1. Genome Editing
AGRY 600
Fall 2016

2. 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)

3. Damage/Disruption vs. Sequence Replacement

4. 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)

5. CRISPR-Cas Clustered Regularly InterSpaced Pallindromic Repeats
Discovered as what prokaryotes have as an immune system
Pallindromic Repeats of 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

6. 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

7. CRISPR-Cas

8. CRISPR-Cas

9. Streptococcus pyogenes has only one Cas protein (Cas9)

10. Streptococcus pyogenes has only one Cas protein (Cas9)

11. Modified CRISPR- Cas9 System

12. CRISPR-Cas9 finds DNA that base pairs with the guide RNA sequence and then cuts on both strands

13. Question then becomes how the cell will repair this DSB?

14. Question then becomes how the cell will repair this DSB?

15. Question then becomes how the cell will repair this DSB?

16. Question then becomes how the cell will repair this DSB?

17. 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

18. 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

19. 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)

20. 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:

21. 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

22. Homologous repair events are extremely rare in plants
Typcally require selection or other tricks to find them
Zhao et al (2016) Scientific Reports
doi: /srep23890

23. 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’

24. Generally cuts much more effectively if guideRNA match is perfect
Generally cuts much more effectively if guideRNA match is perfect. Complex eventually just falls off if not. 3’
However, can see low levels of cutting at mismatched (≤3) sequences
Most sites in human genome have at least one similar sequence that ≤3 mismatches
“Off-target effects” 5’

25. 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’

26. Types of CRISPRs/Cas systems

27. 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 trans- activating 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 double- stranded break with a 4 or 5-nt 5′ overhang, instead of a blunt ended DSB

28. 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.