L13 Genome editing in biomedical science research

how many base pairs are in the human genome

3 billion

what is genome editing

a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using nucleases

why is genome editing useful

• enabling specific targeting of sequences within the genome without impacting the rest of the genomic sequence

• potential to cure genetic disease in a patient specific manner

what does CRISPR/Cas9 stand for

Clustered regulatory interspaced short palindromic repeats and CRISPR associated (Cas) proteins

what is the adaptive immune system of prokaryotes

found in more than 40% of bacterial genomes

describe the three component system which operates as a complex

• cas9

• crRNA

• tracrRNA

the complex cleaves, invading DNA to prevent re-infection by viruses

what are protospacers

fragments made by recognised invading DNA cut into cas1-cas2 protein complexes

what are protospacers integrated into

CRISPR locus located in the bacterial genome

upon viral reinfection, transcription of the protospacers to RNA is activated which bind to…

Cas9

Cas9/RNA duplex is recruited to… on the invading strand of DNA

complementary sequence

Cas9 cuts DNA strands creating a…

strand break

• prevents infection

what is the structure of the CRISPR locus

• transactivating RNA

• operon of cas genes encoding Cas protein components (e.g. cas1/cas2/cas9)

• identical repeat array

• spacer of invading DNA

what is the complex/duplex formed between the transactivating RNA (tracrRNA) and the Protospacer/CRISPR RNA (crRNA) termed

the guide RNA (gRNA)

what does the gRNA do

enables selective binding of Cas9 to invading DNA sequences

what does the Cas operon encode

Cas proteins required for DNA cleavage (Cas9)

what do Protospacer Adjacent Motifs (PAM) enable

Cas9-mediated DNA cleavage

where is the PAM site located

3-4 basepairs downstream of the cut site

how long is the PAM site

2-8 base pair long sequence

Cas9 will not cut invading DNA without a PAM site irrespective of…

Cas/gRNA binding

why arent PAM sequences present in the CRISPR locus

removed during protospacer creation

what does the removal of PAM sequences (during protospacer creation) prevent?

the bacterial CRISPR locus being targeted by Cas proteins to maintain intact protospacers which support immune surveillance

what two components is the gRNA in bacteria made of

• tracrRNA

• crRNA

how is gRNA simplified

adding a linker loop, creating a composite gRNA

what will deposition of the Cas9/gRNA complex at a desired locus of the genome enable

a site-specific cleavage through nuclease activity

what does the repair of the DNA break by endogenous DNA repair pathways enable

specific genomic edits to be introduced

what should the gRNA contain upstream of the PAM site

proto-spacer sequence (target sequence)

what should gRNAs be selective to avoid

off target effects

what is the PAM site for Cas9

NGG

what are cellular DNA repair pathways critical for

desired CRISPR-mediated DNA editing

when DNA is damaged by ionising radiation, what are generated

double stranded breaks

how are double stranded breaks repaired

cellular DNA repair machinary

what are the mechanisms of cellular DNA repair machinary

• Homology-directed repair (HDR)

• Non-homologous end-joining (NHEJ)

what happens when Cas9 cleaves DNA (critically)

a double strand break is introduced

• HDR or NHEJ repair pathways function downstream to repair DNA

• creates the desired gene edit

Non-homologous End Joining enables what

error-prone DNA repair

why is DNA repair by NHEJ error prone

• introduces insertions or deletions (indels) into DNA

• impacts gene function

KNOCKOUT

what is DNA precisely repaired using

sister chromatids during S phase of the cell cycle

KNOCKIN

describe CRISPR-mediated gene knock out via NHEJ

Target Cas9: gRNA complex to gene of interest

• DSB introduced

• Cell repairs the break via NHEJ (error prone)

• Indels introduced generating a frameshift (premature stop codons introduced)

• Normal gene product not expressed

describe CRISPR-mediated gene knock-in via HDR

• DSB introduced by Cas9: gRNA complex

• introduce a template the cell will use to repair the DSB through HDR

• HDR template requires >60 bp homology arms on either side of the point of mutation/insert

• PAM sites are removed from HR template to prevent re-targeting of the region

• inserts of several kilobases are possible