Genet 302 lec 23 & 24
How would you make a knockout mutation of YFG using CRISPR + NHEJ?
Choose an important early coding region of YFG with a unique 20 bp target next to a PAM, deliver Cas9 + gRNA into a zygote, let Cas9 cut the DNA, let NHEJ repair create a small indel, then screen offspring for a loss-of-function YFG allele
Why should a knockout target be in an important early region of YFG?
Because an indel there is more likely to disrupt the protein and make an amorphic allele. late-exon frameshifts are less likely to trigger NMD and may still produce a partly functional truncated protein.
What 3 conditions were given for choosing a CRISPR knockout target?
It should be in an important region of YFG, the 20 bp target should be unique, and it must end with PAM sequence
What PAM was emphasized for regular Cas9?
NGG
What kinds of mutations does CRISPR + NHEJ usually create for knockouts?
Small insertions, deletions, or del-ins
What is delivered to the zygote in the CRISPR knockout method from lecture 23? how is CRISPR tool made?
Cas9 and guide RNA. using the pX330 plasmid that codes for the gRNA (made in nucleus) and the cas9 (made in cytosol then goes to nucleus)
Where can the CRISPR reagents be injected in the zygote?
Male pronucleus or cytosol
What must you do after obtaining some YFG knockout offspring?
Breed them to make a pure-breeding strain
How could you make a mouse mutant for both YFG1 and YFG2 by breeding?
Cross a YFG1 mutant strain with a YFG2 mutant strain, then identify offspring mutant for both genes
How could you make a YFG1 YFG2 double mutant directly with CRISPR?
Inject a wild-type zygote with Cas9 and guide RNAs against both YFG1 and YFG2, then screen offspring for both mutations
How could you make a double mutant if you already have a YFG1 mutant strain?
Inject zygotes from the YFG1 mutant background with Cas9 and a guide RNA against YFG2, then screen for double mutants
How would you make a knock-in mutation in Yfg using CRISPR + HDR?
Choose a target site in Yfg, inject zygotes with Cas9 + guide RNA + donor DNA carrying the desired change, let Cas9 cut the target, let HDR copy the donor DNA into the break, implant embryos into pseudopregnant females, then identify knock-in offspring
What is the key difference between CRISPR knockout and CRISPR knock-in?
Knockout uses NHEJ to make indels, while knock-in uses HDR and supplied donor DNA to make a specific designed change
What extra component is required for CRISPR knock-in compared with knockout?
Donor DNA
What forms of Cas9 can be supplied for CRISPR knock-in?
Gene, mRNA, or protein
What forms of donor DNA can be supplied for CRISPR knock-in?
Double-stranded DNA or single-stranded DNA
Why do HDR enzymes find the donor DNA in knock-in experiments?
Because the donor DNA molecules are small and numerous
What is Phe508Del?
A 3 bp deletion removing phenylalanine at position 508
Why is Phe508Del important?
It is the most common mutant allele causing cystic fibrosis
What does CFTR normally do?
It makes a chloride channel
How does defective CFTR cause thick mucus in the airway example?
Reduced chloride transport reduces water movement into the airway lumen, causing dehydrated thick mucus
Why are knock-in mice useful?
They let you make specific alleles, including hypomorphic alleles or actual human mutations
What kinds of mouse alleles can CRISPR make in these lectures?
Knockout, knock-in, floxed, and other DNA-edited alleles
What kind of allele is usually made by CRISPR knockout methods?
An amorphic loss-of-function allele
What kinds of sequence changes commonly create CRISPR knockout alleles?
Small insertions, deletions, or del-ins
Why is the CRISPR knockout procedure considered simpler than knock-in?
It relies on NHEJ making indels rather than precise HDR using donor DNA
How does CRISPR + NHEJ make a knockout mutation?
Cas9 and guide RNA create a double-strand break, then NHEJ repairs it imperfectly and makes a small indel that disrupts the gene
What is the first rule for choosing a CRISPR knockout target in YFG?
Target an important early coding region, not a late exon
Why are late exons poor CRISPR knockout targets?
Mutations there are less likely to fully disrupt protein function
What is the second rule for choosing a CRISPR knockout target?
The 20 bp target sequence should be unique to YFG
Why must the CRISPR target sequence be unique?
To reduce off-target cutting elsewhere in the genome
What is the third rule for choosing a CRISPR knockout target?
It must lie beside a PAM sequence
What PAM was emphasized for regular Cas9?
NGG
What is the guide RNA target length emphasized in these lectures?
20 bp
What plasmid was used in lecture 23 to make the CRISPR tool?
pX330
What does the pX330 plasmid make?
Both the guide RNA and Cas9
What does the U6 promoter in pX330 do? the black part of the Bbsl after the U6 is what and the red part is what?
It drives guide RNA expression. black: part of gRNA that matches target. red: common part of gRNA.
What do you order to create the target-specific insert for the guide RNA plasmid?
Two single-stranded DNA oligos
How is the target-specific guide sequence inserted into the plasmid?
The 2 oligos are annealed into a short double-stranded insert and ligated into the plasmid at the BbsI sites
What does the Cas9 part of the pX330 plasmid encode? whats its promoter and what comes after it?
Humanized Cas9. CBh: promoter. pA: poly(A)signal come after hCas9.
What does humanized Cas9 mean here?
It has codons optimized for humans and includes a nuclear localization signal
Where can CRISPR reagents be injected into a zygote in lecture 23?
Into the male pronucleus or the cytosol
What is the outcome after injecting zygotes for a CRISPR knockout experiment?
Some offspring carry knockout mutations in the target gene
What must be done after finding knockout founders?
Breed them to make a pure-breeding strain
How can you make a mouse mutant for both YFG1 and YFG2 by standard genetics?
Cross a YFG1 mutant strain with a YFG2 mutant strain, then identify offspring carrying both mutations
How can CRISPR make a YFG1 YFG2 double mutant directly?
Inject a wild-type zygote with Cas9 plus guide RNAs against both genes, then screen offspring for mutations in both
How can CRISPR make a double mutant if you already have a YFG1 mutant?
Inject zygotes from the YFG1 mutant background with Cas9 plus a guide RNA against YFG2, then screen for double mutants
What is a knock-in mutation?
A specific designed sequence change inserted at a chosen genomic site
Why are knock-in mutations especially useful?
They let you make specific alleles such as hypomorphic alleles or actual human disease mutations
What is the key difference between CRISPR knockout and CRISPR knock-in?
Knockout uses NHEJ to make disruptive indels, while knock-in uses HDR and donor DNA to introduce a precise designed change
What is the disease-gene example used in lecture 24?
CFTR and cystic fibrosis
What does the CFTR gene normally encode?
A chloride channel
What does normal CFTR function help maintain in airways?
Hydrated mucus
How can CFTR loss of function contribute to cystic fibrosis symptoms in the airway model?
Reduced chloride movement reduces water movement into the airway lumen, producing dehydrated thick mucus
What mutant CFTR allele was emphasized?
Phe508Del
What is Phe508Del?
A 3 bp deletion that removes phenylalanine at amino acid 508
Why is Phe508Del important?
It is the most common mutant allele causing cystic fibrosis
What pre-CRISPR method was used to make a mouse Cftr Phe508Del model?
The embryonic stem cell method
How does CRISPR + HDR make a knock-in mutation?
Cas9 and guide RNA cut the target site, then HDR copies sequence information from supplied donor DNA into the break
What 3 components are injected into zygotes to make a knock-in by CRISPR + HDR?
Cas9, guide RNA, and donor DNA
In what forms can Cas9 be supplied for knock-in experiments?
As a gene, mRNA, or protein
In what forms can donor DNA be supplied for knock-in experiments?
As double-stranded DNA or single-stranded DNA
Why do HDR enzymes find the donor DNA during knock-in experiments?
Because the donor DNA molecules are small and numerous
What happens after zygotes are injected in a CRISPR knock-in experiment?
The embryos are implanted into pseudopregnant females and the offspring are screened for the desired knock-in allele
How would you make a knockout mutation of YFG using CRISPR + NHEJ?
Choose an important early unique 20 bp target next to a PAM, deliver Cas9 and guide RNA into a zygote, let Cas9 cut YFG, let NHEJ create an indel, then screen offspring for a loss-of-function YFG allele
How would you make a knock-in mutation in Yfg using CRISPR + HDR?
Choose a target at the Yfg site to change, inject zygotes with Cas9, guide RNA, and donor DNA carrying the desired mutation, let Cas9 cut the site, let HDR copy the donor sequence into the chromosome, implant embryos, then screen offspring for the knock-in allele