Sickle Cell Anemia
-the beta-globing gene is mutated
-a single base-pair transversion changes A to T within the 6th codon of exon 1
-the substitution of amino acid valine where glutamic acid is normally found causes the hemoglobin to clump in low O2 environments
-this causes the cells to have a rigid sickle shape, making it extremely difficult for the cells to pass through small blood vessels (capillaries), causing extreme pain and tissue damage
Microbial Immune System
-prokaryotes that survive viral attack can add 40 bp segment of viral DNA to their genome
- The CRISPR array is a library of viral DNA sequences
- a mix of repeated constant DNA sequences and unique (viral) DNA sequences
When Microbes are Infected
-they will transcribe the CRISPR locus to generate crRNA
-express Cas (CRISPR-associated proteins)
-Cas are endonuclease that cut dsDNA
-If one of the crRNA molecules binds incoming viral DNA, the Cas proteins will make a cut, allowing the microbe to degrade the viral DNA
CRISPER- Cas 9 Gene Editing System
-CRISPR/Cas 9 is a simple and fast method to introduce changes to genomes
-uses the Cas 9 endonuclease and synthetic 20-nucleotide RNA sequence (sg RNA- synthetic guide RNA) to target specific sites in any genome
How CRISPR Exploits Natural DNA Repair to Eukaryotic Cells
-sgRNAS can bind to specific locations in the genome and make a double strand break (DSB) to change or edit the genome
- non-homologous end joining
- a natural repair mechanism to fix double stranded breaks
-error prone- often results in INDELS that change the reading frame + disrupts genes - homology-directed repair
- new genes can be added if flanking sequences have homology to the area on either side of the DSB
-if the cell modified is a single-celled zygote, then the whole organism has its own genome edited
Gene Editing with CRISPR
-homologous DNA with desired sequence is injected along with plasmid coding for Cas 9 and sgRNA
-endogenous gene to edit
-DSB- new gene with be inserted
Using CRISPR- Cas 9 to Cure Sickle Cell Anemia
- epsilon is disregualted
- gamma-globin replaces epsilon (gives O2 to fetus)
- gamma-globing decreases, beta-globin takes over
-symptoms only appear when beta-globin takes over
2 Repair Methods:
- CRISPR and DNA template fix the mutation in the adult hemoglobin gene (replace it with wild-type)
- CRISPR reactivates the fetal hemoglobin gene by turning off the BCL11A gene (gamma-globing not turned off)
BCLL1A stops production of gamma-globin
Enhancers
-a noncoding DNA sequence that binds proteins called transcription factors and activates transcription of a nearby gene on the same chromosome
How Enhancer is Inactivated in CRISPR Swap
-indels created by Cas 9 cleavage and NHEJ destroy the GATA-binding site
