Benchmark

Evaluation protocol for CRISPR/Cas9-mediated

CD19 knockout GM24385 cells by flow cytometry

and Sanger sequencing

Sarah L Inwood*,1 , Linhua Tian1 , Kirsten Parratt1 , Samantha Maragh1 & Lili Wang**,1

1Biosystems and Biomaterials Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA; *Author for correspondence:

sarah.inwood@gmail.com; **Author for correspondence: lili.wang@nist.gov

BioTechniques 72: 279–286 (June 2022) 10.2144/btn-2022-0015

First draft submitted: 25 January 2022; Accepted for publication: 6 May 2022; Published online: 15 June 2022

ABSTRACT

Although several genome editing options are available, CRISPR/Cas9 is one of the most commonly used systems for protein and advanced

therapies. There are some long-term data regarding genomic and phenotypic stability, however, information is sparse. Flow cytometry can offer

a method to characterize these edited cells for longitudinal studies. The objective of this work is to describe a protocol for using flow cytometry

to measure the edits from CRISPR/Cas9 on a well-characterized B-lymphoblast cell line, GM24385, with the goal of supporting safe and effective

CRISPR/Cas9-engineered therapies.

METHOD SUMMARY

The objective of this work is to describe a protocol for using flow cytometry to measure the edits from CRISPR/Cas9 on a well-characterized

B-lymphoblast cell line, GM24385, with the goal of longitudinal monitoring to support safe and effective CRISPR/Cas9-engineered therapies. The

protocol methods include cell culture, flow cytometry, CRISPR/Cas9 editing and Sanger sequencing.

KEYWORDS:

CD19 • CRISPR/Cas9 • flow cytometry • genome editing • GM24385 cell line

The CRISPR/Cas9 system is naturally found in bacterial and archaeal immune systems [1]; however, it has been adapted for use in

eukaryotes as a Nobel prize-winning genome editing system [2,3]. The CRISPR/Cas9 system uses gRNA to direct the Cas9 nuclease to

a specific target near an NGG protospacer adjacent motif sequence. The Cas9 then cleaves the DNA and the cell’s native DNA damage

repair machinery repairs the double-strand break.

The two main repair pathways leveraged for knockout and modification of target genes during CRISPR/Cas9 editing are the nonhomologous end joining (NHEJ) and homology-directed repair pathways. NHEJ is the primary choice for repairing double-strand breaks

when the donor DNA template is not available, making it a good option for CRIPSR/Cas9 knockout experiments. Loss and gain of nucleotides are common occurrences during NHEJ-mediated repair, and a knockout comes from a shift in the coding sequence. Homologydirected repair is based on availability of the donor DNA template and is mainly used for custom gene modifications in coding or noncoding sequences [4,5].

The CRISPR/Cas9 system is being used more and more often for advanced therapies, including cell and gene therapies, with great

promise (e.g., CRISPR test for treating sickle cell disease) [6,7]. Many commercial products for enabling CRISPR/Cas9 nuclease RNAguided genome editing are available in a variety of delivery methods. These products include DNA encoded in viruses, plasmids, mRNA

and RNPs. Additionally, CRISPR gRNA can be delivered in two parts, crRNA and tracrRNA, or as an sgRNA. Multiple bacterial species offer

CAS protein options; a commonly used one is Streptococcus pyogenes Cas9. Similarly, there are many options for how these molecules

can be delivered into cells, including viral vectors, such as lentiviral and adeno-associated viral vectors, and chemical methods, such

as lipids, injection and electroporation [8,9]. Although CRISPR/Cas9 is an exciting genome editing tool, there are limited longitudinal

data on genomic and phenotypic stability after using CRISPR/Cas9 to edit cells. There are now several reports providing evidence that

there is also the potential for additional unintended long-term changes [10–12] associated with CRISPR/Cas9 editing. In addition, genetic

bottlenecks can arise during selection of edited cell subpopulations. Hence, it is essential to characterize the edited cells intended for

use in advanced therapies such as cell and gene therapies, where the therapy often involves administering populations of edited cells

to the patient [13].

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Table 1. Flow cytometry reagents used for cell analysis.

B-cell panel

Marker Clone Fluorophore Company Product no. Dilution volume per

0.56 cells, μl

B-cell subtype expression

indicator

Live/Dead Aqua Thermo Fisher Scientific L34957 0.5 Dead cells

CD19 J3-119 PE Beckman Coulter IM1285U 0.5 All B-cell lineages

CD27 1A4CD27 PE-Cy7 Beckman Coulter A54823 7.5 Memory B cells and short-lived

plasma cells

CD20 B9E9 (HRC20) Pacific Blue Beckman Coulter A74777 7.5 Mature B cells and memory B cells

CD24 ALB9 APC Beckman Coulter A87785 7.5 All B cells

CD38 LS198.4.3 APC-AF700 Beckman Coulter B23489 7.5 Regulatory B cells

IgD IA6-2 FITC Beckman Coulter B306527 30 Naive B cells

CD138 MI14 BV711 BD Biosciences 563184 7.5 Bright plasma B cells

Compensation

Marker Clone Fluorophore Company Product no. Dilution volume per

0.56 cells, μl

CD19 SJ24C1 PE-Cy7 BD Biosciences 557835 5

CD19 HIB19 APC BioLegend 302212 5

CD19 J3-119 APC-AF700 Beckman Coulter A78837 5

CD19 HIB19 FITC BioLegend 302206 5

CD19 SJ25C1 BV711 BD Biosciences 563036 5

Intracellular

Marker Clone Fluorophore Company Product no. Dilution volume per

0.56 cells, μl

GAPDH 14C10 AF647 Cell Signaling Technology 3907S 5

AF647: Alexa Fluor 647; APC: Allophycocyanin; APC-AF700: Allophycocyanin–Alexa Fluor 700; BV711: Brilliant Violet 711; FITC: Fluorescein isothiocyanate; PE: Phycoerythrin;

PE-Cy7: Phycoerythrin–Cyanine7.

In biomanufacturing, mammalian cells are often engineered to produce protein products. Common mammalian cell lines for protein

expression include Chinese hamster ovary, mouse myeloma (NS0) and human embryonic kidney 293 cells [14,15]. However, for cell and

gene therapies, a variety of primary human cell types, including B cells and fibroblast cells, are modified [16,17].

In cell therapies, the final therapeutic edited cell product may be a pool of cells or cells that have been sorted for specific characteristics. Since the therapeutic value is often reliant on DNA sequence changes, it is imperative to determine if the edits are persistent [17].

Although it is expected that different cell types may have varying responses to CRISPR/Cas9 editing, there may also be some similar

underlying long-term trends that have yet to be elucidated.

The Genome in a Bottle Consortium has put forth a significant amount of effort in characterizing and developing National Institute of

Standards and Technology reference materials for the genomes of several cell lines [18]. For this reason, one of the Genome in a Bottle

B-lymphoblast cell lines, GM24385, has become a good candidate for characterizing the products of genome editing. A well-known Blymphocyte antigen, CD19, is expressed on the surface of human B cells [19] from early stages to terminally differentiated plasma cells,

making it a good target for editing a B-lymphoblast cell line.

The objective of this protocol is to enable study of the longitudinal genotypic and phenotypic stability of CRISPR/Cas9 using genomics

and flow cytometry. By evaluating the stability of CRISPR/Cas9 genome editing, we will have a better understanding of its long-term

effects on the cell. This will help to further characterize the safety and effectiveness of therapies produced using the CRISPR/Cas9

system.

Protocol

Cells & media

The authors used the GM24385 B-lymphoblast cell line (Coriell Institute for Medical Research, NJ, USA), Roswell Park Memorial Institute

(RPMI) 1640 medium with 2 mM L-glutamine (30-2001; American Type Culture Collection, VA, USA), fetal bovine serum (FBS) (16140071;

Thermo Fisher Scientific, MA, USA) and T flasks or cell culture plates.

Flow cytometry

The authors used an Attune NxT flow cytometer (A24858; Thermo Fisher Scientific), antibodies for B-cell panel (Table 1), phosphatebuffered saline (PBS) (10010049; Thermo Fisher Scientific), FBS, FIX & PERM Cell Fixation and Cell Permeabilization Kit (GAS003; Thermo

Fisher Scientific), Attune performance check beads (4449754; Thermo Fisher Scientific), Attune focusing fluid (100085929CST; Thermo

Fisher Scientific), centrifuge and tubes (1.5, 5 and 15 ml).

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Table 2. gRNAs for CD19 knockout.

Guide Sequence Cut site, hg19 Exon Binds to

1 CGCUGUGCUGCAGUGCCUCA 28,932,373 2 Sense

2 GCUGUGCUGCAGUGCCUCAA 28,932,374 2 Sense

3 UUCCCAGGCCUGGCAGCCCC 28,932,458 2 Antisense

4 UUCAACGUCUCUCAACAGAU 28,932,527 2 Sense

Table 3. Primers for PCR amplification of CD19-edited section.

Direction Sequence

Forward GGGTGTCCTTGGCTGAGTAA

Reverse CCTCTCTCCAGCTCCATTGT

Transfection reagents & instrumentation

The authors used 4D-Nucleofector (AAF-1003B; Lonza, NJ, USA), SF Cell Line Kit (V4XC-2032 or V4XC-2012; Lonza), sgRNA designed to

target (Synthego, CA, USA) (Table 2), S. pyogenes Cas9 (9212-5MG; Aldevron, ND, USA) and tissue culture plates.

Sanger sequencing

The authors used a Veriti thermocycler (A48141; Thermo Fisher Scientific), NanoDrop (ND-ONE-W; Thermo Fisher Scientific), Quick-DNA

Miniprep Plus Kit (D4069; Zymo Research Corporation, CA, USA), Q5 Hot Start High-Fidelity 2× Master Mix (M0494L; New England

Biolabs, MA, USA), PCR primers (Table 3) and DNA Clean & Concentrator kit (D4014; Zymo Research Corporation).

Data analysis

The authors used FlowJo (Becton, Dickinson and Company, NJ, USA) and the inference of CRISPR Edits analysis tool (Synthego) for

statistical analysis (alternative instruments, kits and software may have similar results).

Methods

Cell culture

The Genome in a Bottle B-lymphoblast cell line, GM24385, was selected as the cell line of choice as a result of its extensive use in

genome sequencing [20]. GM24385 cells are anchorage-dependent cells and loosely aggregated when cultured in upright T flasks or

plates. Low passage cells are recommended because growth rate decreases after a number of passages [21]. It should be noted that

one should always be extremely careful to avoid contamination.

The authors maintained cells in RPMI 1640 medium supplemented with 15% FBS. Cells were cultured in a humidified incubator set at

5% CO2 and 37◦C. Cells were passaged when the density reached approximately 1 × 106 cells/ml (a split ratio of 1:4 is recommended).

Prior to passage, cells were dissociated by pipetting. If cells had not reached the splitable density in 3–4 days, the medium was changed

by centrifuging cells (200 × g for 10 min at 25◦C), gently aspirating the supernatant and resuspending cells in prewarmed fresh medium.

CRISPR/Cas9 editing for CD19 knockout

CRISPR/Cas9 is a popular method for creating a DNA double-strand break. When a repair template is unavailable, the cell repairs doublestrand breaks using the NHEJ pathway, during which several nucleotides may be lost at the repair sites, leading to knockout of gene

expression. Of the many delivery methods available, transfection of an RNP complex by electroporation is popular because of its transient

nature, which works for many cell types [22]. In this experiment, because of the possibility of different guide efficiencies, four gRNAs

were tested. To knock out CD19, four gRNAs targeting human CD19 (Table 2) were purchased as modified sgRNAs in a Synthego gene

knockout kit. sgRNAs were selected based on location in the coding region of the gene, location in an exon found in most transcripts,

activity score and base mismatches. sgRNAs were used following the manufacturer’s instructions with the kit-supplied S. pyogenes

Cas9 2NLS nuclease and transfected using 4D-Nucleofector and an SF Cell Line Kit. Prior to scaling up to cuvettes, samples were tested

in nucleofection strips.

When designing transfection experiments, the authors recommend controls such as electroporation only, Cas9 and electroporation

with no gRNA as well as a positive control such as a gRNA targeting the RELA gene with a known knockout effect. Because of the nature

of nucleofection, many cells die during this step, and it takes time for the surviving cells to stabilize and replicate to a sufficient quantity

to analyze. It is important to begin with healthy cells and handle them gently to minimize cell death.

The authors resuspended gRNA sterile nuclease-free water to make a 30-μM stock solution, used the stock solution to form RNP

complexes with 20 μM of Cas9 and nucleofector reagent at a ratio of 9:1 for sgRNA and Cas9 and incubated for 10 min at room temperature. Depending on the size of the cuvette to be used, 1 × 106 cells per cuvette or 2 × 105 cells per well of a 16-well strip cuvette needed

to be prepared. Cells were centrifuged at 100 × g for 10 min at 25◦C and then resuspended in nucleofector reagent to the appropriate

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Table 4. Laser and antibody matrix for flow cytometry analysis.

Attune channel 440/50 512/25 710/50 530/30 585/30 780/60 670/14 720/30

Channel label VL1 VL2 VL4 BL1 YL1 YL5 RL1 RL2

Fluorophore Pacific Blue Aqua BV711 FITC PE PE-Cy7 APC APC-AF700

Comp unstained

Comp Pac Blue CD20

Comp Aqua Live/Dead

Comp BV711 CD19

Comp FITC CD19

Comp PE CD19

Comp PC7 CD19

Comp APC CD19

Comp APC-AF700 CD19

FMO1 Live/Dead CD19

FMO2 CD20 Live/Dead CD138 IgD CD19 CD24

FMO3 CD20 Live/Dead CD19 CD27 CD38

Samples CD20 Live/Dead CD138 IgD CD19 CD138 CD27 CD24

APC: Allophycocyanin; APC-AF700: Allophycocyanin–Alexa Fluor 700; BV711: Brilliant Violet 711; Comp: Compensation; FITC: Fluorescein isothiocyanate; FMO: Fluorescence minus

one; Pac Blue: Pacific Blue; PC7: Phycoerythrin–Cyanine7; PE: Phycoerythrin; PE-Cy7: Phycoerythrin–Cyanine7.

Spillover values are read across rows

Spillover

BL1-A

BL1-A

RL1-A

RL1-A

RL2-A

RL2-A VL1-A

VL1-A

VL2-A

VL2-A VL4-A

VL4-A

YL1-A

YL1-A

YL4-A

YL4-A

100.00

0.19

0.40

0.20

6.82

0.12

0.61

0.49

0.17

100.00

35.31

0.10

4.64

2.05

0.00

0.05

0.06

7.83

100.00

0.00

0.50

14.37

0.00

0.03

0.10

0.00

0.35

100.00

15.55

2.37

0.03

0.00

0.31

0.00

0.12

19.23

100.00

0.04

0.00

0.09

0.24

3.10

24.99

0.64

16.68

100.00

0.43

0.13

0.18

0.00

0.43

0.21

3.13

0.13

100.00

3.03

0.06

2.18

27.82

0.07

0.60

0.80

0.42

100.00

Figure 1. Example compensation matrix for flow cytometry analysis. A compensation matrix was used to ensure proper separation of the fluorescence

signals for cell marker staining.

volume (5 μl of cells at 3 × 104 cells/ml per well). Cells were then transferred to a nucleofector cuvette, with RNP complexes or controls

added to the cells in the cuvettes and wells to a total of 100 μl per cuvette or 30 μl per well of solution. Next, cells were nucleofected

using the SF Cell Line and DN-100 in 4D-Nucleofector. Prewarmed medium was then added to the cuvettes and wells and incubated at

37◦C for 30 min. Finally, cells were transferred to a prewarmed six-well plate and incubated at 37◦C for 72 h until harvesting.

B-cell panel with flow cytometry

To measure the phenotypic effect of knocking out CD19, the authors established a panel for measuring differences and a baseline

phenotype. B lymphoblasts that are immortalized with Epstein–Barr virus express CD19 on the cell surface, so this was used as the

test marker for knockout [23–25]. A panel containing several surface markers common to B cells and a viability marker was designed

based on the expression levels of surface markers and availability of lasers and fluorescence channels (Tables 1 & 4). This panel was

initially measured to create a baseline from the parent GM24385 cells. The panel was used with the parent GM24385 cells to set the

gates (Figures 1 & 2) [26]. The steps for performing the flow cytometry analysis are discussed in the following sections. This analysis

was periodically applied to the edited cells.

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Single cells CD19

CD24

100 101 102 103 104 105 106

IgD

20 60 80 100 40

200

400

600

800

1.0K

0

100 101 102 103 104 105 106

500

1.0K

1.5K

2.0K

0

100 101 102 103 104 105 106

Parent cells Knockout cells

200

400

600

800

1000

0

0 600 800 1000 200 400

200

400

600

800

1000

0

0 600 800 1000 200 400

Lymphocytes

100

200

300

0

100 101 102 103 104 105 106

CD20

100

200

300

400

500

0

100 101 102 103 104 105 106

50

100

150

200

0

100 101 102 103 104 105 106

CD138

100

200

300

400

0

100 101 102 103 104 105 106

CD38

50

100

150

200

0

100 101 102 103 104 105 106

CD27

100

200

300

0

100 101 102 103 104 105 106

Time

200

400

600

0

200

400

600

800

1000

0

0

Time:: Time PBC-A::PBC-A PBC-A::PBC-A

Figure 2. Representative gating strategy for flow cytometry analysis. (A) Representative gating strategy for flow cytometry analysis based on gates set

using FMO controls. Time versus SSC was used to gate the steady-state population. Single cells were then gated using the FSC-A versus FSC-H plot. A

lymphocyte gate was created using the FSC versus SSC plot. The remaining plots are histograms of the respective markers (CD20, CD19, IgD, CD24,

CD27, CD38 and CD138). (B) Example CD19 histogram plot comparing parent cells with knockout cells demonstrating a count of CD19+ cells in the

knockout group.

FMO: Fluorescence minus one; FSC: Forward scatter; FSC-A: Forward scatter area; FSC-H: Forward scatter height; SSC: Side scatter.

Because of the influence of cell passaging time in cell culture, it is important to be consistent with timing of the sample collection

between experiments (e.g., collecting cells of the same passage). Therefore, to minimize variations due to sample preparation and cell

behavior, measurements should be made at the same time point.

For cell sample preparation, cells were divided into three groups: live/dead compensation samples; single fluorophore-conjugated

antibody-stained samples for compensation; and sample cells, including the controls. Additionally, an intracellular staining process was

used with CD19 antibody to determine whether any CD19 proteins were expressed but not translocated on the cell surface. Three groups

of samples were stained in parallel. Approximately 5 × 105 cells were needed for each sample, and this was determined using the flow

cytometry analysis laser and antibody matrix as a guideline for staining (Table 4). It should be noted that the parent cells were used

for compensation and fluorescence minus one controls. Compensation beads can alternatively be used, but they are not the same size

as stained cells. Compensation is a technique used in multifluorophore panels to correct for fluorescence spillover between detectors.

Fluorescence minus one controls are used to identify positive and negative cells to appropriately place gates for marker identification.

With regard to staining cell samples, the aqua compensation tube should possess dead cells to stain for compensation. To obtain

dead cells, cells were placed in a tube and heated to 60◦C for 20 min. Cells were stained with the Live/Dead stain after washing with PBS

or alternatively stained with the compensation stain using PBS containing 2% FBS. The authors added 1 ml of PBS containing Live/Dead

stain and incubated in the dark at room temperature for 30 min. To obtain a compensation matrix, the authors stained one tube for each

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Parent surface + intracellular

Parent surface

Parent intracellular

Edited surface + intracellular

Edited surface

Edited intracellular

100 101 102 103 104 105 106

CD19

100 101 102 103 104 105 106

GAPDH

Figure 3. Sample histograms from intracellular staining. An overlay plot of CD19 histograms comparing the knockout and parental cell lines with

surface staining only, internal staining only and both surface and internal staining shows less CD19 in the knockout cell lines. Using GAPDH as a control

for the staining protocol shows a lack of GAPDH in the surface staining-only cell lines.

of the fluorophores used (in this case with CD19 to get a sharp peak). After coating the tube with PBS containing FBS, the stain was

added to the cells and incubated for 30 min at room temperature.

To remove extra fluorophores, the authors washed cells with PBS containing 2% FBS and then placed the compensation tubes in a

refrigerator until use. Next, antibodies were added for additional markers and incubated for 30 min at room temperature. For intracellular

staining, cells were split into three groups: cells that were stained with both surface and intracellular antibodies, cells that were stained

with only surface antibodies and cells that were stained with only intracellular antibodies (Figure 3). Cells were then washed with PBS

and stained with Live/Dead as previously described. Next, cells were washed with PBS containing 2% FBS and stained with a cocktail of

CD19 antibodies and GAPDH antibodies as a control for 30 min at 4◦C. Finally, cells were washed with PBS with 2% FBS.

The authors fixed and permeabilized the cells using the FIX & PERM Cell Fixation and Cell Permeabilization Kit following the manufacturer’s instructions. Briefly, FIX & PERM buffer A was added to the cells and incubated at room temperature for 15 min. Cells were

washed with PBS containing 5% FBS and 0.1% sodium azide. FIX & PERM buffer B was added to cells with intracellular antibodies, CD19

and GAPDH and incubated at room temperature for 15 min. Excess fluorophores were washed and all cells were then resuspended in

PBS containing 2% FBS.

To measure samples, the authors used a panel designed for the Attune NxT flow cytometer. The steps used included checking

performance and generating a compensation matrix and then measuring the samples. First, a startup and performance check was done.

Following the manufacturer’s instructions, the authors started the instrument, computer and software. Next, three drops of performance

beads were added to 2 ml of PBS, vortexing before and after, and a performance check was run.

For compensation samples, the lasers to be used were selected in the compensation window (Table 4). Using a small sample size

and slow rate, a small amount of each compensation sample was sampled to make sure the voltages were correct (highest peaks

for the positive sample with good separation of positive and negative peaks). After voltages were set, compensation samples were

measured at a sample rate of 100 μl/min, and the authors collected 100,000 lymphocyte events. Finally, a compensation matrix was

applied (Figure 1). With regard to samples, the authors set up plots for single cell, live/dead and lymphocyte (one for the knockout marker

is also recommended). Samples were measured at a sample rate of 200 μl/min, and 200,000 lymphocyte events were collected.

Although several data analysis options are available for flow cytometry data, the authors used FlowJo software. The authors created

a group for compensation and a group for the samples and added the respective flow cytometry standard files. The authors used the

compensation wizard to create a compensation matrix and applied it to the samples. The authors then set up the gates for the samples

using the fluorescence minus one control samples (Figure 2) and used the layout and table editors to generate plots and tables for the

samples.

DNA sequencing

To determine the effect of CRISPR/Cas9 editing on the genome, sequencing analysis of the target site was performed after 72 h and

then at periodic time points after cell harvest. Cells were harvested by collecting up to 1.5 ml of cell culture by centrifuging at 100 ×

g and washing cells with PBS. The remaining cells were maintained in the incubator for flow cytometry analysis as described earlier

and for future sequencing. The authors extracted gDNA using a Quick-DNA Miniprep Plus Kit following the manufacturer’s protocol and

measured DNA concentration using a NanoDrop. The authors used PCR to amplify the CRISPR/Cas9-edited CD19 locus using the primers

listed in Table 3 and Q5 Hot Start High-Fidelity 2× Master Mix; the authors followed the manufacturer’s instructions for preparing PCR

reaction samples. A total of 500 ng of DNA, the Q5 Hot Start High-Fidelity 2× Master Mix, 10 μM of each primer and nuclease-free water

were combined for a 50-ul reaction.

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0

1000

Parental cells

255 260 265 270 315

2000

275 280 285 290 295 300 305 310

0

25

CD19 KO

250 255 260 265 310

75

50

270 275 280 285 290 295 300 305

Control sample 252 to 317 bp

Cut site

SYNTHEGO ICE

T TT CCCC C CCC C G A AA AAA A A A A

A

TT TT T T C C CC CCC CC C CC C C CC C CC C G G GG G G G GG G G GG A AA T

T C C C C G C T T AAA C C C T T TT C A A AA A AA C C CC C T G GG T T G G G GG C C C C T GGG G T C C A C C A T G G A C C

Edited sample 247 to 312 bp Powered by

Figure 4. Example Inference of CRISPR Edits analysis result demonstrating edit near the cut site. The defined peaks prior to the targeted CRISPR/Cas9

DNA cut site in the knockout cell line match the parental cell line. At the cut site, the lower-amplitude peaks demonstrate a mixed population of edited

cells.

The following conditions were used for PCR in a Veriti thermocycler: denaturation at 95◦C for 30 s followed by 35 cycles at 95◦C

for 5 s, 67◦C for 30 s and 72◦C for 30 s and a final extension at 72◦C for 2 min. The PCR products were cleaned using the DNA Clean

& Concentrator kit according to the manufacturer’s instructions and eluted into 25 μl of ultrapure water. The final PCR concentration

was determined using a NanoDrop. Clean PCR products were submitted to Psomagen (MD, USA) for Sanger sequencing using their

protocol for difficult-to-sequence DNA products. DNA sequences were returned as .ab1 files and analyzed using the Synthego Inference

of CRISPR Edits tool to determine editing efficiency (Figure 4). The expected outcome for Sanger sequencing on a mixed population of

edited cells is a lower-amplitude signal and mixed signal near the expected CRISPR/Cas9 DNA cut site. Analytics such as the Synthego

Inference of CRISPR Edits tool may enable disambiguation of mixed Sanger sequence signal around the cut site.

Additional analysis

For long-term stability analysis, Sanger sequencing and flow cytometry are used at multiple time points following editing. To further

analyze the effects of CRISPR/Cas9 editing, transcriptomics, next-generation DNA sequencing and single-cell sequencing should be

considered as follow-ups for stability analysis. To ensure that the cell population contains a 100% knockout, cell sorting can be used

to isolate knockout cells. Ideally, this would occur over a significant period of time, but culture limitations and cell exhaustion will likely

impact this time frame.

Author contributions

S Inwood, with assistance from L Wang and S Maragh, formulated the idea and direction for the manuscript. L Tian and K Parratt worked

with S Inwood to complete supporting experiments.

Disclaimer

To specify an experimental procedure as completely as possible, certain commercial materials, instruments and equipment are identified

in this manuscript. In no case does identification of the manufacturer of particular equipment or materials imply a recommendation or

endorsement by the National Institute of Standards and Technology, nor does it imply that the materials, instruments and equipment

identified are necessarily the best available for the purpose.

Financial & competing interests disclosure

The authors received funding from the US Department of Commerce, National Institute of Standards and Technology and Material

Measurement Laboratory. The authors have no other relevant affiliations or financial involvement with any organization or entity with a

financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

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Open access

This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit

http://creativecommons.org/licenses/by-nc-nd/4.0/.

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