BIOL 2500 - Gene Editing (Topic 10.2)

gene editing

changing the nucleotide sequence at a specific chromosomal locus to any desired sequence

• uses a DNA endonuclease to cleave (cut) the DNA at specific target locations within the genome

• results in double-stranded breaks (DSB) in the DNA at target site

*DSB occur naturally within the cell, occur due to external and internal agents

• DSB allow for genes to be modified

cell can repair DSB DNA damage naturally inside the ______

nucleus

failure to repair DNA results in…

chromosomal instability and incomplete replication in the genome

• can lead to cell death, cancer, and other mutations

nonhomologous end joining (NHEJ) allows for gene knockouts/downs (4)

1. proteins (PKcs, Ku70, Ku80) recognize double-stranded breaks

2. protein complex attaches to the ends of the broken DNA

3. end are trimmed to remove nucleotides until a blunt end is achieved

4. DNA ligase “glues” the blunt ends back together

homology directed repair (HDR) allows for gene insertions and/or modifications (5)

1. a DSB occurs

2. nucleases trim off a portion of the broken strand, Rad51 binds the undamaged chromatid

3. strand invasion occurs resulting in a displacement (D) loop

4. DNA replication within the D loop synthesizes new DNA using the sister chromatid as a template

5. DNA strands are trimmed to blunt ends and are ligated to finish repair

gene editing tools exploit the cell’s natural DNA ______ mechanisms

repair

• translationally fused to a sequence-specific DNA binding domain

• incorporated into a complex with an RNA molecule

what were the first 2 widely available gene editing tools

ZFNs and TALENs

CRISPR/Cas9

first found in prokaryotes

• first discovered in 90s

• “cluster regularly interspersed short palindromic repeats”

• adjacent to the CRISPR, they also found DNA nucleases encoded in the genome

• CRISPR-associated (cas) genes

how does CRISPR/Cas9 work

natural defense mechanism is prokaryotes

1. CRISPR sequences are transcribed into non-coding RNA

2. tracer RNA gene is also transcribed into tracrRNA and Cas9 is produced

3. processed into crRNAs by cas-encoded RNases

tracrRNA and crRNA form a complex with Cas endonucleases to cleave foreign DNA

4. tracrRNA binds to Cas endonuclease

5. crRNA binds to tracrRNA via complementary base pairing

6. tracrRNA-crRNA-Cas complex cleaves the invading DNA into a DBS, deactivating the invader

• bacteria can store cleaved DNA in its genome for future invader recognition or inheritance

• CRISPR can be passed on to progeny!

HNH domain - CRISPR

cleaves the strand complementary to the crRNA

RuvC domain - CRISPR

cleaves the opposite strand of DNA

PAM - CRISPR

protospacer adjacent motif, destabilizes the adjacent sequence for crRNA complementary pairing (in Cas9 = NGG)

agrobacterium mediated transformation

1. T-DNA of the Ti plasmid is transcribed

2. virulence proteins are transcribed and translated

3. conjugative transfer proteins (green) are transcribed and translated

4. the T-DNA will insert itself randomly into the nuclear genome

genetically modified organisms (GMOs)

organisms that have had their DNA changed by artificial means