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CRISPR/Cas9 Nuclease Product Guide

Innovative Science.Consistent Biologics.Experienced Partners.Aldevron makes high quality CRISPR-associated nucleases to support your work: SpyFi™ Cas9 Nuclease

SpCas9 Nuclease

AsCpf1 (Cas12a) Nuclease

SaCas9 Nuclease

Custom Nucleases

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Aldevron makes the highest quality biologics. Aldevron specializes in CRISPR-associated nuclease products, as well as custom protein development and manufacturing services supporting research, clinical, and commercial use. Our inventory of clinical-grade nucleases provides a fast, cost-effective sourcing option for your cell or gene therapy projects.

Research Grade, GMP-Source® and cGMP Products. Aldevron’s GMP-Source® (GMP-S) CRISPR-associated nucleases retain the key hallmarks of cGMP manufacturing while reducing costs and shortening development timelines. You can then transition seamlessly to a cGMP product as you progress toward commercialization. As your manufacturing partner, Aldevron can help you save money and time without putting your plans at risk.

Available Pack Sizes Research Grade GMP-Source® cGMP

SpyFi™ Cas9 Nuclease 0.25 mg 5 mg 1 mg 10 mg 1 mg 10 mg

SpCas9 Nuclease 0.25 mg 5 mg 1 mg 10 mg 1 mg 10 mg

AsCpf1 Nuclease (Cas12a) 0.25 mg 5 mg Custom Request Custom Request

SaCas9 Nuclease 0.25 mg 5 mg Custom Request Custom Request

Available online at aldevron.com/CRISPR

SpCas9SpyFiTM

Cas9

AsCpf1

CustomNuclease

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Assay Specification

Purity by RP-HPLC ≥ 95%

Purity by SDS-PAGE ≥ 95%

Aggregation by aSEC ≥ 95% monomer

Concentration by A280 10.0 mg/mL

Activity (in vitro) > 80% cleavage of a DNA reference

Appearance Clear, colorless

Residual DNase < LOQ

Residual RNase < LOQ

Residual Host Cell Protein ≤ 200 ng/mL

Residual Host Cell DNA < 1% (w/w) (LOQ)

Endotoxin ≤ 10 EU/mg

Bioburden < 2 CFU per sample

Mycoplasma Negative

Aldevron CRISPR-associated nucleases are being used in multiple clinical trials.While many vendors offer research-grade CRISPR-associated nucleases, these are not applicable to clinical programs due to construct design and quality grade. Aldevron now offers nucleases designed for development through commercial applications.

Custom manufacturing of CRISPR-associated nucleases. Aldevron offers custom manufacturing of CRISPR-associated nucleases, including dCas9 (Dead Cas9), fusions and nickases. Manufacturing scale ranges from 10 milligram to multigram lots. Custom nucleases are available at Research Grade, GMP-Source® and cGMP quality grades.

Example Quality Specifications

Gene Editing Nucleases Available from Aldevron

SpyFiTM Cas9 Nuclease

PRODUCT NAME CONSTRUCT DESIGN

sNLS-SpCas9-sNLS

sNLS-AsCpf1-sNLS (Cas12a)

sv40 NLS sv40 NLSSpyFiTM Cas9

sv40 NLS sv40 NLSwt SpCas9

sv40 NLS sv40 NLSAsCpf1

sNLS-SaCas9-SNLS sv40 NLS sv40 NLSSaCas9

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Getting you to the clinic. Faster.Research Grade (RG) vs cGMP Cas9 performance in client’s cell editing system. The data has been normalized to the performance of the RG material, so the blue bars are provided for reference and ease of understanding. No significant difference in either editing efficiency or cell count was observed when testing RG vs cGMP sample vs cGMP product. One of the two assays was performed under standard conditions and the other was targeting an EC50.

FIGURE 1. Client tested RG material then compared to cGMP sample (0.25 mg) to confirm performance in two key assays: gene editing efficiency and cell viability prior to placing an order for 10 mg cGMP vial and then 3 x 50 mg cGMP vials. Once order was placed, client received cGMP product the same month.

1.5

1.0

0.5

0.0Per

form

ance

Rel

ativ

e to

RG

Performance Metric

RG

cGMP

Efficien

cy

Cell C

ount

Research Grade Cas9 vs cGMP

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SpCas9 Activity is Measured Using an in vitro Cleavage Assay

One unit of SpCas9 will cut 50% of a lin-pDNA in 60 minutes at 37°C with: - 6 nM lin-pDNA - 60 nM crRNA - 60 nM tracrRNA

Uncut DNA

Cleavage products

SpCas9 in vivo Activity; T-Cell Editing

human TCR alpha gene

Q1, top: Unmodified (TCR +)

Q3, bottom: Modified (TCR null)

control

78%

competitor SpCas9Aldevron

sNLS-SpCas9-sNLS

40%<1%

SpCas9sv40 NLS sv40 NLS

Data provided by Dr. Mark J. Osborn, University of Minnesota

Aldevron Cas9 Nuclease Technical Data

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FIGURE 2. High-quality, reproducible manufacturing. SpCas9 batches were expressed and purified over the course of several months at different scales. Quality test results show that Aldevron’s manufacturing process consistently yields high-quality SpCas9.

FIGURE 3. Aldevron SpyFiTM Cas9 has reduced off target effects. SpyFi Cas9 has measurably lower frequency of off-target editing compared to wt SpCas9. Aldevron’s SpyFi Cas9 Nuclease is sold under license of patents and/or patents pending from Integrated DNA Technologies, Inc. (IDT). Data shown above was generated in collaboration with IDT.

38509 38087 38285 386360

20

40

60

80

100

% T

7EI C

leav

age

SpCas9 Lot -01 SpCas9 Lot -02 SpCas9 Lot -03

1 unit = 0.09 ± 0.02 pmol

0 2 4 6 8 100

20000

40000

60000

80000

100000

Time (min)

Inte

nsity

(mV)

Analytical SEC

15 16 17 18 19 200

200000

400000

600000

800000

1000000

Time (min)In

tens

ity (m

V)

RP-HPLC

M20752-03

M20653-05

MRD022-02

> 95% purity > 95% monomer (162 kDa)

-3 -2 -1 00

20

40

60

80

100

log10 pmol SpCas9

% C

uttin

g

Activity

On-Target Off-Target Site 1 Off-Target Site 2 Off-Target Sites 3-90

20

40

60

80

100

% IN

DELS

by

NGS

(48

h)

4.00.250.125

4.00.250.125

4.00.250.125

< 1% INDELS

M

Competitor SpCas9M

wt SpCas9

M SpyFi Cas9

High-Quality, Reproducible Manufacturing

SpyFi™ Cas9 Nuclease Reduces Off-Target Effects

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FIGURE 4. Aldevron SpCas9 is stable for 24 months in a DTT-free formulations. SpCas9 was formulated at 10.0 mg/mL in 25 mM Tris, 0.3M NaCl, 0.1 mM EDTA, 50% glycerol, pH 7.4. Quality tests show no loss of protein activity or purity when the nuclease is stored at -20˚C.

SpCas9 is Stable for 24 Months When Stored at -20˚C

Forced Degradation Demonstrates Utility of Quality Control Tests

Time (hours)

0 50 100 150 2000

25

50

75

100

% C

uttin

g

in vitro Activity

Time (hours)

0 50 100 150 2000

25

50

75

100

% C

uttin

g

in vitro Activity

0 50 100 150 2000

5

10

15

Time (hours)

mg/

mL

Concentration

0 50 100 150 2000

5

10

15

Time (hours)

mg/

mL

Concentration

RP-HPLC of tryptic digest

Retention Time (min)

Inte

nsity

(mV)

0 10 20 30 40 50 600

3×105

6×105

9×105

0 h1.5 h

5.5 h

Retention Time (min)

Inte

nsity

(mV)

aSEC of H2O2-treated SpCas9

0 5 10 15 20 250

3×104

6×104

9×104

24 h48 h

120 h168 h

0 h

Plasmid │ RNA │ Protein │ Antibodies 12

RG wt SpCas9 Stability

• Three year study in progress (3, 6, 9, 12, 15, 18, 24 and 36 months)• -20 ˚C (recommended storage), 4 ˚C and 22 ˚C

• Note: Testing concluded for 4 ˚C and 22 ˚C at 18 month timepoint

Research Grade

Nucleases

0 3 6 9 12 15 18 21 240

25

50

75

100

Time (month)

% P

urity

(RP-

HPL

C)

+22°C+4°C-20°C

0 3 6 9 12 15 18 21 240

25

50

75

100

Time (month)

% C

uttin

g

+22°C+4°C-20°C

Plasmid │ RNA │ Protein │ Antibodies 12

RG wt SpCas9 Stability

• Three year study in progress (3, 6, 9, 12, 15, 18, 24 and 36 months)• -20 ˚C (recommended storage), 4 ˚C and 22 ˚C

• Note: Testing concluded for 4 ˚C and 22 ˚C at 18 month timepoint

Research Grade

Nucleases

0 3 6 9 12 15 18 21 240

25

50

75

100

Time (month)

% P

urity

(RP-

HPL

C)

+22°C+4°C-20°C

0 3 6 9 12 15 18 21 240

25

50

75

100

Time (month)

% C

uttin

g

+22°C+4°C-20°C

aSEC of H2O2-treated SpCas9

in vitro Activity

RP-HPLC of tryptic digest

Concentration

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For the last 21 years, Aldevron has been a proud partner of research laboratories through the production of:• Plasmid DNA at any scale, with fast

turnaround times• Proteins in bacterial, insect and

mammalian expression systems• mRNA to your specifications using a

robust and scalable process• Novel antibodies using our genetic

immunization technologyAldevron supports research-use production through commercialization.

Contact us about Aldevron Nucleases 4055 41st Avenue South, Fargo, ND 58104 USA+1 (701) 297-9256 Toll-free (U.S. and Canada): +1 (877) 787-3362Additional facilities in Madison, WI and Freiburg, GermanyVisit: aldevron.com/contact

20-Cas9-1