CRISPR and SRY Gene

transcription

DNA → RNA

• by RNA polymerase

translation

RNA → protein

• by ribosome

bacteriophage

viruses that infect bacteria

transduction

when new phage are assembled within the bacteria and burst out of the bacterium (lysis)

innate immunity in bacteria

Bacteria have enzymes that protect itself.

1. methylases that methylate its own DNA

2. endonucleases that cleave foreign DNA

   • not specific to a specific pathogen

adaptive immunity in bacteria

CRISPR-Cas

• specific to a specific pathogen

CRISPR

genomic locus in the bacterium containing clustered regularly interspaced short palindromic repeats

CRISPR regions are found:

• adaptive immunity

within the genome of the bacteria

Cas

• adaptive immunity

proteins made by the bacteria

• mainly DNAses and RNAses

Cas proteins function

involved in creating or acquiring fragments of phage DNA that will be integrated in the bacterial CRISPR locus

spacer regions

viral sequences which act as a molecular memory of previous viral attacks

leader region

region where RNA polymerase will bind for transcription of the DNA region

• contains promotor sequence

CRISPR locus

evolves with each bacterial generation that survives an infection

CRISPER Cas Mechanism

1. spacer acquisition

2. crRBA biogenesis

   • protection against the next invasion

3. target interference

spacer acquisition

1. invading phage DNA is cleaved into smaller fragments (protospacers)

2. protospacers are inserted into CRISPR loci to become new spacers

crRNA biogenesis

1. CRISPR loci is transcribed from the promotor within the leader sequence.

2. long RNA transcripts → short CRISPR-derived RNAs (crRNAs)

target interference

1. crRNA acts as guide to bring nucleases to invading phage DNA

2. Cas nucleases cleave and destroy the phage DNA.

Mature crRNAs will associate with:

1. Cas nucleases

2. nuclease complexes

3. Cas9

Cas9 functions

1. recognizes protospacer adjacent motif (PAM)

2. cleaves spacer

Cas1/Cas2 complex function

integrates the spacer cleaved by Cas9 into the CRISPR locus

What is involved in Cas9 during crRNA processing?

1. transactivating crRNA (tracrRNA)

2. RNase type III

transactivating crRNA (tracrRNA)

noncoding RNA that binds to crRNA repeat with complementary sequence

RNase type III

cleaves pre-crRNA to form mature crRNA

• specifically requires double-stranded RNA

Cas9 protein contains two different active sites with nuclease activity:

• during targeted inference

1. HNH domain

2. RuvC domain

HNH domain

cleaves viral DNA strand complimenting the crRNA

RuvC domain

cleaves viral DNA strand that is non-complementary to the crRNA strand

Cas9 only cleaves target DNA if:

it is next to a PAM sequence (protospacer adjacent motif)

In order to cleave genomic DNA in mammalian cells, you have to supply:

1. sgRNA (single guide RNA)

2. Cas9 protein for host cell

sgRNA

single guide RNA

• contains a linker loop

• contains tracrRNA region

• ensures Cas9 enzyme cuts at the right point in the genome

methods of plasmid delivery into mammalian cells

1. electroporation

2. microinjection

3. lipid nanoparticles

types of CRISPR-Cas9

1. plasmid-based CRISPR-Cas9

2. Cas9 mRNA and sgRNA

3. Cas9 protein and sgRNA

delivery strategies of plasmid-based CRISPR-Cas9

1. electroporation

2. microinjection

3. lipid nanoparticles

delivery strategies of Cas9 mRNA and sgRNA

1. electroporation

2. microinjection

3. lipid nanoparticles

delivery strategies of Cas9 protein and sgRNA

1. electroporation

2. lipid nanoparticles

electroporation

putting an electric current through a cell in the presence of things you want in the cell

microinjection

injecting plasmids

process of knocking out a gene

1. CRISPR-Cas9 cuts target DNA in specific spot

2. ligation of broken DNA fragments

3. nonhomologous end-joining (NHEJ)

nonhomologous end-joining (NHEJ)

repairs the double-stranded break resulting in small insertions/deletions (indels) leading to non-functional gene product

process of knocking in a gene

1. CRISPR-Cas9 cuts target DNA

2. New DNA is added into the genome (donor template)

The process of knocking in a gene relies on which cellular repair system?

homology-directed repair (HDR)

• can be tricked into using an artificial donor template to make a substitution in the host genome

What is special about Cosmo the calf?

1. All of Cosmo’s cells have the SRY gene on chromosome 17.

2. has two copies of the SRY gene

What is the probability of genes in Cosmo’s offspring?

1. 75% male-like offspring

2. 50% XY

3. 25% XX with the SRY gene