DNA mutations and Inheritance

Sickle cell anemia is a WHAT disease

Sickle cell anemia is a GENETIC disease

Sickle cell is the mist common WHAT in the world with 400,000 infants a year born with the disease

Sickle cell is the mist common INHERITED DISEASE in the world with 400,000 infants a year born with the disease

In sickle cell anemia you get painful WHAT which is cause by WHAT blockage

In sickle cell anemia you get painful VASO-OCCULSIVE CIRSIS which is cause by ARTERIOLE blockage

Sickle cell anemia

• The WHAT gene is mutated in sickle cell anemia

• A single base-pair WHAT changes a HWAT to WHAT with the WHAT codon of exon (close to the start)

• The substitution of the amino acid WHAT where WHAT is normally found causes the hemoglobin to WHAT in WHAT environments

• This causes the cells to have a WHAT shape making it difficult for the cells to pass through WHAT causing extreme pain and tissue damage

Sickle cell anemia

• The B-GLOBIN (B-globin) gene is mutated in sickle cell anemia

• A single base-pair TRANSVERSION changes a A to T with the 6th codon of exon (close to the start)

• The substitution of the amino acid VALINE (Val) where GLUTAMIC ACID (Glu) is normally found causes the hemoglobin to CLUMP in LOW O2 environments

• This causes the cells to have a RIGID SICKLE shape making it difficult for the cells to pass through SMALL BLOOD VESSELS (capillaries) causing extreme pain and tissue damage

Prokaryotes have a WHAT

Microbial immune system

Microbial immune system

• Prokaryotes that survive viral attack can add 40bp segment of WHAT to their genome

• The WHAT array is a library of WHAT

• Clustered Regularly Interspace Short Palindromic Repeat

• A mix of repeated WHAT DNA sequences and WHAT DNA sequences

Microbial immune system

• Prokaryotes that survive viral attack can add 40bp segment of VIRAL DNA to their genome

• The CRISPR array is a library of VIRAL DNA SEQUENCES

• Clustered Regularly Interspace Short Palindromic Repeat

• A mix of repeated CONSTANT DNA sequences and UNIQUE (VIRAL) DNA sequences

Microbial immune system: When infected. microbes will:

• WHAT the CRISPR locus to generate WHAT

• Express WHAT proteins

• Cas are WHAT that cut WHAT

• If, one of the crRNA molecules WHAT incoming viral DNA, the Cas proteins will make a WHAT, allowing the microbe to WHAT the viral DNA

Microbial immune system: When infected. microbes will:

• TRANSCRIBE the CRISPR locus to generate crRNA

• Express Cas (CRISPR-associated) proteins

• Cas are ENDONUCLEASE that cut DOUBLE STRANDED DNA

• 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

CRISPR-Cas9 Gene Editing System:

• Since 2013, the understanding of an adaptive immune system from bacteria called CRISPR/Cas9 has sparked a revolution in the ability to modify or edit the genomes of variety of organisms - including humans

• Compared to earlier methods, CRISPR/Cas9 is a much WHAT and WHAT method to introduce changes into genomes

• CRISPR/Cas9 uses the WHAT and WHAT RNA sequence (WHAT) to target specific sites in ANY genome

CRISPR-Cas9 Gene Editing System:

• Since 2013, the understanding of an adaptive immune system from bacteria called CRISPR/Cas9 has sparked a revolution in the ability to modify or edit the genomes of variety of organisms - including humans

• Compared to earlier methods, CRISPR/Cas9 is a much SIMPLER and FASTER method to introduce changes into genomes

• CRISPR/Cas9 uses the Cas9 ENDONUCLEASE and SYNTHETIC 20-NUCLEOTIDE RNA sequence (sgRNA (synthetic guid RNA)) to target specific sites in ANY genome

sgRNAs can be designed to bind to specific
locations in the genome and make a double
strand break (DSB) to then change or edit the
genome:

1. WHAT

2. WHAT

sgRNAs can be designed to bind to specific
locations in the genome and make a double
strand break (DSB) to then change or edit the
genome:

1. Non-Homologous end joining (NHEJ)

2. Homology-directed repair

1. Non-Homologous end joining (NHEJ)

• A natural repair mechanism to fix WHAT breaks

• Error-prone – often results in WHAT that change the WHAT (insertion) + can disrupt WHAT (deletion)

Use CRISPR in genes you don’t want funtioning

1. Non-Homologous end joining (NHEJ)

• A natural repair mechanism to fix DOUBLE-STRAND breaks

• Error-prone – often results in INDELs that change the READING FRAME (insertion) + can disrupt GENES (deletion)

Use CRISPR in genes you don’t want functioning

2. Homology-directed repair

• New genes can be WHAT if flanking sequences have WHAT to the area on either side of the WHAT

Introduce genes taht were not there before

2. Homology-directed repair

• New genes can be ADDED if flanking sequences have HOMOLOGY to the area on either side of the DSB (double-strand break)

Introduce genes taht were not there before

If the cell modifies is a single-celled zygote, then the whole organism has its WHAT edited

If the cell modifies is a single-celled zygote, then the whole organism has its GENOME edited

Gene editing with CRISPR/Cas9

1. Inject Homologous DNA with desired WHAT along with plasmid coding for WHAT and WHAT

Gene editing with CRISPR/Cas9

1. Inject Homologous DNA with desired SEQUENCE along with plasmid coding for Cas9 and sgRNA

Gene editing with CRISPR/Cas9

2. Allow Cas9 system to cause a WHAT and then use WHAT to switch the old genes with the wanted genes

Gene editing with CRISPR/Cas9

2. Allow Cas9 system to cause a DOUBLE-STRAND BREAK and then use ENZYMES to switch the old genes with the wanted genes

Using CRISPR-Cas9 to cure sickle cell anemia

• When in utero the fetus uses WHAT hemoglobin which is more oxygen prone than the WHAT hemoglobin in adults

• The WHAT hemoglobin gene mutation is what causes sickle cell anemia

• Therefor to cure sickle cell anemia you want to do WHAT

Using CRISPR-Cas9 to cure sickle cell anemia

• When in utero the fetus uses GAMA-hemoglobin (Y-globin)which is more oxygen prone than the BETA-hemoglobin in adults

• The BETA-hemoglobin (B-globin) gene mutation is what causes sickle cell anemia

• Therefor to cure sickle cell anemia you want to do REPLACE ALL B-GLOBIN WITH Y-GLOBIN

Using CRISPR to cure sickle cell anemia

Option A: Fix WHAT and WHAT fix the mutation in the adult hemoglobin gene.

Option B: Swap CRISPR reactivates the WHAT hemoglobin gene by turning off the WHAT gene.

Using CRISPR to cure sickle cell anemia

Option A: Fix CRISPR and DNA template fix the mutation in the adult hemoglobin gene.

Option B: Swap CRISPR reactivates the FETAL hemoglobin gene by turning off the BCL11A gene.

Which option was more affective

Option B

An enhancer is a WHAT DNA sequence that binds proteins called WHAT and activates transcription of a specific nearby gene on the same chromosome

An enhancer is a NON-CODING DNA sequence that binds proteins called TRANSCRIPTION FACTORS and activates transcription of a specific nearby gene on the same chromosome

Option B: Using CRISPR-Cas9 to turn off a gene coding for a repressor of fetal hemoglobin genes

How do they inactivate this enhancer so efficiently?

• Approximately 80% of CRISPR treated cells (with WHAT) have WHAT in this 2019 paper.

• Not necessarily the precise WHAT used in the clinical trial.

Option B: Using CRISPR-Cas9 to turn off a gene coding for a repressor of fetal hemoglobin genes

How do they inactivate this enhancer so efficiently?

• Approximately 80% of CRISPR treated cells (with sgRNA-1617) have INDELs in this 2019 paper.

• Not necessarily the precise sgRNA used in the clinical trial.