BCH 4101 Lecture 8: Genome Editing
28 Apr 2018
School
Department
Course
Professor
October 30, 2017
Genome Editing
A Genomic Revolution
Genomic revolution: leads to transformation of basic science and personalized medicine
Includes:
-Affordable whole-genome sequencing
-Detailed databases (genetic variation)
-Large genome annotation projects
Disease-Causing Genes
To date, ~3445 genes have been identified that are associated with Mendelian diseases (single gene disorders/traits)
~500 genes have been identified that are associated with susceptibility to complex diseases
But, effective therapies are still needed (especially for rare diseases)
Treatments vs. Cures
Protein therapeutics (replacement/augmentation, antibodies)
Viral and non-viral delivery strategies to treat loss-of-function genetic diseases
RNA modification therapies (RNAi, anti-sense oligo nucleotides - ASO)
Issues:
-Delivery barriers: limits clinical application - can only target certain tissues/organs
-Incomplete suppression
-Off-target effects
-Safety
Genome Repair Methods
Non-homologous end-joining
-Method: joins together pieces of DNA
•Ku dimer and DNA PKcs recognize the double strand break, forming a protein complex
that protects the ends
•Processing of the ends by a large complex of proteins to make the ends compatible for
ligation
-Might need to trim the ends
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Natur e 447,"951-958"(21"June 2007)
October 30, 2017
-Might need to add a couple nucleotide
•More proteins are recruited to the complex, including for ligases that allows for the ligation of DNA ends
-Advantage: very quick - doesn’t rely on synthesis of DNA or DNAP
-Disadvantage: error-prone (indels, frameshift are common)
Homology-directed repair: use a template to restore the DNA that has been lost by the DNA
break
-DSB recognized by a complex of RAD proteins
-Resection of ends to create 3’ ssDNA (3’ overhang)
-3’ overhang region initiates pairing with a homologous region in an intact DNA duplex
•The 3’ overhang will then seek out a complementary template in the genome (found on
the homologous chromosome)
-Holliday junction is resolved and DNA ends are rejoined to yield intact dsDNA
•Holliday junction forms
•DNA is copied from the junction
•Resolvases come and resolve the junction
•DNA ends are rejoined and yield intact dsDNA
-Disadvantage: takes longer - need to seek out homologous template, relies on action of DNAP, Holliday junction
needs to be resolved
-Advantage: highly accurate/high fidelity - uses a homology template
Genome Editing Platforms
General method:
-Create a double-stranded break
-Add desired element/change sequence
-Rejoin the ends
•NB: can’t control which cells undergo non-homology end joining vs.
homology-directed repair
•After editing, the population will be very heterogenous
•Will have some cells with insertions, some with deletions, etc.
ZFNs (zing-finger nuclease): recognize specific DNA sequences
-Modular: can change them and direct them to a specific sequence
-Fusion protein: DNA-binding domain of zinc-finger proteins is fused with the
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Nature'Reviews'Genetics'11,'636-646'(September'2010)
October 30, 2017
nuclease domain of Fok1 restriction enzyme
•NB: removed the DNA binding domain of the Fok1 enzyme
-Zinc fingers can be engineered in order to specifically recognize a 3bp sequence that flanks the cleavage site
•Can use different combinations of zinc-finger nucleases (each recognize a 3bp sequence) to make the fusion
protein specific to a certain location
-NB: need to make it so that the zinc-fingers flank the restriction enzyme on both sides
TALENs (transcription activator-like effector nuclease)
-Take Fok1 nuclease domain and fuse it to fuse it to a customizable DNA-binding domain
-Will bind and cleave the sequence as a dimer (13-18 bp spacing)
-Highly modular
-Uses a TALEN-DNA “code”: recognizes highly variable sequences
•NN, NI, HD, and NG (G, A, C, T)
•DNA binding domain is composed of a series of TALE-repeats
-Protein composed of 33-35 aa’s, where aa 12-13 is the hypervariable
region and recognizes a single, specific nucleotide
•Varying the aa’s at position 12 and 13 within these repeats changes what
sequence the TALEN will recognize
CRISPR-CAS (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR
ASsociated system):
-First discovered in bacteria as a defence mechanism
•Invading DNA is cut and incorporated into a CRISPR locus amongst a series of short
repeats (CRISPR repeats, ~20 bps)
-Protospacer is the DNA of interest, placed in between CRISPR repeats
•Loci are transcribed and transcripts processed to produce small RNAs (crRNA)
-Contains the invading sequences and a small repeat
•Transactivating CRISPR RNA (tracrRNA) is also encoded by the system and will
associated with the crRNA to form crRNA-tracrRNA hybrid
-Interaction between the two RNAs only occur within a few nucleotides at the end - interacts with the repetitive
element
-NB: tracrRNA folds into a very specific conformation
•RNA duplex will associated with Cas9 nuclease, activating it
•crRNA-tracrRNA-Cas9 complex will scan DNA for the presence of a PAM
(protospacer adjacent motifs)
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Natur e'Reviews'Molecular'Cell'Biology 14,49–55 (2013)
Document Summary
Genomic revolution: leads to transformation of basic science and personalized medicine. To date, ~3445 genes have been identi ed that are associated with mendelian diseases (single gene disorders/traits) ~500 genes have been identi ed that are associated with susceptibility to complex diseases. But, effective therapies are still needed (especially for rare diseases) Viral and non-viral delivery strategies to treat loss-of-function genetic diseases. Rna modi cation therapies (rnai, anti-sense oligo nucleotides - aso) Delivery barriers: limits clinical application - can only target certain tissues/organs. Might need to add a couple nucleotide. October 30, 2017: more proteins are recruited to the complex, including for ligases that allows for the ligation of dna ends. Advantage: very quick - doesn"t rely on synthesis of dna or dnap. Homology-directed repair: use a template to restore the dna that has been lost by the dna break. Dsb recognized by a complex of rad proteins. Resection of ends to create 3" ssdna (3" overhang)
