the gene
