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Genome Editing
AGRY 600
Fall 2016
Things you can do once you know a genome sequence
• A major goal for many years has been precise alteration of the
genome without unintended, additional damage
– Preferably without the need to search through large numbers of
samples or organism
• Damage/Disruption vs. Sequence Replacement
– Both begin with DNA Double-Strand Breaks (DSBs)
Damage/Disruption vs. Sequence Replacement
Making the DSB Precisely
• Two major methods for providing specificity have
emerged:
– Sequence-specific “Designer” DNA binding proteins
• Zinc-finger proteins
– RNA-guided base pairing
• TRANSIENT (used largely for CHARACTERIZING GENE FUNCTION or IN
SITUATIONS WHERE MUTANT PHENOTYPE IS LETHAL)
• PERMANENT (used largely for GENOME EDITING)
CRISPR-Cas
• Clustered Regularly InterSpaced Pallindromic
Repeats
• Discovered as what prokaryotes have as an
immune system
• Pallindromic Repeats of 20-40 bases,
separated by short sequences that turn out to
be leftover from bacterial viruses that had
previously infected the cell
– Pallindromic DNA, when transcribed make RNA’s
that can base pair with themselves to create
hairpin turns and dsRNA
CRISPR-Cas
• Clustered Regularly InterSpaced Pallindromic
Repeats
• The spacer DNAs are those bits of
bacteriophage
• About the same time that was found, also
found “CRISPR Associated Sequences” (Cas)
genes
• These encode a variety of proteins, typically either
helicases or nucleases
CRISPR-Cas
CRISPR-Cas
Streptococcus pyogenes has only one Cas protein (Cas9)
Streptococcus pyogenes has only one Cas protein (Cas9)
Modified CRISPRCas9 System
CRISPR-Cas9 finds DNA that base pairs with the
guide RNA sequence and then cuts on both strands
Question then becomes how the cell will repair this DSB?
Question then becomes how the cell will repair this DSB?
Question then becomes how the cell will repair this DSB?
Question then becomes how the cell will repair this DSB?
Homologous repair events are extremely rare in plants
Are there ways to favor them?
• Overexpression of yeast Rad54 an ATPase that modifies the
topology of DNA to facilitate HDR, in Arabidopsis increases
HDR efficiency to incorporate a new DNA template by one to
two orders of magnitude (Shaked et al. 2005)
• Knocking out or transiently silencing critical components of
the NHEJ pathway (Qi et al. 2013).
• 5-16-fold increase in HDR a ku70 mutant and 3-4-fold in lig4 mutant.
Also shifted NHEJ to microhomology-dependent alternative version of
NHEJ creating large deletions
Chemical screens identify
compounds that alter outcomes of
CRISPR/Cas9 editing even though
how they do it (or whether they
work in plants is not always clear
Is there a way to favor homology-directed repair?
• One compound, “RS-1”, stimulates HDR by promoting active
presynaptic filaments formed by RAD51 and single-stranded
DNA in vitro and in human cells (Jayathilaka et al. 2008)
• Recently shown to enhance HDR-mediated by CRISPR/Cas9 in rabbits
(Song et al. 2016), not yet tested in plants
• Another strategy: Enhance HDR donor DNA accessibility
• Delivery in the form of incoming T-DNA more effective for HDR than if
donor is already present elsewhere in the genome (Puchta 1999;
Puchta et al. 1996).
•
Excising a chromosome-integrated donor with a nuclease, can
increase HDR to as much as 1 %. (Fauser et al. 2012)
Is there a way to favor homology-directed repair?
• HDR frequency positively correlated with copy number of
freely available donor molecules.
• For donor DNA fragment delivered by geminivirus-based vectors
(replicate up to hundreds of copies per cell) enhances HDR in tobacco
somatic cells (Baltes et al. 2014).
Baltes et al. Plant Cell 2014;26:151-163
Homologous repair events are extremely rare in plants
Typcally require selection or other tricks to find them
Introduction of a kanamycin resistance cassette into the ADH1
gene locus in Arabidopsis thaliana (Schiml et al. 2014).
Introduce a kanamycin resistance cassette upstream of a 35S
promoter in between the ANT1 promoter and open reading
frame (ORF) in tomatoes (Cermak et al. 2015).
Overexpression of ANT1 results in accumulation of purple
anthocyanin providing a convenient screening method
Homologous repair events are extremely rare in plants
Typcally require selection or other tricks to find them
Zhao et al (2016)
Scientific Reports
doi:10.1038/srep23890
CRISPR-Cas finds a binding site by locally opening target
DNA at a frequently found, short sequence (“PAM”) and
testing sequence match
Sequence mismatch more
tolerated if towards the 5’
end of the guide RNA,
away from the PAM
Consecutive mismatches
much more of a problem
then scattered single
mismatches
Binding (base pairing)
occurs from PAM in a 3’ to
5’ direction
3’
5’
Generally cuts much more effectively if guideRNA match is
perfect. Complex eventually just falls off if not.
However, can see low
levels of cutting at
mismatched (≤3)
sequences
3’
Most sites in human
genome have at least
one similar sequence
that ≤3 mismatches
5’
“Off-target effects”
Strategies for reducing off-target effects
Weaken Cas9 binding to force high
stringency guide RNA match
(Kleinstiver et al. 2016)
Deliberately weaken part of the
guide RNA match (two G’s at 5’ end
or shorter sequence to force
remainder of RNA to match
perfectly (Cho et al 2014)
Mutate the Cas9 to make it into a
nickase (ssDNA cleavage) and use
pairs of guide RNAs to make offset
nicks (1500X more accurate (Cong
et al 2013)
3’
5’
Types of
CRISPRs/Cas
systems
Cpf1 v Cas9
• Zetsche et al (2015) Cell 1673:759-771
• Cpf1-containing CRISPR systems have
three features that differ from Cas9.
– Cpf1-associated CRISPR arrays are
processed into mature crRNAs without the
requirement of an additional transactivating crRNA (tracrRNA)
– Cpf1-crRNA complexes efficiently cleave
target DNA proceeded by a short T-rich
protospacer-adjacent motif (PAM), in
contrast to the G-rich PAM following the
target DNA for Cas9 systems.
– Cpf1 introduces a staggered DNA doublestranded break with a 4 or 5-nt 5′ overhang,
instead of a blunt ended DSB
CRISPR-Cas heritable mutations
• To do this requires mutagenesis of gametes in plants (e.g., sperm
nuclei in pollen), or of zygote in animal systems)
• In some plants, mutagenesis of cell lineage that will produce
gametophyte is easy (e.g., Agrobacterium floral dip in
Arabidopsis), BUT…
• …difficulty getting expression of CRISPRs and Cas9 at right time
and place to maximize transmissable to next generation
– In Arabidopsis, CRISPR/Cas9 edits occur mostly in somatic cells, resulting in
a low fqy of homozygous mutations in T1 generation plants.
– Expression of Cas9 by promoters like SPOROCYTELESS or YAO target
expression level and timing. For SPOROCYTELESS , T1 mutant plants still
relatively rare but T2 mutants are significantly more frequent than for Cas9
expressed from ubiquitous promoters. For YAO promoter T1 edited plants
went from 4.3% to 90%
• In rice, T0 transgenic plants and segregation patterns are
Mendelian.
• In wheat, all three MLO homolog alleles have been knocked out by
CRISPR/Cas9, conferring broad-spectrum resistance to powdery
mildew.