Ribonucleoprotein delivery of CRISPR-Cas9 reagents for increased gene editing efficiency

Rolf Turk PhD, Staff ScientistIntegrated DNA Technologies

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Outline—Alt-R™ CRISPR-Cas9 System

• Ribonucleoprotein complex (RNP)– Generation of RNP complex– Stability of RNP complex– Editing efficiency of RNP

• Cas9 electroporation enhancer– Increases editing efficiency

• RNP delivery using the Amaxa® Nucleofector® System (Lonza)– Optimization

• RNP delivery using the Neon® System (Thermo Fisher)– Optimization

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CRISPR genome editingDNA incorporation or gene knockout• Homology-directed repair—add or

replace gene sequences• Non-homologous end joining—

destroy a gene

CRISPR guide RNAs

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Cas9 proteinRNA-directed dsDNA

endonuclease

Implementing CRISPR-Cas9 gene editing

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CRISPR-Cas9 products from IDT

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3-step transfection using Alt-R™ CRISPR-Cas9 System

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+

+

gRNA complex formation

RNP complex formation

RNP delivery

Step 1

Step 2

Step 3

15 minutes

10–20 minutes

30–60 minutes

Cas9 protein is rapidly degraded—fewer off-target effectsWestern blot Off-target indel production

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Liang X, Potter J, et al. (2015) Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol, 208:44–53.

Benefits of RNP: Cas9 protein rapidly degraded, fewer OTEs

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05

101520253035404550

0.1 µM 0.3 µM 1 µM 3 µM

T7EI

cle

avag

e (%

)

HEK-293–Cas9 cellsNucleofection, EMX1 targeting gRNA, 48h

On target Off target

• Sustained expression of Cas9 allows for OTEs. Even low-expression HEK-293–Cas9 cells allow for off target editing.

• RNP is “fast on and fast off,” and has fewer OTEs

05

101520253035404550

0.1 µM 0.3 µM 1.0 µM 5.0 µM 10 µM

T7EI

cle

avag

e (%

)

RNP, HEK-293 cellsNucleofection, EMX1 targeting gRNA, 48h

On target Off target

Ribonucleoprotein ease of use—highly stable Cas9 system

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0102030405060708090100

FreshComplex

–80°C –20°C 4°C FreshComplex

–80°C –20°C 4°C

T7EIcleavage(%

)

5monthRNPcomplexstability—RNPstoredat[1µM]HEK293cells,10nM RNP(1.2µL),RNAiMAX(1.2µL), 48hr

Cas9Buffer OptiMEM PBS

• Loss in activity seen at –20°C (likely due to freeze–thaws)• Premade complexes are stable at 4°C and –80°C with no loss in activity

HPRT site 1 HPRT site 2

CRISPR workflow

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AssembleRNP

DesigngRNAs

LipofectionEasier to transfect cells

ElectroporationHarder to transfect cells,primary cells

MicroinjectionMouse model generationand other research organisms

Collect genomic

DNA

Analyze

Delivery of RNP by electroporation

Amaxa® Nucleofector® (Lonza)• 1-, 16-, or 96-well format• Online database

– Cell lines– Primary cells

• Expensive• Electroporation parameters

unknown à hard to optimize

Neon® Transfection System (Thermo Fisher)• Single cuvette• Online database

– Cell lines• Known parameters• Easy optimization• Expensive

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Considerations when doing electroporation

• Requires higher ribonucleoprotein concentrations [2–4 µM] compared to lipofection

• Cas9 ribonucleoprotein complex is not encapsulated– More accessible to nucleases– More accessible to proteinases

• Electroporation parameters differ per cell line– Amaxa Nucleofector

• Cell database• Optimization protocol (96-well format)

– Neon Transfection System• Optimization protocols

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Optimization strategy for electroporation

• Find optimal electroporation parameters– Nucleofector System: Electroporation program– Neon Transfection System: Voltage, pulse width, number of pulses

• Pay close attention to cell viability– Use cells with low passage numbers– Use appropriate cell density (should be sub-confluent)

• Follow recommendations from IDT user guides and protocols• If necessary, test a range of RNP concentrations• Include positive and negative controls• Use Cas9 electroporation enhancer to boost editing efficiency

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Electroporation—verify reagents free of RNases

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EMEM EMEM+FBS EMEM EMEM+FBS EMEM EMEM+FBS EMEM EMEM+FBS

T7EIcleavage(%

)Electroporation—optimaldissociationmethodAlt-R™CRISPRcrRNA[4µM]—HEK293Cas9Cells

No PBS wash PBS wash No PBS wash PBS wash

Cell dissociation method

Cell resuspension media

• Testing shows that commercially purchased trypsin contains RNases • RNaseAlert® Assay (IDT)

Trypsin Non-enzymatic celldissociation solution

Electroporation enhancer DNA improves gene editing efficiency• Use of electroporation enhancer DNA improves outcome in RNP

electroporation• Effect varies with cell type and electroporation protocol

(Nucleofector > Neon)

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Cas9 electroporation enhancer

• IDT custom Ultramer® Oligonucleotide; available atwww.idtdna.com/ultramer

• Sequence:TTAGCTCTGTTTACGTCCCAGCGGGCATGAGAGTAACAAGAGGGTGTGGTAATATTACGGTACCGAGCACTATCGATACAATATGTGTCATACGGACACG (100 nt)

• Note: This oligo does not have significant homology to the human, mouse, or rat genomes, and has been tested as carrier DNA in multiple human cell lines, including HEK-293, Jurkat, and K562.

• Before use in other species, verify that this oligo does not have similarity to your host cell genome to limit participation of the oligo in the double-stranded DNA break repair process.

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RNP Nucleofection—effect of electroporation enhancer

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0 µM 1 µM 4 µM 10 µM 1 µM 4 µM 10 µM 1 µM 4 µM 10 µM 1 µM 4 µM 10 µM

No Carrier

20 nt 95 nt 200 nt tRNA

T7EIto

taleditin

gefficiency(%

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3.3 µM RNP30 µL volume= 100 pmol (16 µg)

Cas9 RNP, HEK-293

Positive and negative controls

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• Amaxa Nucleofection• Nucleofection Solution SF• Protocol 96-DS-150• 2 x 105 cells/Nucleofection• HPRT locus: 38285• crRNA:tracrRNA:Cas9 =

1.2:1.2:1• RNP concentrations,

0.125–4 µM• Electroporation enhancer DNA,

4 µM• 48 hr incubation, following

Nucleofection• 3X genomic DNA dilution• Digest with T7EI, 2 U• Fragment Analyzer™

(Advanced Analytical Technologies)

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0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

T7EIto

taleditin

gefficiency(%

)

Ribonucleoproteincomplexconcentration(µM)

Nucleofection—RNPdilutionseries—HEK-293cellsTheeffectofcarrierDNAandsequenceoneditingefficiency

4µMelectroporationenhancer Noelectroporationenhancer

Conditions:

• Amaxa Nucleofection• Nucleofection Solution SE• Protocol 96-CL-120• 5 x 105 cells/Nucleofection• HPRT locus: 38285• crRNA:tracrRNA:Cas9 =

1.2:1.2:1• RNP concentrations,

0.125–4 µM• Electroporation enhancer DNA,

4 µM • 48 hr incubation, following

Nucleofection• 3X genomic DNA dilution• Digest with T7EI, 2 U• Fragment Analyzer™

(Advanced Analytical Technologies)

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0102030405060708090100

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

T7EIto

taleditin

gefficiency(%

)

Ribonucleoproteincomplexconcentration(µM)

Nucleofection—RNPdilutionseries—JurkatcellsTheeffectofcarrierDNAandsequenceoneditingefficiency

4µMelectroporationenhancer Noelectroporationenhancer

Conditions:

• Amaxa Nucleofection• Nucleofection Solution SF• 96-FF-120 protocol• 5 x 105 cells/Nucleofection• HPRT locus – 38285• crRNA:tracrRNA:Cas9 =

1.2:1.2:1• RNP concentrations,

0.125–4 µM• Electroporation enhancer DNA,

4 µM • 48 hr incubation, following

Nucleofection• 3X genomic DNA dilution• Digest with T7EI, 2 U• Fragment Analyzer™

(Advanced Analytical Technologies)

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Conditions:

0102030405060708090100

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

T7EIto

taleditin

gefficiency(%

)

Ribonucleoproteincomplexconcentration(µM)

Nucleofection—RNPdilutionseries—K562cellsTheeffectofcarrierDNAandsequenceoneditingefficiency

4µMelectroporationenhancer Noelectroporationenhancer

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Workflow

• HEK-293 cells: 1 x 105 cells per electroporation• HPRT 38285• RNP—crRNA:tracrRNA:Cas9 = 1.8:1.8:1.5 µM• 12 µL volume (10 µL electroporated)• Electroporation enhancer, 1.8 µM• 24 different protocols

– Voltage– Pulse width– Pulse length

• gDNA isolation 48 hr after electroporation• T7EI assay

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HEK-293 electroporation optimization—Neon®

System

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Test -Enhancer +Enhancer -Enhancer +Enhancer -Enhancer +Enhancer Voltage(V) Width(ms) NumberProt_01 2 1 0 1 1Prot_02 31.2 52.0 0.2 3.0 3 3 1400 20 1Prot_03 39.0 63.2 0.7 8.1 4 2 1500 20 1Prot_04 44.8 59.9 0.5 3.7 4 2 1600 20 1Prot_05 44.5 59.9 1.3 3.2 2 2 1700 20 1Prot_06 23.3 42.4 0.8 1.6 3 3 1100 30 1Prot_07 33.8 49.3 1.4 1.2 3 3 1200 30 1Prot_08 41.5 58.8 1.0 1.2 3 3 1300 30 1Prot_09 44.6 61.1 0.8 1.6 3 3 1400 30 1Prot_10 22.3 39.9 1.5 2.3 3 4 1000 40 1Prot_11 34.9 53.9 0.7 2.4 3 3 1100 40 1Prot_12ü 46.2 62.4 0.8 4.8 4 4 1200 40 1Prot_13 30.2 48.9 0.7 2.1 3 4 1100 20 2Prot_14 42.6 51.4 0.9 1.8 4 3 1200 20 2Prot_15 50.5 66.2 0.5 2.1 4 2 1300 20 2Prot_16 61.1 67.4 1.0 2.7 2 1 1400 20 2Prot_17 12.3 24.9 0.5 2.2 3 3 850 30 2Prot_18 27.3 37.5 0.7 0.6 4 4 950 30 2Prot_19 41.4 46.4 1.0 1.8 4 4 1050 30 2Prot_20 53.0 60.4 0.5 1.8 4 3 1150 30 2Prot_21 36.2 52.6 1.3 1.8 4 4 1300 10 3Prot_22ü 47.4 57.2 0.8 1.2 4 4 1400 10 3Prot_23 51.5 59.7 1.0 1.0 3 3 1500 10 3Prot_24 60.9 61.6 1.0 1.4 2 2 1600 10 3

EditingEfficiency(%) SD(%) CellDensity Pulse

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lowcelldensity

highcelldensity

highediting

lowediting

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T7EIeditin

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nen

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T7EIediting(%):Absenceofelectroporationenhancer

Neon®electroporationoptimization—HEK-293Effectofelectroporationenhancer

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Workflow

• Jurkat cells: 2 x 105 cells per electroporation• HPRT 38285• RNP—crRNA:tracrRNA:Cas9 = 1.8:1.8:1.5 µM• 12 µL volume (10 µL electroporated)• Electroporation enhancer, 1.8 µM• 24 different protocols

– Voltage– Pulse width– Pulse length

• gDNA isolation 72 hr after electroporation• T7EI assay

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Jurkat electroporation optimization—Neon®

System

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Test -Enhancer +Enhancer -Enhancer +Enhancer -Enhancer +Enhancer Voltage(V) Width(ms) NumberProt_01 0.0 0.0 0.0 0.0 3 4 0 1 1Prot_02 53.9 64.4 2.7 1.8 2 3 1400 20 1Prot_03 59.0 71.0 0.5 4.5 2 3 1500 20 1Prot_04 0.0 78.2 0.0 1.3 3 1 1600 20 1Prot_05 60.1 64.7 3.1 13.2 2 2 1700 20 1Prot_06 22.0 49.1 0.6 16.3 1 4 1100 30 1Prot_07 41.9 62.9 3.2 6.8 3 4 1200 30 1Prot_08 57.7 63.7 0.9 15.2 2 4 1300 30 1Prot_09 60.2 74.4 2.4 3.1 3 2 1400 30 1Prot_10 14.2 29.8 1.4 0.4 3 2 1000 40 1Prot_11 34.6 53.6 0.9 0.1 4 1 1100 40 1Prot_12 27.0 73.5 1.7 3.8 2 2 1200 40 1Prot_13 22.7 54.5 1.3 4.6 2 2 1100 20 2Prot_14 51.6 69.4 1.9 0.8 2 2 1200 20 2Prot_15 59.9 74.2 3.0 0.7 2 2 1300 20 2Prot_16 68.0 62.0 2.6 11.2 3 4 1400 20 2Prot_17 0.0 12.7 0.0 0.3 4 4 850 30 2Prot_18 23.7 35.1 0.9 2.0 3 2 950 30 2Prot_19 9.7 56.7 0.6 3.1 3 3 1050 30 2Prot_20 53.9 73.7 3.6 7.2 3 2 1150 30 2Prot_21 41.9 59.2 1.4 1.4 3 4 1300 10 3Prot_22 12.2 66.2 1.1 0.2 3 4 1400 10 3Prot_23 61.1 71.1 1.7 2.2 3 2 1500 10 3Prot_24ü 37.0 75.7 2.8 0.4 2 4 1600 10 3

EditingEfficiency(%) SD(%) CellDensity Pulse

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lowcelldensity

highcelldensity

highediting

lowediting

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T7EIeditin

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T7EIediting(%):Absenceofelectroporationenhancer

Neon®electroporationoptimization—JurkatEffectofelectroporationenhancer

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Genome editing in mouse primary T-cells

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Collaboration with Jennifer Barr & Scott Lieberman, Dept of Pediatrics, University of Iowa

IDT collaborator protocols available on the web

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• Amaxa Nucleofection• Nucleofection Solution SF• Protocol 96-DS-150• 3.5 x 105 cells/Nucleofection• HPRT locus• crRNA:Cpf1 = 1.2:1• RNP concentration, 5 µM• Electroporation enhancer

DNA, 3 µM • 48 hr incubation, following

Nucleofection• 3X genomic DNA dilution• Digest with T7EI, 2 U• Fragment Analyzer™

(Advanced Analytical Technologies)

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0102030405060708090100

T7EIto

taleditin

gefficiency(%

)Nucleofection—Cpf1 RNP(5µM),HPRT12,HEK-293

Effectofelectroporationenhanceroneditingefficiency

0µM 3µM

Conditions:

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