Genet 302 lec 19 & 20
What are the two core components of the CRISPR-Cas9 system?
Cas9 protein and guide RNA
What does the guide RNA do in CRISPR?
It base-pairs with the target DNA sequence and directs Cas9 to the correct site
What length of DNA target does the guide RNA typically recognize in this lecture?
About 20 bp
What does Cas9 do once it is guided to the target DNA?
It cuts both DNA strands and creates a double-strand break
What is step 1 of the CRISPR system?
Get Cas9 protein and guide RNA(s) into the cell or organism
What is step 2 of the CRISPR system?
CRISPR makes a double-strand DNA break at the target site
What is step 3a of the CRISPR system?
Repair by the cell’s NHEJ system
What usually happens when a CRISPR break is repaired by NHEJ?
A small deletion or other small indel is made at the target site
Why is NHEJ useful in CRISPR experiments?
It often disrupts the target gene and can create a knockout
What does NHEJ stand for?
Non-homologous end joining
What is step 3b of the CRISPR system?
Repair by the cell’s HDR system using supplied DNA
What does HDR stand for?
Homology-directed repair
What can HDR do after a CRISPR cut?
Replace target DNA with new DNA you supply
What template does HDR use for repair?
A homologous DNA template such as a sister chromatid, homologous chromosome, or experimentally supplied donor DNA
How is CRISPR different from classic restriction enzymes in this lecture?
Restriction enzymes are protein-only enzymes that recognize short fixed sequences and cut DNA in vitro, while CRISPR uses Cas9 plus guide RNA to target chosen sequences and cut DNA in vivo
What are restriction enzymes used for naturally?
Bacteria use them to cut up bacteriophage DNA
What are restriction enzymes used for in molecular genetics?
To cut DNA in vitro
What is a key difference in target recognition between restriction enzymes and CRISPR?
Restriction enzymes recognize short fixed sequences, while CRISPR can be programmed by changing the guide RNA
In the goat CRISPR example, which two genes were targeted?
MSTN and FGF5
What is the effect of MSTN activity?
It negatively regulates muscle cell growth
What is the effect of FGF5 activity?
It negatively regulates hair growth
Why target MSTN and FGF5 in goats?
To produce goats with more muscle and more hair
In the goat example, was the goal NHEJ or HDR?
NHEJ
Why were two cuts made in each target gene in the goat example?
To promote deletion of the region between the cuts and disrupt the gene
How can Cas9 specificity be improved?
By changing amino acids in Cas9 to reduce off-target cutting
How can CRISPR be used to regulate genes without cutting DNA?
Use inactive Cas9 linked to a transcription factor to activate or repress a target gene
How can CRISPR be used to change a base without making a double-strand break?
Use CRISPR-linked base editing enzymes (mutagens) targeted to the DNA site
What is the full testable CRISPR workflow from this lecture?
Deliver Cas9 plus guide RNA, make a double-strand break, then repair by NHEJ for small deletions or by HDR for precise replacement with a piece of dsDNA supplied by you
What is the diploid chromosome number of mice?
2n = 40
What is the haploid genome size of the mouse in this lecture?
About 2600 Mb
What is the general structure of mouse autosomes?
They are telocentric
What does telocentric mean?
The centromere is at or very near one end of the chromosome
How do mouse sex chromosomes compare with human sex chromosomes? evolutionarily what is closest to humans, dogs, cats or mice?
They look similar to those in humans and other mammals. mice- 75 MYA
What are common lab mouse strains mentioned in this lecture?
BALB/c (albino) and C57BL/6J (black 6)
What coat color is typical of wild mice?
Agouti
Why are mice useful as laboratory organisms?
They are small, cheap to maintain, and reproduce quickly
What is a typical litter size for lab mice?
About 5 to 10 pups
What is the mouse egg-to-egg generation time in this lecture?
About 9 weeks. gestastion- nursing- maturation
What major mouse stock center was emphasized in this lecture?
Jackson Laboratory
What is MGI?
Mouse Genome Informatics, a major website/database for mouse genetics information
How are mouse gene symbols written compared with human gene symbols?
Similar to humans, but mouse symbols are not all capitals
How is the mouse gene symbol for the CECR2 ortholog written?
Cecr2
How is the human gene symbol for the same gene written?
CECR2
What is a genetic map based on?
Meiotic recombination
What units are used on a genetic map?
Centimorgans, cM
What is a sequence map based on?
DNA sequence position in base pairs
What is a cytogenetic map based on?
Chromosome banding patterns seen microscopically
Why is the cytogenetic map less commonly used in mice?
Because mouse chromosomes are small and mostly telocentric, so cytogenetic detail is less useful
What is the mouse abbreviation for a deletion chromosome rearrangement?
Del ( ) vs 46, XX, del ( ) - humanns
What is the mouse abbreviation for a duplication chromosome rearrangement?
Dp
What is the mouse abbreviation for an inversion chromosome rearrangement?
In
What is the mouse abbreviation for a translocation chromosome rearrangement?
T
How are mouse chromosome rearrangements generally named? Are mutations in mice induced or spontaneous?
By rearrangement type, chromosome involved, and discovery designation. induced mutation
How would a deletion on chromosome 9 that was the fourth deletion isolated at Harwell be written?
Del(9)4H orDel4H - need to be able to read this not write it
What is the mouse abbreviation for trisomy?
Ts
What is the mouse abbreviation for monosomy?
Ms vs 45, XX, - (in humans)
How is a mouse trisomic for chromosome 16 written?
41,Ts16
Why is the total chromosome number 41 in Ts16?
Because trisomy means one extra copy of chromosome 16 added to the normal 40
Which mouse chromosome is often compared to human chromosome 21 in this lecture?
Mouse chromosome 16. MMU16 ~ HSA21
Why are lab mice not the same as wild mice behaviorally?
Lab mice have undergone artificial selection, inbreeding, and adaptation to captivity
According to the lecture, CCAC considers vertebrates and which invertebrates to be animals? but not..?
Cephalopods. not insects or nematodes (c. elegans)
What is the diploid chromosome number of mice? What is the general structure of mouse autosomes?
2n = 40. They are telocentric.
What is a genetic map in mouse nomenclature based on?
Meiotic recombination
What is a sequence map in mouse nomenclature based on?
DNA sequence position in base pairs
Why is the cytogenetic map less commonly used in mice?
Because mouse chromosomes are small and mostly telocentric, so banding patterns are less useful