This document summarizes a presentation on DNA-free genome editing with CRISPR enzymes. It discusses the evolution of genome editing techniques from random mutagenesis to targeted methods using ZFN, TALENs, and CRISPR/Cas9. The presentation focuses on a DNA-free approach using purified Cas9 protein and sgRNA to assemble CRISPR ribonucleoproteins (RNPs) for direct delivery into plant cells. It shows this method can induce targeted mutations in Arabidopsis, rice, and lettuce without leaving foreign DNA. RNPs generated mutations with high efficiency and specificity, and mutations were heritable after plant regeneration. Compared to other methods, DNA-free RNP delivery avoids issues like trans

1. 1
DNA-free genome editing
with CRISPR enzymes
Sunghwa Choe
Seoul National University
Thursday, 27 June, 2018
14:20 - 14:40
OECD CONFERENCE ON GENOME EDITING: APPLICATIONS IN AGRICULTURE – IMPLICATIONS FOR
HEALTH, ENVIRONMENT AND REGULATION

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Gene discovery by T-DNA random mutagenesis

3. 3
Functional genomics with random mutagenesis
T-DNA tagging methods

4. 4
Evolution of site-specific genome editing
ZFN, TALEN, Cas9
ZFN (Zinc Finger Nuclease)
TALEN (Transcription Activator-Like Effector
Nuclease
CRISPR/Cas9 nuclease (Clustered Regularly
Interspaced Short Palindromic Repeats/Cas9
nuclease)
Fok I
Fok I
Fok I
Fok I
Yu et al., 2016. DOI: 10.5772/6433

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Conventional way of CRISPR/Cas9 genome editing

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Why DNA-free genome editing?
From deregulation to non-regulation

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CRISPR/Cas9 ribonucleoprotein rather than DNA
in vitro assembly of functional CRISPR/Cas9
Cas9 effector protein single guide RNA

8. 8
Procedures to prepare CRISPR enzymes
Purification of the CRISPR/Cas9 and Cas9 Plus
[HiTrap Ni-chelating profile]
CRISPR/Cas9
CRISPR/Cas9 PLUS

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Procedures to prepare CRISPR enzymes
sgRNA preparation and holoenzyme

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Scheme of DNA-free genome editing

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What genes to edit?
Arabidopsis brassinosteroid mutants

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556
187 244 165
Does RNP of Cas9-sgRNA enter and edit protoplasts?
(-) 20 60 (-) 20 60
Day 1 Day 3
(ug/200ul)
T7E1 (+)
T7E1 (-)556
312
One day treatment of 200K cells in 200 ml volume with 20 mg RNP with PEG induced indels
(-) 12 30 12 30 no PEG (mg)
Leaf age : 2 weeks 4 weeks
Heteroduplex-specific cutting by T7E1
BRI1 (brassinosteroid insensitive 1)

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Did the double target method work to delete the intervening DNA?
TGGGTTTGGAGATGTTTACAAAGCGATTTTGAAAGATGGAAGCGCGGTGGCTAT........... 190 bp ..........GCTGGGGTGAAACTAAACTGGTCCACACGGCGGAAGATTGCGATAGGATCAGCTAG (wt)
TGGGTTTGGAGATGTTTACAAAGCGATTTTGAAAGATGGAAGCGCGGTGGCTAT........... 190 bp ..........GCTGGGGTGAAACTAAA-------ACACGGCGGAAGATTGCGATAGGATCAGCTAG (-7)
TGGGTTTGGAGATGTTTACAAAGCGATTTTGAAAGATGGAAGCG---------------------------------------------------------------ACACGGCGGAAGATTGCGATAGGATCAGCTAG (-224)
TGGGTTTGGAGATGTTTACAAAGCGATTTTGAAAGATGGAAGCG--------------------------------------------------------------CACACGGCGGAAGATTGCGATAGGATCAGCTAG (-223)
TGGGTTTGGAGATGTTTACAAAGCGATTTTGAAAGATGGAAGCGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCACACGGCGGAAGATTGCGATAGGATCAGCTAG (-223+62)
1. DNA spanning the 2 target sites was PCR amplified
2. Cloned into a TA-cloning vector
3. Clones were randomly selected and Sanger sequenced: 23/40 = 57.5 (%)

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Are other loci of the Arabidopsis genome accessible?
PhyB
- +
COI1
- +
Protoplast 1*10^5 cell
Cas9 protein 20 mg (final concentration : 50 ng/ml)
sgRNA 15 mg (final concentration : 38 ng/ml)
PEG incubation condition 20 %, 15 min
Total volume 400 ml
5’-ACGGTGCAGCATTTCTTTACCACGGGAAGTATTACCCGTT-GGGTGTTGCTCCTAGTGAAGTTCAGATAAAAGATGTTGTG WT
5’-ACGGTGCAGCATTTCTTTACCACGGGAAGTATTACCCGTT--GGTGTTGCTCCTAGTGAAGTTCAGATAAAAGATGTTGTG -1 bp (3/10)
5’-ACGGTGCAGCATTTCTTTACCACGGGAAGTATTACCCGTTaGGGTGTTGCTCCTAGTGAAGTTCAGATAAAAGATGTTGTG +1 bp (1/10)
5’-ACGGTGCAGCATTTCTTTACCACGGGAAGTATTACCCGTTtGGGTGTTGCTCCTAGTGAAGTTCAGATAAAAGATGTTGTG +1 bp (4/10)
5’-ACGGTGCAGCATTTCTTTACCACGGGAAGTATTACCCGT--GGGTGTTGCTCCTAGTGAAGTTCAGATAAAAGATGTTGTG -1 bp (2/10)
PhyB
BRI1-TS1
5’-GTGGGTTTGGAGATGTTTACAAAGCGATTTTGAAAGATGGAAGCG-CGGTGGCTATCAAGAAACTGATTCATGTTAGCGGT WT
5’-GTGGGTTTGGAGATGTTTACAAAGCGATTTTGAAAGATGGAAGCGgCGGTGGCTATCAAGAAACTGATTCATGTTAGCGGT +1 bp (1/4)
5’-GTGGGTTTGGAGATGTTTACAAAGCGATTTTGAAAGATGGAAG---CGGTGGCTATCAAGAAACTGATTCATGTTAGCGGT -2 bp (2/4)
5’-GTGGGTTTGGAGATGTTTACAAAGCGATTTTGAAAGATGGAAGCGtCGGTGGCTATCAAGAAACTGATTCATGTTAGCGGT +1 bp (1/4)
BRI1
- + + RNP

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Off-target effects are negligible in Arabidopsis

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Do the assembled RNPs work for rice protoplasts?
DWD1
3-1 3-2 3-3
CYP724
3-1 3-2 3-3
5’-TGGTTGATCCCGTCTGCATCGTCCAAGCGCACAGTGGCCCGGCCTACGACGTCAGGTTCT----ACCCGGATTCGCAGCAGC WT
5’-TGGTTGATCCCGTCTGCATCGTCCAAGCGCACAGTGGCCCGGCCTACGACGTCAGGTTCT-//-ACCCGGATTCGCAGCAGC +33 bp(4/5)
5’-TGGTTGATCCCGTCTGCATCGTCCAAGCGC-----------------------------T----ACCCGGATTCGCAGCAGC -29 bp(1/5)
DWD1
CYP724
5’-CAGGAACCTTGCTCTAGCACTGGTCACCTCCACAAAGCTCAAGCCCAGCTACCTTGGCGACATTGAGAAGATTGCACTGCATATAGTTGGGTCATGGCATGGCAAGAGCAAGG WT
5’-CAGGAACCTTGCTCTAGCACTGGTCACCTCCACAAAGCTCAAGCCCAGCTACCTTGGCGACATTGAGAAGATTGCACTGCATATAGTTGGGTCAT-GCATGGCAAGAGCAAGG -1bp (2/2)
Protoplast 3*10^4 cell
Cas9 protein 20 mg (final concentration : 50 ng/ml)
sgRNA 15 mg (final concentration : 38 ng/ml)
PEG incubation condition 20 %, 15 min
Total volume 400 ml

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Does the Cas9-sgRNA complex work in lettuce?
Arabidopsis BIN2
(brassinosteroid insensitive2):
gain-of-function mutants
WT het homo
T7E1
Ctrl 1 2 3 4 5 * (culture)
sgRNA (ug) 0 5 10 20 40 80
CAS9 (ug) 0 2.5 5 10 20 40
Indel (%) 10 14 8 11
LsBIN2
5’-AATTTTTCGGGTTTTAAAGCATCACAGTGATGCTCGTCAAAGGATGCCTCTCAT
||||||||||||||||||||||||||||||||||||||||||||||||||||||
3’-TTAAAAAGCCCAAAATTTCGTAGTGTCACTACGAGCAGTTTCCTACGGAGAGTA
LsBIN2-RG4

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Ribonucleoproteins (RNPs) – mediated genome editing followed by lettuce plant
regeneration
1 2 3
4 5 6

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Regeneration of plantlets after GE in lettuce
Growth and Division of lettuce Protoplasts
(LsBIN2 (RG4) editing line) (Deep sequencing analysis : NGS)

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Regeneration of whole plants from edited protoplasts

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Can we tell edited clones from non-edited ones?
 Yes by RNA guided endonuclease (RGEN)
Kim JM, Kim D, Kim S, Kim JS (2014) Genotyping with CRISPR-Cas-derived RNA-guided endonucleases. Nature Commu 5: 3157
Arrows: cut
Red: deletion
Yellow: insertion
PCR
Digestion
Run

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What are the nature of editing in the regenerating
plantlets?
WT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
ATCACAGTGATGCTCGT-CAAAGG WT
#08 ATCACAGTGATGCTCGTTCAAAGG +1
#09 ATCACAGTGATGCTCGTTCAAAGG +1
#10-A1 ATCACAGTGATGCT----CAAAGG -3
-A2 ATCACAGTGATGCTCGTTCAAAGG +1
#11-A1 ATCACAGT----------CAAAGG -9
-A2 ATCACAGTGATGCTCGTTCAAAGG +1
ATCACAGTGATGCTCGT-CAAAGG WT
#12 ATCACAGTGATGCTCGTTCAAAGG +1
#14-A1 ATCACAGT----------CAAAGG -9
-A2 ATCACAGTGATGCTCGTTCAAAGG +1
#18-A1 ATCACAGTGATGCTCGTTCAAAGG +1
#20-A1 ATCACAGTGATGCTCGT---AAGG -2
-A2 ATCACAGTGATGCTCGTTCAAAGG +1
#21-A1 ATCACAGTGATGCTCGTTCAAAGG +1
ATCACAGTGATGCTCGT-CAAAGG WT
#24-A1 ATCACAGTGATGCTCGTTCAAAGG +1
-A2 ATCACAGTGATGCTCG--CAAAGG -1
#25-A1 ATCACAGTGATGCTCGTTCAAAGG +1
-A2 ATCACAGTGATGCTCGT-CAAAGG WT
#29-A1 ATCACAGTG-------T-CAAAGG -7
-A2 ATCACAGTGATGCTCGT-CAAAGG WT
#30-A1 ATCACAGTGATGCTCGTTCAAAGG +1
#31-A1 ATCACAGTGATGCTCGTTCAAAGG +1
#33-A1 ATCACAGTGATGCTCGTTCAAAGG +1
#34-A1 ATCACAGTGATGCTCGTTCAAAGG +1
#28 (WT) #24 (bi-allelic) #30 (bi-allelic , homo)
-RGEN
+RGEN

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Does the biallelic heterozygous line exhibit any
phenotype?
ATCACAGTGATGCTCGT-CAAAGG WT
#24-A1 ATCACAGTGATGCTCGTTCAAAGG +1
-A2 ATCACAGTGATGCTCG--CAAAGG -1
#26 (WT) ATCACAGTGATGCTCGT-CAAAGG

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Germline transmission of the edited mutations
Bar = 10 cm

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Number of potential off-target sites in the lettuce
genome (Cas-OFFinder www.regenome.net)

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Negligible Indel frequencies at the on-target and 91
potential off-target sites
:
:
:

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8. phyB sgRNA (ggX20) 60 mg + cas9 protein 30 mg
9. phyB sgRNA (gtX20) 60 mg + cas9 protein 30 mg
10. phyB sgRNA (gX19) 60 mg + cas9 protein 30 mg
Indels (%)
NGS 0.020 0.046 0.021 1.7 4.6 4.5 4.2 27 8.7 48
1 2 3 4 5 6 7 8 9 10
12 hours
**
Indels (%)
NGS 0.0066 0.042 0.016 4.0 9.6 9.6 9.5 36 14 49
1 2 3 4 5 6 7 8 9 10
24 hours
**
Plasmid DNA method RNP method
1. Control (PEG treatment only)
2. 35Sp-cas9 plasmid only 12 mg
3. U626p-phyB sgRNA plasmid only 12 mg
4. 35Sp-cas9 plasmid 3 mg + U626p-phyB sgRNA plasmid 3 mg
5. 35Sp-cas9 plasmid 6 mg + U626p-phyB sgRNA plasmid 6 mg
6. 35Sp-cas9 plasmid 12 mg + U626p-phyB sgRNA plamisd 12 mg
7. 35Sp-cas9 plasmid 24 mg + U626p-phyB sgRNA plamisd 24 mg
2*105 cell
RNP method
Comparison of RNP vs DNA methods: time course efficiency

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Cas9 plasmid 3 mg + sgRNA plasmid 3 mg
CACTAGGAGCAACACCC-----------------------------------AACGGG WT
CACTAGGAGCAACACCTGATGATCAGGTCCTTCTTCACCTCCTTGTAGCCCTAACGGG + 35bp (2/90518)
DNA vs RNP method: Unwanted DNA integration issue

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DNA-free genome editing procedure

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Comparison of different genome editing methods

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Comparison of different genome editing methods
Comparison
Way of CRISPR/Cas delivery
Transgenic Transient
T-DNA DNA mRNA RNP
Mutation efficiency ++ ++ + +++
Specificity + + + ++
Duration 10-16 weeks 6-8 weeks 6-8 weeks 6-8 weeks
Foreign DNA integration Yes Yes/No No Never
Antibiotic selection Yes No No No

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Pros and cons of the DNA-free RNP method in plants
• Pros
• Cas9 protein expression and sgRNA processing in vivo not needed
• No foreign DNA remained in the genome
• Homozygous mutant for multiple genes at single generation
• Cons
• Possibility of accompanying somaclonal variation during tissue culture
• Difficulty regeneration of whole plants from some crop protoplasts like maize and soybean
• Safe and efficient delivery of RNPs

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Working together with
• Prof. Jin-Soo Kim
• Jungeun Kim
• Seung Woo Cho, Ph.D.
• Sang-Gyu Kim
• Je Wook Woo Seoul National University
• Soon Il Kwon , Ph.D.
• Claudia Corvalán
• Jongjin Park G+FLAS LIFE SCIENCES
• Sunmee Choi