nature protocols: doi:10.1038/nprot.2017 › original › nature-assets › nprot ›...

Supplementary Figure 1

Gating scheme for identifying and quantifying the number of HSPCs with targeted integration of a GFP reporter gene.

FACS plots show the gating strategy for identifying CD34+ HSPCs targeted with a GFP reporter gene. The figure shows data from cord

blood-derived CD34+ HSPCs that were electroporated two days after isolation with Cas9 RNP targeting the HBB locus. Immediately

after electroporation, cells were transduced at an MOI of 50,000 using an AAV6 donor vector carrying a UbC-GFP expression cassette. Four days after electroporation and transduction, cells were stained for CD34 (and propidium iodide to identify live cells) and then ana-lyzed on a FACS Aria (BD). Cells are identified in a forward/side scatter plot (FCS-A and SSC-A) and single cells discriminated from doublets using SSC-W/SSC-H and FSC-W/FSC-H plots. Live cells are discriminated based on propidium iodide, and in the final GFP/CD34 plot, the targeted GFP+CD34+ cells are identified. Cell frequencies within gates are shown.

Nature Protocols: doi:10.1038/nprot.2017.143


Gating strategy to identify stem and progenitor subpopulations within the CD34+ cells.

Representative FACS plots are shown from the analysis of CD34+ cells freshly isolated from cord blood. Cells were stained for human

lineage markers (CD2, CD3, CD4, CD8, CD16, CD19, CD20, CD56, CD235a, and CD14), as well as CD34, CD38, CD90, CD45RA, and CD123. The gating strategy identifies cells in a forward/side scatter plot (FCS-A and SSC-A) and then single cells are discriminated from doublets using SSC-W/SSC-H and FSC-W/FSC-H plots. Live cells are discriminated based on propidium iodide. Subpopulations are shown in red gates and identified as follows: HSCs (Lin-/CD34

+/CD38

-/CD45RA

-/CD90

+), MPPs (Lin

-/CD34

+/CD38

-/CD45RA

-/CD90

-

), LMPPs (Lin-, CD34

+, CD38

-, CD90

-, CD45RA

+), CMPs (Lin

-/CD34

+/CD38

+/CD45RA

-/CD123

+), GMPs (Lin

-

/CD34+/CD38

+/CD45RA

+/CD123

+), and MEPs (Lin

-/CD34

+/CD38

+/CD45RA

-/CD123

-). HSCs: hematopoietic stem cells, MPPs: multipo-

tent progenitors, LMPPs: lymphoid-primed multipotent progenitors, CMPs: common myeloid progenitors, GMPs: granulocyte-macrophage progenitors, MEPs: megakaryocyte-erythrocyte progenitors.



Gating strategy for analyzing human engraftment in NSG mice.

Representative FACS plots show the gating strategy upstream of the FACS plots shown in Figure 4D. Cells are identified in a for-ward/side scatter plot (FCS-A and SSC-A) and single cells discriminated from doublets using SSC-W/SSC-H and FSC-W/FSC-H plots. Live cells and murine red blood cells (RBCs) are discriminated based on propidium iodide and an anti-murine Ter119 antibody. Out of the live non-RBCs, human cells can be gated based on huCD45 and HLA-ABC expression as depicted in Figure 4D. Adapted with permission from ref. 15, Springer Nature.


Supplementary Methods

CRISPR/Cas9 Genome Editing in Human Hematopoietic Stem Cells

Rasmus O. Bak *, Daniel P. Dever

* & Matthew H. Porteus

Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.

*Co-first author

Correspondence should be addressed to M.H.P. ([email protected])

Identification and plasmid cloning of a panel of sgRNAs

1. Identify 4-8 sgRNA target sites near the genomic locus of interest (preferably within

20bp of the intended genomic change) using the MIT CRISPR Designer website

(crispr.mit.edu) and/or CRISPOR (http://crispor.tefor.net). The algorithms rank the

sgRNAs and predict off-target sites. For a more thorough off-target analyses of high-

ranked sgRNAs, we recommend using COSMID (https://crispr.bme.gatech.edu/).

2. Design complementary sense and antisense oligonucleotides for each of the sgRNAs

for cloning into the px330 plasmid (AddGene plasmid #42230). Design oligos with

overhangs compatible to BbsI-digested px330 as depicted in Fig. 4c. of Ran et al.1,

and order from Integrated DNA Technologies (IDT) as standard desalted 25nmole

DNA oligos. Resuspend oligos to a final concentration of 100µM.


3. Phosphorylate and anneal complementary oligo pairs by mixing:

T4 ligation buffer (10X): 1µL

sgRNA (s) (100µM): 1µL

sgRNA (as) (100µM): 1µL

Nuclease-free H2O: 6µL

T4 Polynucleotide Kinase: 1µL

Total: 10µL

4. Incubate the reaction in a thermocycler:

Phosphorylation: 98°C - 30 min

Annealing: 95°C - 5 min, then ramp down to 25°C at 5°C per min.

5. Set up the following digestion of the px330 plasmid for 2 hrs at 37°C with BbsI:

CutSmart Buffer (10X): 3µL

px330 plasmid: 3µg

Nuclease-free H2O: to 30µL

BbsI-HF enzyme: 1µL

Total: 30µL


6. Column-purify the digested plasmid using the GeneJET Gel Extraction and DNA

Cleanup Micro Kit according to manufacturer’s instructions.

7. Dilute the annealed oligos to a total volume of 200µL using nuclease-free H2O and

ligate into the purified px330 backbone by incubating the following reaction for 5 min

at room temperature:

Purified px330 backbone: 100ng

Annealed oligos: 2µL

Instant Sticky-end Ligase Master Mix: 5µL


Total: 10µL

8. Transform the ligation mix into NEB 5-alpha competent E. coli by mixing 2µL of the

ligation reaction with 25µL NEB 5-alpha. Flick the tube 4-5 times and incubate on ice

for 5 min, heat-shock at 42°C for 30 s, then place on ice for 2 min. Add 200µL SOC

Outgrowth Medium and spread onto a selection LB plate with 100µg/mL ampicillin.

Incubate the plate overnight at 37°C.

9. For each px330-sgRNA construct, use sterile pipette tips to inoculate four colonies

into four 14mL Falcon tubes containing 5mL LB media with 100µg/mL ampicillin.

Incubate the culture in a shaking incubator at 37 °C for 12-16 hrs at 200-250 rpm.


10. Purify plasmids using the GeneJET Plasmid Miniprep Kit according to manufacturer’s

instructions. Sequence the plasmids with the U6 primer (see materials sections) to

identify plasmid with the correct insert. PAUSE POINT. The plasmids can be stored at

4°C for months or -20°C for years.

Functional screening of sgRNA panel

11. For functional screening of a panel of 4-8 px330-sgRNA constructs (in triplicates),

transfer 5µg of each of the plasmids into three separate eppendorf tubes. Also, transfer

5µg of the empty px330 plasmid into three tubes as negative control.

12. Pellet 10 million K562 cells at 300 x g. CRITICAL STEP. Cells should be in log

growth phase when used. Resuspend the cell pellet in 1mL K562 electroporation

solution for nine electroporations + one extra (see materials section for preparation).

Transfer 100µL of the cell suspension into nine of the eppendorf tubes with plasmid

(one replicate). Mix well, and transfer to electroporation cuvette. Tap the cuvettes to

make sure suspension is at bottom of cuvette.

13. Electroporate cells. If using the Amaxa IIb nucleofector use program T-016. If using

the Lonza 4D nucleofector use program FF-120. We have found that both

nucleofectors work comparably. Directly after electroporation, add 500µL pre-warmed

media and use a sterile transfer pipette to gently transfer contents to a 12-well plate

and add 2.5mL pre-warmed media.


14. Repeat Steps12 and 13 twice for triplicate electroporations of each sgRNA plasmid.

This way, electroporations are performed with a maximum of ten samples at a time

minimizing the handling time and the duration that cells will be exposed to the

electroporation buffer.

15. Culture cells for at least four days. Pellet 200,000 cells at 300 x g in a microcentrifuge

tube, aspirate media and wash once in PBS, pellet cells again and aspirate the PBS

taking care not to disturb the pellet.

16. Extract the genomic DNA by adding 50µL QuickExtract DNA Extraction Solution,

vortex at high speed for 15 s, incubate in a 65°C heat block for 6 minutes, vortex for

15 s., then incubate for 10 min. in a 100°C heat block. PAUSE POINT. The extracted

gDNA can be stored at -20°C for years.

17. Design and order primers to amplify the targeted genomic locus. The amplicon should

be 500-700bp with the sgRNA target sites centered in the amplicon. Set up a PCR

reaction on the extracted gDNA samples to assay INDEL levels using the online

software Tracking of Indels by Decomposition (TIDE)2. Use the manufacturer’s

recommended thermocycling conditions for the Phusion PCR Master Mix:

Phusion PCR Master Mix (2X): 12.5µL

Primers (25µM stock): 2 x 0.5µL

Template DNA: 1µL

Nuclease-free water: 10.5µL


Total volume: 25µL

18. Run the PCR reactions on a 1% agarose gel and purify excised gel fragments

containing the appropriate PCR bands using the GeneJET Gel Extraction Kit

according to manufacturer’s instructions. PAUSE POINT. The purified PCR products

can be stored at 4°C for months.

19. Sanger-sequence the purified PCR amplicons using both PCR primers. CRITICAL

STEP. Obtained Sanger sequences must be of high quality for accurate INDEL

analysis by TIDE.

20. Go to the TIDE website (https://tide.nki.nl) and input the sgRNA sequence (the 20nt

sequence preceding the PAM) in the ‘Guide sequence’ form. Upload the sequence file

(.ab1 or .scf file format) obtained from the sample electroporated with the empty

px330 plasmid as ‘Control Sample Chromatogram’. Upload the sequences obtained

from electroporation of the px330-sgRNA plasmids as ‘Test Sample Chromatogram’.

Click ‘update view’ and inspect the ‘Quality control’ view. CRITICAL STEP. Make

sure that there is no aberrant sequence signal before the expected cut site. If INDELs

are present in the test sample, aberrant signal from the test sample only should be

evident right at and after the predicted cut site. The region of decomposition (grey line

above the quality control window) may need to be adjusted under ‘Advanced settings’

as this should not cover regions of poor sequence quality (high aberrant sequence

present in the control sample) that might be observed at the end of the sequences. The

INDEL frequency and the spectrum can be found in the ‘INDEL Spectrum’ window.


https://tide.nki.nl/

The R2 values are generally >0.9 for high-quality sequence reads. Low R

2 values

indicate suboptimal settings or poor sequence quality. Though most sgRNA create

small INDELs, it might be necessary for some sgRNAs to increase the ‘INDEL size

range’ for accurate total INDEL frequency analysis. High activity sgRNAs generally

result in INDEL frequencies of >20% in K562 cells and can often be much higher.

Choosing and ordering synthetic chemically modified sgRNA

21. Pick a sgRNA with high activity. If several high-activity sgRNAs are identified in the

functional screen in K562 cells, the choice may be based on proximity to the genomic

site to be changed, off-target profile, and INDEL spectrum depending on the desired

functional outcome of INDEL events.

22. Order the sgRNA from TriLink BioTechnologies (1 mole minimal starting synthesis

scale) or Synthego (3nmole minimal synthesis) as single guide RNAs with three

terminal nucleotides modified with 2'-O-methyl 3' phosphorothioate (see reagents

section). Resuspend per manufacturer’s recommendation, aliquot, and store at -80°C.


Production and purification of AAV6 HR donor template

Cloning AAV donor plasmid

1. Design primers to PCR-amplify the fragments needed for the donor plasmid.

Fragments are to be Gibson-assembled into either BstXI+XbaI-digested pAAV-GFP

or NotI-digested pAAV-MCS (see Reagents), which carry the plasmid backbone and

the ITRs from AAV serotype 2. Homology arms should be designed to be at least

400bp and following guidelines presented in the ‘donor design’ section and depicted in

Fig. 1. Template DNA for the PCR should preferably be genomic DNA extracted from

CD34+ HSPCs (to avoid artificial polymorphisms that might be generated in highly

mutagenic cancer cell lines). We have not examined whether making homology arms

from a specific individual’s genomic DNA improves homologous recombination-

mediated editing in an individual’s own cells. SNP donors can be created using

overlap extension PCR with the introduced SNPs present in the overlapping regions of

the two amplicons. An expression cassette can be amplified from an existing plasmid

or elements can be amplified from different plasmids with overlaps compatible with

Gibson assembly. Alternatively, the full donor or parts of the donor can be ordered as

synthesized DNA from either GenScript or Integrated DNA Technologies (IDT) or

other gene synthesis companies. We recommend using the human UbC or viral SFFV

promoter as these are both expressed well after targeted integration in vitro and in vivo

in both CD34+ HSPCs and primary human T cells. Primers can be designed using the

online available Primer3 algorithm and can be added linkers (up to a total oligo size of


60bp) that create overhangs compatible with Gibson assembly. Primers are ordered

from Integrated DNA Technologies (IDT) as standard desalted 25nmole DNA oligos.

2. PCR-amplify elements for the AAV donor plasmid. Set up the following PCR reaction

format for each of the donor elements:

Phusion PCR Master Mix (2X): 25µL

Primers (25µM stock): 2 x 1µL

Template DNA: 10ng plasmid or 250ng genomic DNA

Nuclease-free water: to 50µL

Total volume: 50µL

Run the PCR reactions on a thermocycler programmed with the following thermal

cycling parameters:

Initial denaturation 98°C – 30 s

35 cycles of:

Denaturation 98°C – 10 s

Annealing 3°C above the Tm of the lower Tm primer – 30 s

Extension 72°C – 30 s/kb

Final extension 72°C - 5 min

3. Set up the digestion of either pAAV-GFP or pAAV-MCS:


CutSmart Buffer (10x): 3µL

pAAV-GFP or pAAV-MCS plasmid: 3µg


BstXI+XbaI (pAAV-GFP) or NotI (pAAV-MCS): 2x1µL or 1µL

Total: 30µL

Incubate for 2 hrs at 37°C.

4. Run the PCR reactions and the digestion on a 1% agarose gel and purify excised gel

fragments containing the appropriate DNA bands using the GeneJET Gel Extraction

Kit according to manufacturer’s instructions.

5. Measure the concentration of DNA in the gel-purified samples by UV absorption

spectroscopy on a NanoDrop One and set up a Gibson assembly reaction according to

manufacturer’s recommendations.

6. Transform Gibson assembly reaction into NEB Stable Competent E. coli by mixing

2µL of the ligation reaction with 200µL NEB 5-alpha. Flick the tube 4-5 times and

incubate on ice for 30 min, heat-shock at 42°C for 30 s, then place on ice for 5 min.

Add 950µL room-temperature SOC Outgrowth Medium and place the tube at 30°C for

1 hr while shaking horizontally at 250 rpm. Meanwhile, pre-warm a selection LB plate

with 100µg/mL ampicillin to 30°C. After 1 hr, add and spread 100µL of the

transformation mix on the plate and incubate overnight at 37°C.


7. Use sterile pipette tips to inoculate 4-8 colonies into separate 14mL Falcon tubes

containing 5mL LB media with 100µg/mL ampicillin. Incubate the culture in a

shaking incubator at 37°C for 12-16 hrs at 200-250 rpm.

8. Purify plasmids using the GeneJET Plasmid Miniprep Kit according to manufacturer’s

instructions. PAUSE POINT. The plasmids can be stored at 4°C for months or -20°C

for years.

9. To identify correct plasmids, use a plasmid map to identify suitable restriction sites for

RFLP analysis following the general digestion reaction presented in 3. Identify correct

colonies by running the digestions on a 1% agarose gel and visualizing bands under

UV light. Depending on the number of fragments in the Gibson assembly and the

specific plasmid to be cloned, it may be necessary to screen more than eight colonies

by RFLP analysis or by setting up a PCR screening approach to identify rare positive

colonies. Note that both cloning orientations of the insert between the ITRs are

expected to produce equivalent levels of functional AAV vectors.

10. Sequence one or more of the correct plasmids identified in 9. Note that the ITRs are

prone to recombination and deletions. However, due to their strong secondary

structure they cannot be sequenced, but separate digestions with SmaI and AhdI

restriction enzymes, which both cut within both ITRs, can confirm a gross ITR

integrity analysis.


11. Set up a midi culture of the correct colony and make a plasmid midi-prep of the

culture using the NucleoBond Xtra Midi EF kit following manufacturer’s guidelines.

Repeat 10 to validate integrity of the new plasmid prep.

Production of AAV by Iodixanol Gradient purification

12. Expand HEK293T cells to a total of at least 110 million cells in DMEM containing

10% fetal bovine serum, 100mg/mL streptomycin, 100unit/mL penicillin and 2 mM L-

glutamine media. CRITICAL STEP. Do not let cells reach full confluency during

expansion. Passage cells by trypsinization by first aspirating media, then washing cells

gently with PBS, and then applying TrypLE to cover the cells, followed by incubation

at 37°C for 2 min. Gently tap the flask to dislodge cells if necessary and pipette cell

suspension up and down to make a single cell suspension. Add complete DMEM

medium and count the number of viable cells using a hemocytometer and Trypan blue

dye exclusion. Plate ten 15cm TC-treated dishes with 11 million cells per dish making

sure to distribute the cells equally in the dishes. Let the cells adhere to the dishes

overnight.

13. Before transfection, change media to 20mL media containing 1mM sodium butyrate.

Transfect the cells by mixing 10mL Opti-MEM with 60µg of the AAV donor plasmid

and 220µg of the helper plasmid pDGM6. Add 1120µL PEI and quickly vortex or

shake vigorously for 30 s. Incubate the transfection reaction at room temperature for

15 min (do not exceed 20 min). Carefully distribute the PEI-complexed DNA

dropwise to the ten dishes (approx. 1.1mL per dish) and swirl dishes gently to


distribute. Incubate for 48-72 hrs. CRITICAL STEP. Cells should be 70-80%

confluent when transfected.

14. If plasmids have a fluorescent reporter, transfection efficiencies can be evaluated by

fluorescence microscopy or flow cytometry of cells scraped of a small area of a culture

dish using a pipette tip wile aspirating with a pipette. Detach all HEK293T cells using

a cell scraper, and collect cells and media in 50mL conical tubes. Pellet the cell

suspension by centrifugation at 1,400 x g for 15 minutes at 4°C. Resuspend the cell

pellets in AAV lysis buffer. Combine in one tube and adjust total volume to 10mL

using lysis buffer. PAUSE POINT. The cell suspension can be stored at -80°C for

weeks.

15. Prepare a dry ice/ethanol bath. Lyse the cells by three freeze-thaw cycles by

alternating between 15 min incubation in a 37°C water bath and 10 min in the dry ice-

ethanol bath. Mix intermittently and don’t incubate at 37°C for longer than necessary

to completely thaw the cells. At the end of each thaw, vortex for 10 s.

16. After the third thaw, add Benzonase to the lysate to a final concentration of 200U/mL.

Mix well and incubate in a 37°C water bath for 45 – 60 min while mixing every 15

min.

17. Centrifuge the lysate at 2,500 x g for 20 min at 4°C to pellet debris. Transfer

supernatant to a 50mL conical tube and add PBS or AAV lysis buffer to a total of

14mL. PAUSE POINT. The crude virus can be stored at -80°C for weeks.


18. Prepare iodixanol (OptiPrep) solutions at 15%, 25%, 40%, and 58% iodixanol. For

building two iodixanol gradients (purification of two virus preps) prepare four 50mL

conical tubes (10% extra is added per solution):

15% 25% 40% 58%

OptiPrep 5.3mL 5.8mL 7.9mL 17.0mL

10X Gradient Buffer 2.1mL 1.4mL 1.2mL 0.58mL

5M NaCl 4.3mL 0mL 0mL 0mL

UltraPure H2O 9.6mL 6.9mL 2.8mL 0mL

Phenol Red (0.5%) 0mL 0.03mL 0mL 0.04mL

Final 21.3mL 14.1mL 11.9mL 17.6mL

Phenol red is added to the 25% and 58% solutions to aid visualization of the layers

within the tube.

19. Build the Iodixanol gradient in a Quick-Seal ultracentrifuge tube using a 10mL syringe

with a long 18 gauge spinal needle. Start by adding the 14mL virus solution to the

bottom of the tube. Add the subsequent layers underneath the virus layer by extending

the needle to the bottom of the tube and slowly injecting the iodixanol solutions in the

following order and volumes: 15% (9.7mL), 25% (6.4mL), 40% (5.4mL), and 58%

(8mL). Be very careful to rid the syringe and needle of air before injection and not to

introduce bubbles while building the gradient, which can lead to collapse of the tube


during ultracentrifugation. CAUTION. Take care when handling needles and discard

into sharps container without recapping needle.

20. Top off the tubes with PBS or AAV lysis buffer using a 20-21 gauge needle.

CRITICAL STEP. Add the PBS very slowly to avoid disruption of the gradient layers.

If bubbles are present in the tube, use the tip of a Kim wipe to remove liquid in the

stem of the tube top and to disrupt the bubbles. CAUTION. Take care when handling

needles and discard into sharps container without recapping needle.

21. Seal the top of the tube using the Quick-Seal Cordless Tube Topper kit. Attach the

Aluminum Spacer to the top of the tubes, balance the tubes pairwise on a precision

scale by applying tape on top of the Aluminum Spacer. Load balanced tubes pairwise

in opposing slots in the Beckman Type 70Ti rotor, fasten the rotor lid, and load the

rotor into the Optima L-80 XP ultracentrifuge or any ultracentrifuge compatible with

the Type 70Ti rotor. Apply vacuum and program the ultracentrifuge to maximum

acceleration, minimum deceleration (but not ‘no brake’), 48,000 rpm (237,000 x g), 2

hrs, and 18°C.

22. Carefully remove the tubes from the rotor and mount on a ring stand with a utility

clamp. Insert an 18-gauge needle near the top of the tube as airflow vent. Place a

50mL conical tube directly underneath the tube to catch any leakage. Using a syringe

with an 18 gauge needle, puncture the side of the tube 1-2mm below the interface

between the 40% and 58% gradient layers with the bevel of the needle facing up. With

the syringe, gently extract 3-4mL of the virus-containing layer with the needle bevel


facing up during the first half of the extraction and then facing down for the other half.

Transfer the virus to a 15mL conical tube. CAUTION. Take care when handling

needles and discard into sharps container without recapping needle.

23. Equilibrate a 10kDa molecular weight cut-off dialysis cassettes in PBS for 5 min.

Then load the extracted virus into the dialysis cassette and place the cassette in a float

buoy and dialyze overnight in 1L of PBS on a magnetic stirrer in a cold room. The

next day, change the PBS and dialyze for another 2 hrs. Then change the PBS to PBS

with 5% D-Sorbitol and dialyze for an additional 2 hours. Remove the dialyzed virus

from the cassette using a 10mL syringe with an 18 gauge needle. The dialysis chamber

may have swollen and the extracted volume can be up to 7mL. CAUTION. Take care

when handling needles and discard into sharps container without recapping needle.

24. Add Pluronic F-68 to a final concentration of 0.001% as a surfactant to prevent loss of

vector particles due to plastic adhesion, aliquot into the desired number of tubes, and

store at -80°C until use.

Titration of AAV donor vectors

25. To make a standard curve for absolute quantification of viral vector genomes, set up a

digestion of either pAAV-GFP or pAAV-MCS with PvuII similar to the reaction

presented in Step 3. Gel-purify the fragment containing the ITRs and very carefully

measure the precise concentration of extracted DNA by UV absorption spectroscopy

on a NanoDrop One. Use online algorithms (e.g.


http://www.endmemo.com/bio/dnacopynum.php) to calculate the molecular weight of

the extracted double-stranded DNA fragment containing two ITRs. Use the calculated

molecular weight to prepare a 1mL solution with 1E9 molecules/µL. From this stock,

prepare 10-fold serial dilutions using nuclease-free H2O as diluent to make up a

standard series ranging from 1E3 to 1E9 molecules/µL.

26. Extract vector genomes from the AAV by mixing 10µL AAV with 40µL QuickExtract

DNA Extraction Solution. Vortex at high speed for 15 s, incubate in a 65°C heat block

for 6 minutes, vortex for 15 s., then incubate for 10 min. in a 100°C heat block. Dilute

the extracted vector genomes 200-fold in nuclease-free water for a total dilution of

1,000-fold. PAUSE POINT. The extracted vector genomes can be stored at -20°C for

months.

27. Set up qPCR reactions on the 1,000-fold diluted extracted AAV genomes, the standard

curve samples ranging from 1E3 to 1E9 copies/µL, and water as non-template control.

Per qPCR reaction:

SsoAdvanced Supermix (2X): 7.5µL

AAV ITR qPCR assay (20X): 0.75µL

Precision Blue Real-Time PCR Dye: 0.075µL

Template DNA: 6.67µL

Total volume: 15µL


Run the qPCR reactions on a LightCycler 480 II Real-Time PCR Thermal Cycler set

up for detection of the Fluorescein (FAM) hydrolysis probe and programmed with the

following thermal cycling parameters:

Initial denaturation 95°C – 15 min

40 cycles of:

Denaturation 95°C – 60 s

Annealing/extension 60°C – 60 s

28. When the run is complete, input the concentrations of the plasmid standards and perform

the standard curve calculation in the ‘Absolute Quantification Analysis’ mode in the LightCycler

software. The PCR efficiency should be between 90% and 105% (amplification factor between

1.90 and 2.05). Transfer the calculated concentrations of the unknown AAV samples to an Excel

spreadsheet and calculate the physical titer as vector genomes per mL (vg/mL). CRITICAL

STEP. Adjust for the 1,000-fold dilution of the vector genomes and the 2-fold difference in ITR

copies between plasmid and single-stranded AAV. Titers typically range between 5E11 – 1E12

vg/mL. We recommend that functional AAV titration be performed in HR experiments in CD34+

HSPCs to identify the lowest MOI that yields maximum HR frequencies and high viabilities. We

typically find this to be around an MOI of 50,000-100,000 vg/cell.

Reagents

sgRNA cloning

T4 DNA Ligase Reaction Buffer (10X) (NEB, cat. no. B0202S)


UltraPure DNase/RNase-Free Distilled Water (Fisher Scientific, cat. no. 10977-015)

T4 Polynucleotide Kinase (NEB, cat. no. M0201S)

BbsI-HF restriction enzyme (NEB, cat. no. R3539S)

CutSmart Buffer (NEB, cat. no. B7204S)

px330 plasmid (pX330-U6-Chimeric_BB-CBh-hSpCas9, AddGene, plasmid #42230)

GeneJET Gel Extraction and DNA Cleanup Micro Kit (Fisher Scientific, cat. no. K0831)

Instant Sticky-end Ligase Master Mix (NEB, cat. no. M0370S)

NEB 5-alpha Competent E. coli (High Efficiency) (NEB, cat. no. C2987I)

NEB Stable Competent E. coli (High Efficiency) (NEB, cat. no. C3040I)

SOC Outgrowth Medium (NEB, cat. no. B9020S)

Fisher BioReagents LB Broth, Miller (Granulated) (Fisher Scientific, cat. no. BP9723-

500)

Agar (Sigma-Aldrich, cat. no. A1296)

Ampicillin solution, 100mg/mL (Teknova, cat. no. 101414-068)

14mL Falcon Round-Bottom Polypropylene Tubes (Corning, cat. no. 352059)

GeneJET Plasmid Miniprep Kit (Fisher Scientific, cat. no. K0502)

U6 primer, forward: 5’-GAGGGCCTATTTCCCATGATTCC-3’

Phusion Green Hot Start II High-Fidelity PCR Master Mix (Fisher Scientific, cat. no.

F566S)

AAV6 cloning, production and purification

pAAV-MCS (Agilent Technologies, cat. no. 240071)

pAAV-GFP (Addgene, plasmid #32395)


pDGM6 AAV6 Helper/Cap/Rep plasmid (Russell Lab, University of Washington. Need

MTA)

pDP6 AAV6 Helper/Cap/Rep alternative plasmid (Plasmid Factory, cat. no. PF436)

NotI-HF restriction enzyme (NEB, cat. no. R3189S)

CutSmart buffer 10x (NEB, cat. no. B7204S)

Gibson Assembly Master Mix (NEB, cat. no M5510A)

GeneJET Gel Extraction Kit (Fisher Scientific, cat. no. K0692)

Spinal Needles 16 G x 6 in (BD, cat. no. 408360)

Quick-seal cordless tube topper kit (Beckman Coulter, cat. no. 358313)

Tube rack, blue, 25.4mm, 1 in (Beckman Coulter, cat. no. 348124)

Heat sink tube topper (Beckman Coulter, cat. no. 348117)

Guide large seal (Beckman Coulter, cat. no. 348643)

Seal Former (domed top) (Beckman Coulter, cat. no. 348120)

Spacer, Aluminum, Tube, 25mm (Beckman Coulter, cat. no. 342699)

Ultracentrifuge tube, Quick-Seal (Beckman Coulter, cat. no. 344326)

10mL syringe (BD, cat. no. 309604)

HEK293T cell line (HEK293FT) (Fisher Scientific, cat. no. R70007). CAUTION: The

cell lines used in your research should be regularly checked to ensure they are authentic

and are not infected with mycoplasma

T225 tissue culture treated flasks (USA Scientific, cat. no. CC7682-4822)

15cm tissue culture treated plates (Cellstar, cat no. 639160)

Penn/Strep (Fisher Scientific, cat. no. 15070063)

DMEM, high glucose (GE, cat. no. SH302423.01)


TrypLE Express (Fisher Scientific, cat. no. 12605010)

L-Glutamine (Fisher Scientific, cat. no. 25030081)

10K MWCO Slide-A-Lyzer G2 Dialysis Cassette (Fisher Scientific, cat. no. 88251)

OptiPrep 60% w/v Iodixanol (Sigma Aldrich, cat. no. D1156)

Benzonase Nuclease (Bio Vision, cat. no. 7680-25)

Disposable Transfer Pipets (VWR, cat. no. 414004-018)

BstXI restriction enzyme (NEB, cat. no. R0113S)

XbaI restriction enzyme (NEB, cat. no. R0145S)

SmaI restriction enzyme (NEB, cat. no. R0141S)

AhdI restriction enzyme (NEB, cat. no. R0584S)

PvuII-HF restriction enzyme (NEB, cat. no. R3151S)

NucleoBond Xtra Midi EF kit (Macherey-Nagel, cat. no. 740420.10)

Cell Scrapers (Fisher Scientific, cat. no. 12-565-59)

Sodium Butyrate (Sigma-Aldrich, cat. no. B5887-5G). Make a 2,500mM solution by

mixing 5g Sodium Butyrate with 18.17mL PBS. Mix well, filter-sterilize using a 0.22µm

vacuum filter (EMD Millipore, cat. no. SCGP00525), aliquot, and store at -20°C.

PEI (Polyethylenimine, linear) 22 kDa (Polysciences, cat. no. 23966). Add 50 mg of

linear PEI to 50 mL of pH 4.5 PBS (made with HCl) in a 50 mL conical tube. Place the

tube in a 70°C waterbath and vortex every 10 min until the PEI is completely dissolved.

Cool to room temperature and filter sterilize using a 0.22 µm filter. Aliquot and store at -

80°C.

Phenol red, 0.5% solution (Sigma-Aldrich cat. no. P0290-100ML)

Opti-MEM I Reduced Serum Medium (Fisher Scientific, cat. no. 31985070)


AAV lysis buffer (milliQ water with final concentration of 2mM MgCl2 and 10mM Tris,

adjust pH to 8.0, filter-sterilize using a 0.22µm vacuum filter (EMD Millipore cat. no.

SCGP00525), and store solution at 4°C.

10X Gradient Buffer (GB). Mix 10mL 1M Tris (pH 7.6), 30mL 5M NaCl, 10mL 1M

MgCl2, 50mL milliQ water. Filter-sterilize using a 0.22µm vacuum filter (EMD Millipore

cat. no. SCGP00525) and store solution at 4°C.

Kim wipes (Fisher Scientific, cat. no. 06-666A)

Utility Clamp (VWR, cat. no. 89083-218)

Ring stand (Fisher Scientific, cat. no. 11-474-207)

Needles, 18 gauge (Fisher Scientific, cat. no. 14-821-15A)

Float buoys for dialysis cassettes (Fisher Scientific, cat. no. PI66430)

Pluronic F-68 (Fisher Scientific, cat. no. 24040032)

D-Sorbitol (Sigma Aldrich, cat. no. S1876)

SsoAdvanced Universal Probes Supermix (Bio-Rad, cat. no. 172-5280)

Precision Blue Real-Time PCR Dye (Bio-Rad, cat. no. 1725555)

AAV ITR qPCR assay from Aurnhammer et al.3. Order primers and probe from IDT.

Forward primer: 5′-GGAACCCCTAGTGATGGAGTT-3′, reverse primer: 5′-

CGGCCTCAGTGAGCGA-3′, hydrolysis probe labeled with 5’ FAM, internal ZEN, and

3’ BFQ: 5’-CACTCCCTCTCTGCGCGCTCG-3′). A 20X assay is made of 6.8µM

reverse primer, 2µM forward primer, and 2µM probe (final working concentrations are

340, 100, and 100nM, respectively).


1. Ran, F.A. et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8, 2281-2308 (2013).

2. Brinkman, E.K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42, e168 (2014).

3. Aurnhammer, C. et al. Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Hum Gene Ther Methods 23, 18-28 (2012).


nature protocols: doi:10.1038/nprot.2017 › original › nature-assets › nprot ›...

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