CRISPR-EZ

He Lab Protocol

Modzelewski - 2016

CRISPR-EZ

Clustered Regularly Interspaced Short Palindromic repeats RNP Electroporation of Zygotes

CRISPR (clustered, regularly interspaced, short palindromic repeat)/Cas9 (CRISPR-associated 9) system has been developed and used recently to efficiently edit the genomes of various model organisms. The current standard method of editing mouse embryos is by co-injection in vitro transcribed CAS9 DNA or mRNA and a small guide (sgRNA) into mouse zygotes to target a genomic loci for cleavage (1). Microinjection is technically demanding, expensive and requires a skilled technician to perform. Even under ideal conditions, embryo viability is poor and transgenics facility are typically booked many weeks or months in advanced. To reduce these burdens, this protocol describes a method to introduce pre-assembled cas9 protein in complex with an in vitro transcribed sgRNA, forming a RiboNucleoProtein (RNP) Complex that quickly and efficiently edits zygotic genomes with 100% delivery during in vivo experiments. The use of a preloaded RNP complex has been shown to be extremely quick acting, high delivery and results in significantly higher degrees of Homology Directed Repair (HDR) that that DNA or mRNA based methods (2). Our recent experiences with this technique has resulted in high viability and efficiency in terms of insertion/deletion (indel), knockout and knock-in experiments. The protocol presented here is aimed at helping you design, introduce and assay sgRNA editing efficiency in mouse zygotes with in vitro assays prior to animal derivation.

REFERENCES

(1) Qin. W, et al. “Efficient CRISPR/Cas9-Mediated Genome Editing in Mice by Zygote Electroporation of Nuclease” Genetics. 2015 Jun 200(2): 423-430

(2) Lin. S, et al. “Enhanced homology-directed human genome engineering by control timing of CRISPR/Cas9 delivery” eLife 2014,3:e04766

Designing single guide RNA (sgRNA):

Candidate sgRNA designs can be selected from a number of available algorithms. For most experiments, we recommend selecting three to four candidate sgRNAs based on the predicted scores and proximity to desired target sites.

Successful guides have been generated from the following online resources:

-Sequence scan for CRISPR: http://crispr.dfci.harvard.edu/SSC/

-Gene Perturbation Platform: http://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design

-Chop-Chop: https://chopchop.rc.fas.harvard.edu/

-CRISPR Design: http://crispr.mit.edu/

1. Use online resource to generate sgRNA sequence.

2. Input target DNA sequence.

3. Chose sgRNA with highest ranking (Based on Online Resource Scoring method)

4. Not all resources provide off-target analysis, therefore be sure to also BLAT the sgRNA sequence when necessary.

NOTE: We generally find the most successful sgRNAs have coincident high scores across multiple algorithms. Therefore, we recommend inputting your sequence of interest into multiple online resources prior to generating In Vitro Transcribed sgRNAs.

Cloning Free In vitro T7 template synthesis and transcription:

Reagent

Source

Phusion High Fidelity DNA Polymerase

New England Biolabs, Cat#: M0530

HiScribe T7 High Yield RNA Synthesis Kit

New England Biolabs, Cat#: E2040

DNAse

New England Biolabs, Cat#: M0303

Oligos (Described Below)

Vendor must offer Ultramer Purification

T7FwdVar: (Variable Oligo designed by user)

5’-GGATCCTAATACGACTCACTATAG---20nt-guide-sequence---GTTTTAGAGCTAGAA

T7RevLong: (ULTRAMER PURIFICATION)

5’-AAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTT

AACTTGCTATTTCTAGCTCTAAAAC

Amplification Primers:

T7FwdAmp: 5’-GGATCCTAATACGACTCACTATAG

T7RevAmp: 5’-AAAAAAGCACCGACTCGG

PCR Amplification of IVT Template (per individual sgRNA):

Reagent

Volume (µl)

Final Concentration

H2O

35.5

-

5x Phusion HF Buffer

10

1X

10 mM dNTPs

1

0.2 mM

T7FwdVar (1 µM)

1

0.02 µM

T7RevLong (1 µM)

1

0.02 µM

T7FwdAmp (100 µM)

0.5

1 µM

T7RevAmp (100 µM)

0.5

1 µM

Phusion HF (2Units/µl)

0.5

0.02 Units

Total

50

-

ThermoCycler Conditions:

Temperature

Duration

Cycles

95˚

30s

1x

95˚

10s

30x

57˚

10s

72˚

10s

72˚

2m

1x

4˚

Hold

1x

NOTE: Once complete, it is recommended that 10uL of the 50uL reaction be run on a 1% Agarose gel to confirm size and equal reaction efficiency between samples. Below is an example of a successful template amplification reaction.

T7 Transcription (NEB E2040S):

Reagent

Volume

Final Concentration

10x Buffer

2 µl

1X

ATP (100 mM)

2 µl

10 mM

GTP (100 mM)

2 µl

10 mM

CTP (100 mM)

2 µl

10 mM

UTP (100 mM)

2 µl

10 mM

DNA template (85 ng/µl)

8 µl

25 ng/µl

T7 RNA polymerase mix

2 µl

-

Total

20 µl

-

· Mix by gentle pipetting.

· Incubate In Vitro Transcription Reaction mixture for 18hrs+ at 37˚ in a thermocycler.

· Add 1 µl of RNase-free DNase (NEB M0303S).

· Incubate for 20 minutes at room temperature.

· Proceed immediately to RNA Cleanup.

RNA Isolation and Purification of sgRNA:

Reagent

Source

SeraMag Speedbeads

GE Healthcare, Cat#: 65152105050250

Magnetic Stand

Invitrogen, Cat#: 12321D

· Bring volume of IVT reaction (above) to 150uL with 100% Ethanol.

· Add 100 uL 5X SPRI (we use homemade SeraPure beads for RNA binding).

· Pipette gently to mix (10 times)

· Incubate 5 minutes at room temperature.

· Place on magnetic stand and let sit for 5 minutes.

· Discard supernatant

· Add 200 uL, 80% EtOH. Wait 2 min. Remove EtOH. (Do not disturb pellet)

· Repeat Wash Step.

· Air dry 5-10 min. Wait until pellet goes from Glossy to Matte.

· Elute RNA with 20 uL of water or TE.

· Pipette to mix 10 times, making sure to break up pellet.

· Incubate 2 minutes at room temperature.

· Place on magnetic stand for 5 min.

· Transfer supernatant to a RNASE free tube.

Note: Run on 1% gel to ensure presence of product. Be sure to heat to 95oC for 5min, then snap cool on ice prior to loading to disrupt secondary structure. Below is a representative image of a successful IVT reaction. Lane 3 shows incomplete heating and lane 5 shows inefficient IVT product.

*Note: A “homemade” SPRI Bead mixture can be used as an even more cost effective IVT reaction cleanup alternative. (MagNa Beads: Refer to Rohland et al. 2012. Genome Res. 22, 939–46). Other IVT reaction cleanup kits, such as Ambion’s Megaclear (AM1908) are also effective.

Embryo Electroporation:

Required Material

Source

Stock

Working

Male Mice (3-8mo)

C57BL/6J JAX 000664

Female Mice (3-4wk)

C57BL/6J JAX 000664

1. Pregnant Mare Serum Gonadotropin (PMSG)

2. Millipore: Cat# 367222

Lyophilized

1mg = 1000IU

100 IU

Human Chorion Gonadotropin (HCG)

Millipore: Cat# 230734

Lyophilized

1mg = 3000IU

100 IU

Phosphate Buffered Saline (PBS)

Gibco: Cat# 14190-144

1X

1X

2. Prepare RNP Buffer:

Reagent

Source

1X

5X

1M HEPES ph7.5

Sigma, Cat# H3375

0.2mL [20mM]

1.0mL [100mM]

3M KCl

Sigma, Cat# P9333

0.5mL [150mM]

2.5mL [750mM]

1M MgCl2

Sigma, Cat# M8266

0.01mL [1mM]

0.05mL [5mM]

100% Glycerol

Fisher, Cat# BP229

1.0 mL [10%]

5.0mL [50%]

100mM TCEP*

Sigma, Cat# C4706

0.1 mL [1mM]

0.5mL [5mM]

H20 Nuclease Free

Ambion, Cat# AM9937

To 10mL

To 10mL

3. Prepare Culture Reagents:

Reagent

Source

Storage

M2

Zenith Biotech -ZFM2-050

4oC

BSA

Sigma A3311

4oC

1. 0.2uM filter

2. Millipore SLGV033RB

3. RT

Hyaluronidase/M2

Millipore MR-051-F

-20oC

15-inch aspirator tube

Sigma A5177

RT

capillary tubes

Sigma P0674

RT

KSOM+AA

Zenith Biotech ZEKS-050

4oC

mineral oil

Millipore ES-005C

4oC

4. Filter Sterilize all reagents (0.2uM Filter).

5. Prepare Culture Plates (Previous night, to allow equilibration)

Use cell culture tested culture plates (Falcon)

Use 35mm Plate.

Evenly distribute 10-20uL of KSOM+AA+BSA per plate (5-6 droplets)

Overlay with 2-3mL Mineral Oil.

6. Embryo Collection:

1. Refer to Superovulation and ProNuclear Collection Protocol.

2. Remove Cumulus Oocyte Complex with M2+Hyal (1min).

3. Wash embryos in five separate 100uL droplets of M2+BSA.

4. Weaken Zona Pellucida with Acid Tyrode Treatment (30-60s).

5. Wash embryos in five separate 100uL droplets of M2+BSA.

CUT HERE

OVIDUCT

OVARY

FAT PAD

AMPULLA

SEGMENT

OF UTERUS

CUMULUS OOCYTE COMPLEXES

Figure: Images of Ovary, ampulla, oviduct and uterus. The top images show stereo-microscope images of a newly dissected set of reproductive organs (Left Image). In order to aid in the collection of the Cumulus Oocyte Complex, it is recommended to orient the organs in a way so that a single, quick movement of fine forceps will be able to release the complex from the ampula (Right Image)

CUMULUS CELLLS

OOCYTES

OOCYTES

OOCYTES

Figure: Phase contrast images of released complex. If performed correctly, the entire complex mass should be released from the ampulla intact (Left Image). A zoomed in image to show the clear presence of oocytes in conjunction with their support cumulus cells (Right Image).

7. Electroporation:

Reagent

Source

0.1cm Electroporation Cuvette

Biorad

BioRad Gene Pulser II

Various (Described Below)

1. Embryos are transferred to 10uL of pre-warmed Optimem.

2. Embryos are passed through 2+ drops of Optimem to reduce M2 volume.

3. Embryos are them transferred to 10uL Cas9 Droplet:

Reagent

Volume

5x RNP Buffer

2 uL

40uM Cas9 Protein

4 uL

2ug/uL sgRNA

4 uL

TE Buffer pH 7.5

To 10 uL

8. Entire 20ul mixture is pipetted into 1mm Electroporation cuvette.

9. BioRad GenePulser II: (Square Wave Protocol)

Condition

Value

Voltage

30V

Pulses

4-8

Pulse Length

3 msec

Pulse Interval

100 msec

10. Embryos are recovered by flushing the cuvette with 100uL Prewarmed KSOM+BSA.

11. A small volume pipette is used to retrieve embryos.

12. A second flush of KSOM+BSA is used to recover remaining embryos.

13. Embryos are washed with 3 passages of M2+BSA.

14. Under Mineral Oil conditions, embryos are washed in KSOM+BSA 3 times.

15. Embryos are allowed to culture at 37oC, 5% CO2 until blastocyst formation.

16. If generation of Founder Mice is desired, embryos are instead transferred to recipient pseudo pregnant female (Please refer to Embryo Transfer protocol)

Note: Embryos are extremely sensitive to culture conditions. This includes temperature, oxygen levels and media conditions. Be sure to test incubator conditions and media expiration dates once a month to ensure proper in vitro development.

Single Embryo Lysis

Embryos are typically allowed to culture to the morula or blastocyst stage in order to ensure all repair events have completed as well as provide additional genomic DNA templates used in the subsequent PCR assay. Therefore, a slightly modified proteinase K based lysis reaction is employed.

Embryo Lysis Buffer (ELB)

Reagent

Source

Working Stock

Potassium Chloride

Fisher, Cat# P217-3

50mM

Tris-HCl

Fisher, Cat# BP1531

10mM pH 8.5

MgCl2

Fisher, Cat# M33-500

2.5mM

Gelatin

Fisher, Cat# G7-500

0.1mg/mL

Nonidet P-40

Fluka, Cat# 74385

0.45%

Tween 20

Sigma, Cat# P7949-500

0.45%

Proteinase K*

Fisher, Cat# BP1700-100

0.2mg/mL

*Note: Proteinase K is added immediately prior to Lysis Reaction.

1. From the in vitro culture plate, late stage embryos are first washed by passing through two droplets of PBS using a glass mouth pipette.

2. In a third PBS droplet, embryos are arrayed and placed away from one another in order to ensure single embryos are collected per lysis reaction.

3. Using a small volume hand held pipette, single embryos are collected in 1uL of PBS and deposited into a single well of a PCR tube containing 10uL of ELB.

4. Once all embryos have been collected, load embryos and ELB into thermalcycler.

5. Program and run the following reaction conditions:

Temperature

Duration

55˚

4hr

95˚

10m

4˚

Hold

6. Lysis can either be used immediately or stored at -20oC. Avoid multiple rounds of Freeze Thaw.

IN/DEL Assay by RFLP (NHEJ)

Tyrosinase sgRNA (sgTyr):

5’-GGATCCTAATACGACTCACTATAGGGGTGGATGACCGTGAGTCCGTTTTAGAGCTAGAA

sgTyr F1: TCTTTTCGGAGACACTCAAATCA

sgTyr F2: TCTGTACAATTTGGGCCCCC

sgTyr R1: GCTTTCAGGCAGAGGTTCCT

HDR Assay by RFLP

Modification to protocol for HDR: Simply include the ssDNA oligo into the RNP mixture prior to electroporation.

Reagent

Volume

5x RNP Buffer

2 uL

40uM Cas9 Protein

4 uL

2ug/uL sgRNA

3 uL

200 pmol ssDNA Oligo

1 uL

TE Buffer pH 7.5

To 10 uL

Tyr ssDNA:

5’GTGCACCATCTGGACCTCAGTTCCCCTTCAAAGGGGTGGATGACCGTGAATTCCTGGCCCTCTGTGTTTTATAATAGGACCTGCCAGTGCTC

Reagent

Source

GoTaq

Promega, Cat# M712

HinfI

NEB, Cat# R0155S

Note: Above is a representative diagram of a typical assay to determine NHEJ and HDR outcomes. Note the position and selection of Restriction Enzyme in the Donor Oligo (EcoRI) to aid in genotyping.

1. Following single embryo DNA isolation, perform the following PCR:

Reagent

Volume (µl)

H2O

7.5

2x GoTaq

12.5

Single Embryo Lysate

3

sgTyr F1 10uM

1

sgTyr R1 10uM

1

Total

25

Temperature

Duration

Cycles

95˚

2min

1x

95˚

30s

30x

57˚

30s

72˚

30s

72˚

10m

1x

4˚

Hold

1x

2. It is essential for the success of this assay to ensure that only a single product is produced, therefore a nested PCR strategy is suggested to be developed/

3. Create a 1:10 dilution of the above PCR product to serve as template of the following PCR.

4. Using the same conditions stated above, use 1uL of the PCR Product dilution

5. Perform the following PCR:

Reagent

Volume (µl)

H2O

9.5

2x GoTaq

12.5

1:10 Diluted Product

1

sgTyr F2 10uM

1

sgTyr R1 10uM

1

Total

25

Temperature

Duration

Cycles

95˚

2min

1x

95˚

30s

30x

57˚

30s

72˚

30s

72˚

10m

1x

4˚

Hold

1x

6. The PCR product from this reaction is then used as substrate for RFLP analysis using the Hinf1 Restriction Enzyme. Prepare the following reaction:

Reagent

Volume (µl)

H2O

5

Hinf1 or EcoR1

1

Cutsmart 10X Buffer

1

PCR Product

3

Total

10

7. Incubate this reaction for 4hrs at 37oC.

8. Following digest, run product on 2% agarose gel and image RFLP result to determine editing efficiency per embryo. An equal amount of the non-digested nested PCR product can also be electrophoresed as a loading control, to ensure that the starting material in a single band, making the RFLP result interpretation more straightforward.

Note: Above is a representative image of a typical assay results to determine NHEJ and HDR outcomes.