L8: Genome Editing Flashcards
define genome editing
- precise deletion, insertion or replacement of DNA
- facilitated by site-specific nucleases (known as “molecular scissors”)
what is an advantage of genome editing?
Desirable DNA modifications can be introduced rapidly and precisely
what are molecular scissors?
- used to create double-stranded break (DSB) at specific site in genome
- and stimulates DNA repair mechanisms
molecular scissors - what DNA repair mechanism does it stimulate?
- Non-homologous end-joining (NHEJ)
- Homology directed repair (HDR)
molecular scissors - non-homologous end-joining (NHEJ)
- can create insertions/deletions (indels)
- and cause gene mutation
molecular scissors - homology directed repair (HDR)
- requires template homologous to broken region
- can be used to repair DNA or introduce foreign insertion
- more persice than NHEJ
what are three genome editing techniques
1) Zinc finger nucleases (ZFNs) (1996)
2) Transcription activator-like effector nucleases (TALENs) (2010)
3) Clustered regularly-interspaced short palindromic repeats (CRISPR/Cas9) (2012)
zinc finger nucleases (ZFNs)
- looks at DNA-protein interaction
- the zinc finger domains each recognize 3 bases of DNA.
- Linking together 4-6 zinc finger proteins creates 12-18 base pair specificity within the enzyme FokI
zinc finger nucleases (ZFNs) - explain the enzyme Fokl
- non-specific endonuclease that cuts DNA
- FokI must dimerize (two molecules come together) to cut, ensuring specificity and reducing off-target effects
Transcription activator-like effector nucleases (TALENs)
- based on a natural infection process by the plant pathogen Xanthomonas
- it makes transcription factors that regulate plant genes
- transcription factor ex: TALE (Transcription Activator-Like Effector)
Transcription activator-like effector nucleases (TALENs) - TALE
- has modules and 2 amino acids that dictate what the module binds to
- can use this to design a TALE to target a specific gene to get double stranded breaks
what are clustered regulatory interspaced short palindromic repeats (CRISPRs)
- first identified in the E. coli genome: 1987
- CRISPR has unique spacer sequences that match known plasmid and phage genomes
- spacer sequences could be a possible defense mechanism against foreign DNA
CRISPRs - where are spacers located
upstream of a PAM sequence (any nucleotide followed by NGG)
CRISPR/Cas9 - natural system (CRISPR)
- a new spacer sequence is added to an array when encountered on a foreign piece of DNA
- CRISPR locus is transcribed into pre-crRNA (precursor CRISPR RNA)
- pre-crRNA is processed into mature crRNAs
- crRNA binds to the Cas9 protein, forming a crRNA-Cas9 complex which then degrades (cuts) foreign DNA
CRISPR/Cas9: natural system (CRISPR) - number of spacers in pre-crRNA vs crRNA
- pre-crRNA: multiple, each one matching the DNA of what is attacking the cell
- crRNA: one in each that is produced
CRISPR/Cas9: natural system (CRISPR) - how is this related to the bacterial “immune system”
bacteria do not have an immune system but this method cuts foreign DNA to avoid infection and thus acts like an immune system
CRISPR-Cas9 system
- uses nucleic acid interactions (w each other)
- the natural CRISPR system has been engineered to make RNA-guided Nucleases (RGNs)
- based on CRISPR-associated protein 9 (Cas9) nuclease from Streptococcus pyogenes
CRISPR-Cas9 system - advantages
- RGNs use base pairing between an engineered RNA and the target DNA site
- expression of a single Cas9 with an array of gRNAs simultaneously targets multiple genomic loci
- considered to be most efficient, least expressive, and most user-friendly of the three strategies
CRISPR-Cas9 system - exploit base pairing
- can design guide RNA (gRNA) to associate to a specific region of DNA
- gRNA then (1) interacts with DNA and (2) complexes with the Cas9 nuclease to cut DNA
- can then use NGEJ or HDR to seal in break
CRISPR/Cas9 system - targets multiple genomic loci
- can use multiple gRNAs simultaneously
- will then result in a large deletion rearrangement once the DNA fuses together
comparison of ZFN and TALEN vs CRISPR/Cas9 - ZFN and TALEN
- protein-DNA interaction (interface is complex)
- complicated and expensive construction
- difficult to target multiple nuclei
comparison of ZFN and TALEN vs CRISPR/Cas9 - CRISPR/Cas9
- RNA-DNA (base pairing is simple)
- simple and inexpensive (can be done in days)
- array of gRNAs possible
genome editing application - introduction of mutation
gene therapy
introduction of mutation - gene therapy
- introduce a beneficial mutation
- need a healthy donor and use CRISPR/Cas9 or ZFN to create a mutation
- transplant the mutation into patient
introduction of mutation: gene therapy - Huntington’s Disease
- use donor DNA to correct the extended repeat region
- use homology directed repair
technique to detect genome editing
T7 endonuclease I (T7EI) cleavage assay
T7 endonuclease I (T7EI) cleavage assay - explain the T7EI enzyme
structure-sensitive and will only cut DNA if there are structural deformities
T7 endonuclease I (T7EI) cleavage assay process
- apply PCR
- after PCR: apply heat then bring it back to a lower temp so everything zips back up
- T7EI cuts the gene
- run results on gel
T7 endonuclease I (T7EI) cleavage assay - why heat/cool it up?
- once the gene is edited, an indel will appear on both strands of DNA bc PCR will duplicate the edit into the second strand
- heating and cooling allows the DNA to possibly zip back up wrong so a structural deformity so T7EI can cut it
T7 endonuclease I (T7EI) cleavage assay - what do the results look like?
- T7EI was successful if you see multiple bands in gel results
- represents mismatched DNA
