Chapter 12
Highly Multiplexed Analysis of CRISPR Genome Editing
Outcomes in Mammalian Cells
Soh Ishiguro and Nozomu Yachie
Abstract
CRISPR–Cas-based genome editing has enabled efficient genetic engineering of a range of organisms andsparked revolutions in many fields of biology. After Streptococcus pyogenes Cas9 was first demonstrated for
mammalian genome editing, many CRISPR-associated (Cas) protein variants have been isolated fromdifferent species and adopted for genome editing. Furthermore, various effector domains have been
fused to these Cas proteins to expand their genome-editing abilities. Although the number of genomeediting tools has been rapidly increasing, the throughput of cell-based characterization of new genomeediting tools remains limited. Here we describe a highly multiplexed genome editing and sequencing library
preparation protocol that allows high-resolution analysis of mutation outcomes and frequencies induced byhundreds to thousands of different genome-editing reagents in mammalian cells. We have successful
experiences of developing several key genome-editing tools using this protocol. The protocol also isdesigned to be compatible with robotic liquid handling systems for further scalability.
Key words Genome editing, CRISPR–Cas9, Base editing, High-throughput sequencing, Ampliconsequencing

1

Introduction
A number of CRISPR genome-editing systems have been developed. Streptococcus pyogenes Cas9 (SpCas9) was the first to be
demonstrated for mammalian genome editing [1, 2] and it has
been the most widely used in a range of fields, including
genome-wide gene knockout screens and development of genome
editing-based therapeutic approaches [3]. In CRISPR–Cas9
genome editing, a chimeric single guide RNA (gRNA) recruits
Cas9 to the target genomic sequence that is adjacent to a 30 protospacer adjacent motif (PAM) through sequence complementarity(Fig. 1). The two endonuclease domains of Cas9 then induce a
double-stranded DNA break (DSB) at the target genomic region.
This has been used to induce either mutations (mostly deletions) intarget genes through error-prone nonhomologous end-joining

Ryosuke Kojima (ed.), Mammalian Cell Engineering: Methods and Protocols, Methods in Molecular Biology, vol. 2312,
https://doi.org/10.1007/978-1-0716-1441-9_12, © Springer Science+Business Media, LLC, part of Springer Nature 2021

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Cas9
M
PA

3
5
3

3
5

gRNA

5

Target-AID
H2 O

C

ABEmax
H 2O

NH3

H 2O

NH3

A

U

C

I

nCas9

5

gRNA

5
3

3

nCas9
A1
CD C
3
5

Ta
dA
5

gRNA

5
3

A

3

M
PA

A
3
5

I

UGI

M
PA

3

NH3

A

U

nCas9

M
PA

5
3

H 2O

NH3

UGI

UGI
A1
CD C

Target-ACEmax

3
5

Ta
dA
5

gRNA

Fig. 1 CRISPR–Cas9 genome editing and base editing. Conceptual diagrams of genome editing by wild-type
Cas9 and three base editors. The base editing tools, Target-AID [11], ABEmax [17], and Target-ACEmax [35],convert C·G!T·A, A·T!G·C, and simultaneously C·G!T·A and A·T!G·C, respectively

DNA repair or transgene insertions through homology-directed
DNA repair in eukaryotic cells. While wild-type SpCas9 has been
adopted in diverse applications, many protein engineering efforts
have further enhanced its genome-editing ability. For example,
recent studies have expanded the targeting scope of SpCas9 byrelaxing its wild-type PAM restriction (50 -NGG-30 or 50 -NGA-30 ).
Such PAM-altered Cas9 variants include SpCas9-NG [4] andxCas9-3.7 [5] for 50 -NG-30 PAM, and near PAM-less SpG andSpRY [6]. Other efforts have focused on minimizing undesired
off-target edits that usually occur through mismatch-tolerant
annealing of gRNA to nontargeting genomic regions, and several
high-fidelity Cas9 variants, such as SpCas9-HF1 [7], eCas9(1.1)[8], and evoCas9 [9], have been derived.
The DSB-based genome editing by wild-type SpCas9 is particularly effective in gene knockout, but has a drawback in precision
genome editing, where mutation outcomes are not always predictable. Induction of a single-base substitution has been proposedwith the transgene insertion approach to replace target sequencewith a donor template sequence by homology-directed DNA
repair, but its efficiency is limited. Furthermore, Cas9-derived
DSBs are cytotoxic in cells [10, 11]. Harnessing a nucleosidedeaminase domain to catalytically inactive Cas9 (dCas9) or nickase
Cas9 (nCas9), base editing technologies have enabled targetedHigh-Throughput Analysis of CRISPR Genome Editing Outcomes

195

nucleotide substitutions to be induced without DSB [12]. The
current base editors are categorized as cytosine base editors(CBEs), for targeted C·G!T·A substitution, and adenine baseeditors (ABEs), for targeted A·T!G·C substitution (Fig. 1). The
CBEs use a cytidine deaminase such as rAPOBEC1 or PmCDA1 toconvert cytidines into uridines in the nontargeting DNA strand ofgRNA and a uracil glycosylase inhibitor to prevent base excision
repair of uracil bases [11, 13]. Following DNA replication, uracils
are converted into thymines. The ABEs instead use an evolvedtRNA adenosine deaminase (TadA) that converts adenosines toinosines on single-stranded DNA (ssDNA). Although none of thenatural adenosine deaminases have been found to have catalytic
activity for ssDNA, a directed protein evolution approach derived
the evolved TadA heterodimer for A·T!G·C base editing[14]. The CBEs and ABEs both have narrow base editing windows
within the gRNA target site and are suited for targeted sequence
alternation. The idea of base editing has been rapidly applied to theengineered SpCas9 variants to expand their targeting scopes [15]and enhance their targeting fidelities [16]. Furthermore, several
studies have reported that codon optimization, linker optimization,and other accessory domains, such as nuclear localization signals
and ssDNA-binding domains, can be used to derive more efficientbase editors if they are fused in an optimal order [17–20]. Non-specific C!U and A!I edits of the cellular transcriptome have been a concern for CBEs using rAPOBEC1 and ABEs
using TadA, the original wild-types of which have catalytic activity
for RNA (this has been reported not to be the case for PmCDA1based CBEs). However, protein engineering approaches also havederived deaminase variants that minimize such nonspecific RNA
off-target activities [21–24].
Although the delivery efficiency of genome-editing reagents tocells and nuclei is important, SpCas9 is 1358 aa long, which limits
the delivery efficiency of SpCas9 to cells by, for example, adenoassociated viral vectors. To resolve this issue, other compact Cas9
orthologues have been explored and reported for genome editing,such as SaCas9 from Staphylococcus aureus (1053 aa) [25] andCjCas9 from Campylobacter jejuni (984 aa) [26]. These Cas9
orthologues also have been fused to deaminase domains to obtaincompact base editing tools [27, 28]. Furthermore, other CRISPR–
Cas systems from outside the CRISPR–Cas9 family have beencharacterized for efficient genome editing [29]. For example,
LbCas12a from Lachnospiraceae bacterium (formally characterized
as LbCpf1) catalyzes site-specific cleavage of DNA with T-rich
50 -TTTV-30 PAM adjacent to the 50 -end of the gRNA-targeting
sequence [30]. rAPOBEC1 and TadA also have been fused tocatalytically inactive LbCas12a for base editing [31].

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Accordingly, a range of Cas proteins, mutations, and different
effector and linker domains have been reported for genome editingand expanding the design space to develop new genome-editing
tools. However, the number of possible design combinations toderive a new tool has encountered a combinatorial explosion,
making it difficult to derive an optimal-best tool for a specific target
of application because the editing outcome patterns of each tool
need to be thoroughly evaluated for a range of genomic on-target
and predicted off-target sites in cells. Recently, along with threeother similar studies [32–34], we have developed a novel dualfunction base editor Target-ACEmax for the simultaneous introduction of C·G!T·A and A·T!G·C substitutions by fusing both
cytidine and adenosine deaminases to nCas9 [35]. We tested
genome-editing outcome patterns of 47 endogenous target sites
for three dual-function base editor candidates, six single-function
base editors, and four single-function base editor mixes, as well as a
non-functional enzyme control in triplicates by amplicon sequencing (a total of 1833 assays). In this methodology article, we
describe a modified protocol that enables genome-editing outcome
assays of 48 different gRNA target sites in human embryonickidney 293 T (HEK293T) cells for genome-editing tools as well
as a control enzyme in triplicate (Fig. 2). The protocol has fivesections: (1) screening of gRNA target sites and target amplification
primers, (2) preparation of a gRNA expression plasmid library,(3) culturing of cells, (4) massively parallel transfection of
genome-editing reagents, (5) template genomic DNA preparation,and (6) generation of a multiplexed amplicon sequencing library.
This protocol produces a single multiplexed amplicon sequencing
library for hundreds to thousands of genome-editing assay samples
and enables analysis of their genome-editing outcomes by one-shot
massively parallel sequencing (Fig. 3). This basic protocol can easily
be scaled up with increased numbers of gRNA on-target and predicted off-target sites. It can also be modified for different cell lines
with optimal cell culture and transfection protocols. (We have successful experiences of performing similar genome-editing assays for
HeLa, HCC827, and mouse embryonic stem cells with similar
library-scale protocols.) Furthermore, we designed many of theprocess modules in the protocol to be compatible with robotic
liquid handling systems for more systematic characterization of alarge number of genome-editing tools. We believe that this step-bystep protocol will be of great use for researchers to facilitate therapid development of new genome-editing tools in mammaliancells.

High-Throughput Analysis of CRISPR Genome Editing Outcomes

197

Fig. 2 Overview of the highly multiplexed amplicon sequencing protocol to analyze CRISPR genome editing
outcomes and frequencies. First, the genome-editing enzyme plasmids and gRNA plasmids are assembled to
produce all possible combinatorial transfection reagents. Then, highly parallel genome-editing assays are
performed in triplicates by cell transfection. Next, cell samples are lysed and direct PCR is used to amplify thegRNA target regions with primers that have common adapter sequences (PS1.0 and PS2.0). The first-round
PCR products are then pooled for each genome-editing enzyme of each replicate and the second-round PCR is
performed to attach custom Illumina index adapters. After sample normalization, the amplicon sequencing
libraries are pooled into a single multiplexed library and subjected to an Illumina sequencing

2

Materials

2.1 General
Considerations

All the experimental steps described here use PCR-grade ultrapure
distilled water (not autoclaved distilled water) to minimize thepotential risks of nucleic acid and DNase/RNase contaminations
that may affect the genome-editing assays and the quality of thesequencing libraries. We also recommend performing all the experiments using filtered tips on a clean bench. In the following sections,
we provide a basic protocol to measure mutation outcomes of given
genome-editing tools and a nonfunctional control enzyme (EGFP)
condition for 48 different endogenous gRNA targeting sites of
HEK293T cells in triplicates (hundreds to thousands of genomeediting assays). However, this protocol can be easily scaled up for an
increased number of gRNAs and target sites, and the cell line also
can be replaced with others with their own cell culture and transfection protocols.

Outcome patterns

Soh Ishiguro and Nozomu Yachie

EMX1

Outcome patterns

NEAT1

198

A T G C

TargetPAM

Fig. 3 Data analyses of two arbitrary selected genome-editing assays in triplicates multiplexed with a total ofnearly two thousand assays. Base editing of NEAT1 and EMX1 encoding regions by Target-ACEmax
2.2 Screeningof gRNA Target Sites
and Target
Amplification Primers

1. DNase/RNase-free ultrapure distilled water.
2. 8-strip individually capped PCR tubes.
3. Hard-Shell® 96-Well PCR Plates, low profile, thin wall, skirted,
white/clear (Bio-Rad HSP9601).
4. Microseal® ‘C’ PCR Plate Sealing Film (Bio-Rad MSC1001).
5. 5 Phusion HF Buffer (NEB B0518S).
6. Phusion DNA Polymerase (NEB M0530S).
7. dNTPs (25 mM each).
8. 1/100 diluted SYBR Green: mix 10 μL of SYBR™ Green I
Nucleic Acid Gel Stain (Invitrogen S7563) and 990 μL of 100%
dimethyl sulfoxide. Wrap with aluminum foil to protect fromlight and store at 4  C.
9. 6 loading dye.
10. Target amplification of forward primers with PS1.0 adapter
sequence: 50 -TAACTTACGGAGTCGCTCTACG XXXXXXXXXXXXXXXXXXXX -30 (Xs: annealing sequence of forward
primer for target amplification).
11. Target amplification of reverse primers with PS2.0 adapter
sequence: 50 -GGATGGGATTCTTTAGGTCCTG XXXXXXXXXXXXXXXXXXXX -30 (Xs: annealing sequence of reverse
primer for target amplification).

High-Throughput Analysis of CRISPR Genome Editing Outcomes

2.3 Preparationof gRNA Expression
Plasmid Library199

1. DNase/RNase-free ultrapure distilled water.
2. DH5-alpha Competent E. coli.
3. Super Optimal broth with Catabolite repression (SOC) outgrowth medium.
4. T4 DNA Ligase (NEB).
5. 10 T4 Ligase Buffer with 10 mM ATP (NEB).
6. T4 Polynucleotide Kinase (NEB).
7. BsmBI (NEB R0580S).
8. 1 mg/mL BSA: mix 10 μL of 10 mg/mL BSA and 90 μL of
ultrapure distilled water.
Store at 20  C.
9. gRNA cloning backbone plasmid (pSI-356 v2; available at
Addgene https://www.addgene.org/158431/) (see Note 1).
10. DNA miniprep kit.
11. 100-mL disposable DNase/RNase-free reagent reservoirs.
12. 96-well DNase/RNase-free 2-mL round bottom deep-wellplates.
13. 96-well DNase/RNase-free PCR plates.
14. Microseal® ‘C’ PCR Plate Sealing Films (Bio-Rad MSC1001)
or any alternative that can perfectly seal up the wells.
15. Lysogeny broth (LB) medium: add 25 g of LB powder todistilled water, top up to 1 L, autoclave and store at 4  C.
16. LB-Ampicillin medium: add 100 μL of 100 mg/mL ampicillin
to 100 mL of LB medium.
17. LB-Ampicillin agar plate: add 15 g of agar, and 25 g of LB
powder to distilled water in a glass bottle, top up to 1 L,
autoclave, cool down to 55  C, then add 1000 μL of
100 mg/mL ampicillin, and pour to bacterial petri dishes.
18. 15-mL bacterial culture tubes.
19. Sanger sequencing primer to validate gRNA spacer sequences.
Use 50 - TTTCCCATGATTCCTTCATATTT -30 for thegRNA cloning backbone plasmid above (compatible with any
human U6 promoter-gRNA expression plasmid).

2.4

Culturing of Cells

1. Human embryonic kidney cells 293T (HEK293T cells).
2. 0.05 w/v% Trypsin-0.53 mmol/L EDTA-4Na Solution with
Phenol Red.
3. Heat-inactivated fetal bovine serum (FBS): thaw FBS (Invitrogen 10437-028) at 4  C overnight, incubate for 30 min in
56  C water bath, and aliquot to 50-mL tubes. Store at20  C.

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4. 1 Phosphate-Buffered Saline (PBS): mix 100 mL of 10
D-PBS() and 900 mL of DNase/RNase-free ultrapure distilled water in a 1 L glass bottle. Store at 4  C.
5. 100 penicillin–streptomycin.
6. Cell culture medium: mix 500 mL of DMEM, 50 mL of heatinactivated FBS, and 5 mL of 100 penicillin–streptomycin.
Store at 4  C.
7. 5 mM Acetic Acid (AcOH): add 500 μL of 1 M AcOH to
99.50 mL of DNase/RNase-free ultrapure distilled water in
200 mL glass bottle and autoclave. Store at 4  C.
8. Collagen-I solution: mix 40 μL of Collagen Type I and11.96 mL of 5 mM AcOH in a 15-mL tube. Store at 4  C.
9. 96-well cell culture plates.
10. 10-cm cell culture dishes.
2.5 Massively
Parallel Transfectionof Genome-Editing
Reagents

1. Genome-editing enzyme expression plasmids. For example,
PpX165 is a wild-type SpCas9 nuclease expression plasmid for
mammalian genome editing (Addgene 48137). A range of baseeditor plasmids also can be obtained from Addgene (https://www.addgene.org/crispr/base-edit/).
2. EGFP expression plasmid as non-functional enzyme control(pLV-eGFP, Addgene 36083, or any alternative).
3. Plasmid DNA midiprep Kit.
4. Plasmid DNA miniprep Kit.
5. 96-well DNase/RNase-free 1.1 mL round-bottom deep wellplates.
6. Microseal® ‘C’ PCR Plate Sealing Film (Bio-Rad MSC1001).
7. 1 mg/mL Polyethylenimine (PEI) MAX solution: dissolve
10 mg PEI MAX (Polysciences 49553-93-7) in 10 mL ofultrapure distilled water, filter through a 0.22-μm filter, andaliquot 1000 μL into each 1.5-mL microcentrifuge tube withscrew cap. Store at 20  C for up to 12 months.

2.6 Template
Genomic DNA
Preparation

1. 50 mM NaOH: add 500 μL of 5 N NaOH to 49.5 mL of
DNase/RNase-free ultrapure distilled water in a 50-mL tube.
Store at 4  C (see Note 2).
2. 1 M Tris–HCI, pH 8.0.
3. Hard-Shell® 96-Well PCR Plates, low profile, thin wall, skirted,
white/clear (Bio-Rad HSP9601).
4. Microseal® ‘C’ PCR Plate Sealing Film (Bio-Rad MSC1001).

High-Throughput Analysis of CRISPR Genome Editing Outcomes

2.7 Generation of aMultiplexed Amplicon
Sequencing Library
and Sequencing

201

1. DNase/RNase-free ultrapure distilled water.
2. 8-strip individually capped PCR tubes.
3. Hard-Shell® 96-Well PCR Plates, low profile, thin wall, skirted,
white/clear (Bio-Rad HSP9601).
4. Microseal® ‘C’ PCR Plate Sealing Film (Bio-Rad MSC1001).
5. 5 Phusion HF Buffer (NEB B0518S).
6. Phusion DNA Polymerase (NEB M0530S).
7. dNTPs (25 mM each).
8. 1/100 diluted SYBR Green: mix 10 μL of SYBR™ Green I
Nucleic Acid Gel Stain (Invitrogen S7563) and 990 μL of 100%
dimethyl sulfoxide. Wrap with aluminum foil to protect fromlight and store at 4  C.
9. 6 loading dye.
10. Agencourt AMPure XP (Beckman Coulter A63881).
11. 1.5 mL DNA LoBind tubes (Eppendorf 0030122348).
12. 70% Ethanol: add 30 mL of 100% Ethanol to 70 mL of ultrapure distilled water. Store at 4  C.
13. 1 Tris-EDTA (TE) buffer.
14. KAPA Library Quantification Kits (Illumina) (KAPA Biosystems KK4824, or any alternative).
15. Illumina MiSeq v3 600 cycles kit (Illumina MS-102-3003) orIllumina MiSeq v2 Micro 300 cycles kit (Illumina
MS-103-1002).
16. Illumina PhiX control v3 (Illumina FC-110-3001).
17. Target amplification forward primers with PS1.0 adapter
sequence: 50 -TAACTTACGGAGTCGCTCTACG XXXXXXXXXXXXXXXXXXXX -30 (Xs: annealing sequence of forward
primer for target amplification).
18. Target amplification reverse primers with PS2.0 adapter
sequence: 50 -GGATGGGATTCTTTAGGTCCTG XXXXXXXXXXXXXXXXXXXX -30 (Xs: annealing sequence of reverse
primer for target amplification).
19. Custom Illumina P5 index primer: 50 -AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNYYYYYYYYY
T
AACTTACGGAGTCGCTCTACG-30 (Ns: random sequence
for better flow cell clustering; Ys: 9-bp custom index).
20. Custom Illumina P7 index primer: 50 -CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT NNNNN YYYYYYYYY G
GATGGGATTCTTTAGGTCCTG-30 (Ns: random sequence
for better flow cell clustering; Ys: 9-bp custom index).

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Soh Ishiguro and Nozomu Yachie

List of Equipment

1. Micropipettes (0.2–2 μL, 2–20 μL, 20–200 μL, and
100–1000 μL).
2. 12-channel micropipettes
20–200 μL).

(0.2–2

μL,

2–20

μL,

and

3. 5–50-mL pipete.
4. Bench-top centrifuge.
5. Centrifuge with 96-well plate and 50-mL tube rotors.
6. Heat block.
7. 96-well aluminum block.
8. 1.5-mL tube aluminum block.
9. Thermal cycler machine.
10. Agarose gel imager.
11. Agarose gel electrophoresis bath.
12. Quantitative PCR machine.
13. Vortex mixer.
14. Bacterial incubator.
15. Shaking incubator.
16. Human cell culture incubator (including CO2 controller andaspirator).
17. Cell culture hoods (bacterial and human cell culture).
18. Automated cell counter.
19. Water baths (37  C for human cells and 42  C for bacterialtransformation).
20. Plate seal roller.
21. Spectrophotometer.
22. Magnet stand (1.5-mL tube or 96-well plate format).
23. Illumina sequencer.

3

Methods

3.1 Screeningof gRNA Target Sites
and Amplification
Primers

Because target genomic regions need to be stably amplified by PCR
and the following genome editing assay procedures need to alignwith such target regions, we first screen two or three primer pairs
for each candidate gRNA target region (Fig. 4), and discard candidate gRNAs with no stable PCR amplification with any of the tested
primer pairs. To secure a certain number of gRNAs to be used in
the genome editing assays, primer pairs for an excess number ofgRNA target sites can be screened in this step. The PCR efficiencies
and expected band sizes of primer pairs are evaluated by qPCR andagarose gel electrophoresis. This section describes screening of
three primer pairs for 48 genomic gRNA target sites (a total of
144 primer pairs).

High-Throughput Analysis of CRISPR Genome Editing Outcomes203

Fig. 4 Example of PCR primer screening for stable amplification of gRNA target
regions. PCR products with the expected band sizes are marked with white dots
3.1.1 Primer Preparation

1. Design three PCR primer pairs for each of 48 selected gRNAtargeting regions and order DNA oligos (144 forward and
144 reverse primer oligos). See Subheading 2.2 for the adapter
sequences added to the target amplification primers. Using
Primer3 or any alternative, design primer pairs each to have amelting temperature of 55–65  C without homopolymer orpalindrome sequences and to produce a PCR product of
150–230 bp for the pooled library preparation (see Subheading
3.6.1 for details).
2. Mix 5 μL of 100 μM forward primer and 5 μL of 100 μM
reverse primer to prepare a 50 μM primer mix in each well offresh 96-well plates using a 12-channel pipette.
3. Prepare 10 mL ultrapure distilled water in a disposable regent
reservoir.
4. Transfer 40 μL of ultrapure distilled water from the reservoir toeach sample well of the primer mix plates using a 12-channel
pipette to obtain 10 μM primer mixes.
5. Seal the primer mix plates with optically clear adhesive PCRfilms (we recommend the Bio-Rad films described in Subheading 3 throughout the study for good sealability). The primer
mix plates can be stored at 20  C.

3.1.2 Template Genomic
DNA Preparation

1. Place a DMEM bottle in a 37  C water bath to warm up.
2. Thaw one vial of HEK293T cells in a 37  C water bath andtransfer the cells into 5 mL of the prewarmed DMEM in a
15-mL sample tube and centrifuge the cells at 200  g for
5 min.
3. Aspirate and remove the supernatant.
4. Add 10 mL of the prewarmed DMEM into the sample tube.
5. Gently mix the cells by pipetting several times.

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Soh Ishiguro and Nozomu Yachie

6. Transfer the cells into a 10-cm cell culture dish.
7. Incubate the cell culture dish for 2–3 days in a cell culture
incubator at 37  C and 0.05% CO2 to allow cell proliferation
to 80–90% confluency (see Note 3).
8. Remove the media using an aspirator and apply 1000 μL of 1
PBS to the cell culture dish.
9. Remove the reagent using an aspirator and apply 1000 μL of
0.05% trypsin-EDTA to the cell culture dish.
10. Incubate the cell culture dish for 4 min in a cell culture incubator at 37  C and 0.05% CO2.
11. Remove the reagent using an aspirator and add 1000 μL of
50 mM NaOH to the cell culture dish for cell lysis.
12. Transfer the sample to a 2-mL tube and incubate at 95  C for
15 min on a heat block, place on ice for 5 min, and neutralize
the sample with 100 μL of 1 M Tris–HCl (pH 8.0). This celllysate sample can be stored at 20  C.
3.1.3 Primer Screening
by qPCR

1. To perform the following quantitative PCR (qPCR) for each of
the 144 primer pairs, prepare 180 qPCR master mix in a
5 mL sample tube (see Note 4):1 reaction unit
Component

Final concentration

Volume (μL)

5 Phusion HF buffer

1

4

2 mM dNTPs

200 μM2

10 μM primer mix

500 nM each2

DNA template (lysed cell sample)

2

Phusion DNA polymerase

0.2

1/100 diluted SYBR green

0.06

Ultrapure distilled water

9.74

Total

20

180 qPCR master mix
Component

Volume (μL)

5 Phusion HF buffer

720

2 mM dNTPs

360

DNA template (lysed cell sample)

360

Phusion DNA polymerase

36(continued)

High-Throughput Analysis of CRISPR Genome Editing Outcomes

205

Component

Volume (μL)

1/100 diluted SYBR green

10.8

UltraPure distilled water

1753.2

Total

3240

2. Transfer the qPCR master mix into a disposable reagent reservoir placed on ice.
3. Using a 12-channel pipette, transfer 18 μL of the qPCR mastermix from the reservoir to each well of fresh 96-well PCR plates.
4. Using a 12-channel pipette, add candidate primer pair mixes,
2 μL each, to the qPCR reaction plates.
5. Seal the qPCR reaction plates with optically clear adhesive PCRfilms.
6. Centrifuge the qPCR reaction plates at 1000  g for 2 min tospin down and remove air bubbles.
7. Perform qPCR with the following thermal cycle conditions:
Step

Temperature

Time

1

98  C

0’30

2



98 C

0’10

3



60 C

0’10

4

72  C

1’00

Go to 2(29 times)


6

72 C

7



4 C

5’00
1

8. Save the qPCR results with Ct values for the primer pairs.
9. Centrifuge the qPCR reaction plates at 1000  g for 2 min andgently remove the plate seals.
10. Prepare 6 loading dye in a disposable reagent reservoir andtransfer 4 μL to each sample well of the qPCR reaction plateusing a 12-channel pipette and mix well.
11. Run a gel for the PCR products, 5 μL each, using 2% agarose
gel for 20 min with 135 V.
12. Take gel images and evaluate PCR band sizes and amplification
efficiencies with Ct values.
13. Select the best primer pair for each of the gRNA target sites andrearray the curated 10 μM primer pair mixes into a fresh
96-well PCR plate.

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Soh Ishiguro and Nozomu Yachie

14. Seal the curated primer mix plate with optically clear adhesive
PCR films.
15. Centrifuge the curated primer mix plate at 1000  g for 2 min
and store at 20  C.
3.2 Preparationof gRNA Expression
Plasmid Library

After determining the gRNA target sites and their corresponding
primers, gRNA expression plasmids are prepared according to thefollowing procedure. Two ssDNA oligos encoding the gRNA
spacer region are prepared for each target site, annealed, and phosphorylated (Fig. 5a). The dsDNA insert is then ligated into adigested plasmid backbone. The assembled DNA is transformed
into bacterial cells, and the plasmid clones are obtained by colony
isolation (Fig. 5b), followed by miniprep and validated by Sanger
sequencing. This gRNA library preparation protocol allows fast
preparation of 48–96 gRNA expression plasmids within a week.
gRNA libraries can be prepared for other CRISPR–Cas systems bychanging the dsDNA insert design and plasmid backbone.

3.2.1 Preparationof gRNA Spacer Inserts

1. Spin down forward and reverse ssDNA oligo plates (100 μMeach) (see Note 5). The ssDNA pairs need to be designed to
produce sticky 50 (CACC), and 30 (AAAC) ends after
annealing.
2. Add 10 μL of forward oligo and 10 μL of reverse oligo into a
fresh 96-well PCR plate using a 12-channel pipette. The
ssDNA mix plate can be stored at 20  C.
3. To perform the following annealing and phosphorylation reactions for 48 samples, prepare 50 reaction master mix.
1 reaction unit
Component

Volume (μL)

ssDNA oligo mix (50 μM each)

2.0

10 T4 ligase buffer with 10 mM ATP

0.5

T4 polynucleotide kinase

0.25

Ultrapure distilled water

2.25

Total

5.0

50 reaction mix
Component

Volume (μL)

10 T4 ligase buffer with 10 mM ATP

25

T4 polynucleotide kinase

12.5

Ultrapure distilled water

112.5

Total

150

High-Throughput Analysis of CRISPR Genome Editing Outcomes

207

Fig. 5 Cloning of gRNA expression plasmids. (a) A dsDNA insert encoding a gRNA spacer is prepared byannealing of ssDNA oligos and cloned to a plasmid backbone. (b) Colony isolation to obtain clones of theassembled gRNA products

4. Aliquot 3 μL of the reaction mix into each well of a fresh
96-well PCR plate.
5. Add the ssDNA mixes, 2 μL each, to the reaction plate andassemble with the reaction mix.
6. Seal the reaction plate with an optically clear adhesive PCR film.
7. Centrifuge the reaction plates at 1000  g for 30 s.
8. Incubate the reaction plate in a thermal cycler with the following conditions.
Step

Temperature

Time

1

37  C

30’00

2



95 C

5’00

3

95  C, 1  C/cycle

0’12

Go to 3
4

(69 times)


25 C

1

9. Prepare 10 mL ultrapure distilled water in a disposable reagent
reservoir.
10. Remove the optically clear adhesive PCR films.
11. Add 95 μL of ultrapure distilled water to each reaction wellusing a 12-channel pipette and mix well.
12. Seal the plate with an optically clear adhesive PCR film.
13. Proceed to the next step, or store the dsDNA insert plate at
20  C.
3.2.2 Ligation Assembly

1. To perform the ligation assembly for each dsDNA insert, prepare 50 reaction mix.

208

Soh Ishiguro and Nozomu Yachie1 reaction unit
Component

Volume (μL)

1/10 diluted annealed oligo

2.5

10 T4 ligase buffer with 10 mM ATP

0.625

1 mg/mL BSA

0.31

T4 DNA ligase

0.2

BsmBI

0.2

25 ng/μL backbone plasmid (pSI-356 v2)

0.25

Ultrapure distilled water

2.165

Total

6.25

50 reaction mix
Component

Volume (μL)

10 T4 ligase buffer with 10 mM ATP

31.25

1 mg/mL BSA

15.5

T4 DNA ligase

10.0

BsmBI

10.0

25 ng/μL backbone plasmid (pSI-356 v2)

12.5

Ultrapure distilled water

108.25

Total

187.5

2. Add 2.5 μL of insert dsDNA each into a fresh 96-well PCR
plate.
3. Add 3.75 μL of the ligation assembly mix into each well andslowly mix with the insert dsDNA by pipetting up and down.
4. Seal the reaction plate with an optically clear adhesive PCR film.
5. Spin down the reaction plate.
6. Incubate the reaction plate in a thermal cycler with the following conditions.
Step

Temperature

Time

1



37 C

5’00

2

20  C

5’00

Go to 1

(14 times)


3

55 C

30’00

4

4 C

1

7. Proceed to the next step, or store the reaction plate 20  C.

High-Throughput Analysis of CRISPR Genome Editing Outcomes
3.2.3 Bacterial
Transformation

209

1. Thaw chemically competent E. coli cells on ice (a total volumeof 480 μL for 48 reactions).
2. Aliquot 10 μL of the competent cells into a fresh 96-well PCR
plate.
3. Put a 96-well aluminum block on ice to cool down.
4. Transfer 2 μL of the ligation assembly product into each sample
well of the transformation reaction plate using a 12-channel
pipette.
5. Seal the transformation reaction plate with an optically clear
adhesive PCR film.
6. Place the transformation reaction plate on the ice-chilled
96-well aluminum block and incubate on ice for 30 min.
7. Heat-shock the transformation samples at 42  C for 30 s byfloating the reaction plate on a preheated water bath.
8. Immediately place the transformation reaction plate on theice-chilled aluminum block and incubate for 2 min.
9. Remove the optically clear adhesive PCR film.
10. Add 50 μL SOC outgrowth medium into each sample well of
the reaction plate using a 12-channel pipette.
11. Seal the reaction plate with an optically clear adhesive
PCR film.
12. Place the reaction plate in a 37  C bacterial incubator andincubate for 10 min.
13. During the incubation, airdry 5–6 LB+Amp plates on a clean
bench.
14. Remove the optically clear adhesive PCR film and briefly mix
the samples by pipetting up and down several times using a
12-channel pipette.
15. Spot 10 μL of the transformation sample each on an LB+Amp
agar plate (we usually spot 12 samples per 10-cm petri dish).
16. Incubate the LB+Amp sample plates overnight in a 37  Cbacterial incubator.
17. The bacterial plates should be stored at 4  C until the obtainedclones are validated in the following quality check procedure.

3.2.4 Plasmid
Purification and Sanger
Sequencing

1. Isolate two to three colonies for each gRNA and inoculate eachto 5 mL LB+Amp liquid media in a 15-mL culture tube (seeNote 6).
2. Incubate the sample tubes overnight at 37  C using a tube
rotator or shaker.
3. Purify plasmid DNAs by miniprep.

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Soh Ishiguro and Nozomu Yachie

4. Validate the inserts by Sanger sequencing. The sequencing
primer sequence is provided in Subheading 2.3.
5. Store the validated plasmid DNAs at 20  C.
3.3

Culturing of Cells

3.3.1 Collagen Coatingof Cell Culture Plates

Here we describe the culturing of HEK293T cells that are used forthe genome-editing assay (described in Subheading 3.4).
HEK293T cells are expanded and seeded into the wells of 96-well
cell culture plates. One and a half 96-well cell culture plates are
required for each genome-editing enzyme (or control enzyme) toperform 48 genome-editing assays with unique gRNAs in
triplicates.
1. Prepare a 20-mL collagen-I coating solution in a disposable
reagent reservoir.
2. Apply 50 μL of collagen-I coating solution into each well offresh 96-well cell culture plates using a 12-channel pipette.
3. Incubate 30 min in a cell culture incubator at 37  C and 0.05%
CO2.
4. Remove the solution from the cell culture plates using a
12-channel pipette.
5. Add 50 μL 1 PBS to each well of the cell culture plates towash the collagen-I coating solution and remove the reagent
using a 12-channel pipette.
6. Proceed to the next step, or the collagen-coated 96-well cell
culture plates can be stored for at least 1 week at room temperature, wrapped with aluminum foil.

3.3.2 Seeding Cells into
96-Well Collagen-Coated
Cell Culture Plates

1. Place a DMEM bottle in a 37  C water bath to warm up.
2. Thaw 2-mL HEK293T cell stock vials in a 37  C water bath.
Make sure to have enough cells for a target screening space.
3. Transfer the cells from each stock vial to 5 mL of the prewarmed DMEM in a 15-mL sample tube and centrifuge at
200  g for 5 min.
4. Aspirate and remove the supernatant.
5. Add the prewarmed DMEM, 10 mL each, into the sample
tubes.
6. Gently mix the cells by pipetting several times.
7. Transfer the cells from each sample to a 10-cm cell culture dish.
8. Incubate the cell culture dishes for 2–3 days in a cell culture
incubator at 37  C and 0.05% CO2 to allow cell proliferation to
80–90% confluency (see Note 3).
9. Remove the medium using an aspirator.

High-Throughput Analysis of CRISPR Genome Editing Outcomes

211

10. Apply 1 PBS, 1000 μL each, to the cell culture dishes.
11. Remove the reagent from the cell culture dishes using anaspirator.
12. Apply 0.05% trypsin-EDTA, 1000 μL, each to the cell culture
dishes.
13. Incubate the cell culture dishes for 4 min in a cell culture
incubator at 37  C and 0.05% CO2.
14. Apply DMEM, 1000 μL each, to the cell culture dishes andslowly mix by pipetting to obtain a cell suspension.
15. Transfer the cells from each cell culture dish into a 15-mL tube
containing 5 mL of DMEM.
16. Quantify cell concentrations using a cell counter and adjust to
25 cells/μL using DMEM.
17. Transfer the cells from the sample tubes into a sterile disposable
reagent reservoir.
18. Transfer cell sample, 200 μL each (5000 cells), to the collagencoated 96-well cell culture plate wells using a 12-channel
pipette. For each of the genome-editing or control enzymes,
three four-row units (i.e., three units of rows A–D or E–H of
96-well cell culture plates) are required to allow genome-editing assays with 48 gRNAs in triplicates.
19. Incubate the assay plates for 12–15 h in a cell culture incubator
at 37  C and 0.05% CO2 and immediately proceed with thenext step (see Note 7).
3.4 Massively
Parallel Transfectionof Genome-Editing
Reagents

Here we describe massively parallel transfections of genome-editing
reagents to HEK293T cells in the 96-well plates prepared as
described in Subheading 3.3 with an efficient strategy for assembling different gRNAs and different genome-editing enzymes
using a 12-channel pipette (Fig. 6). Each reaction transfects
120 ng of a genome-editing (or control) enzyme expression plasmid and 40 ng of a gRNA expression plasmid to the cells using PEI
Max transfection reagent. After transfection, the cells are incubatedfor 3 days to induce genome editing. The assay conditions mayneed to be modified for other cell lines by changing input plasmid
amounts, transfection reagent, cell density, and/or incubationtime.

3.4.1 gRNA Reagent
Preparation

1. Measure the DNA concentration of each gRNA expression
plasmid using a spectrophotometer immediately before thetransfection assay.
2. Adjust concentrations of gRNA plasmids to 25 ng/μL using
ultrapure distilled water and assemble, 100 μL each, in a fresh
96-well PCR plate. Fill rows A–D of the PCR plate for212

Soh Ishiguro and Nozomu Yachie

Fig. 6 Assembly and transfection of genome editing reagents for each enzyme

48 gRNAs, so that the well positions are in concordance withthe well positions of the target amplification primers in the
curated primer mix plate.
3. Seal the gRNA library plate with an optically clear adhesive
PCR film.
4. Centrifuge the gRNA library plate at 1000  g for 2 min. The
plate can be stored long term at 20  C.
3.4.2 Transfection

1. Prepare genome-editing enzyme plasmids and a control
enzyme plasmid by midiprep.
2. Measure and adjust the DNA concentrations of each enzyme
plasmid to 500 ng/μL (see Note 8).
3. To perform genome-editing assays for 48 gRNAs in triplicates,
prepare 200 enzyme mix in a 15-mL sample tube for each of
the genome-editing and control enzymes. Note that a sufficient excess of the reagent mix is necessary for the following
two layers of aliquoting procedures.
1 reaction unit
Component

Volume (μL)

500 ng/μL genome-editing or control plasmid

0.24

25 ng/μL gRNA plasmid

1.6

1 mg/mL PEI Max

0.48

1 PBS

50.0

Total

52.32

High-Throughput Analysis of CRISPR Genome Editing Outcomes

213

200 reaction mix
Component

Volume (μL)

500 ng/μL genome-editing or control plasmid

48

1 mg/mL PEI max

320

1 PBS

10,000Total

10,368

4. Mix the enzyme mixes by vortexing.
5. For each genome-editing (or control) enzyme, prepare a fresh
96-well round-bottom deep-well plate as the transfection mix
plate and aliquot 800 μL of the enzyme mix to every well in row
H (H1–H12).
6. Take gRNA expression plasmids, 5.5 μL each, from the gRNAlibrary plate (48 samples) and apply to the corresponding positions of the transfection mix plates (rows A–D) for eachgenome-editing (or control) enzyme using a 12-channel pipette.
7. For each genome-editing (or control) enzyme, using a
12-channel pipette, take 180 μL of the enzyme mix from every
well of row H in the transfection mix plate and apply it to row Aof the same transfection mix plate and mix well by pipetting.
Repeat the same procedure for destination rows B–D.
8. Seal the transfection mix plates with optically clear adhesive
PCR films and incubate for 15 min at room temperature.
9. Retrieve the assay plates prepared in Subheading 3.3.2 from thecell culture incubator.
10. Apply transfection mixes, 52.32 μL each, to the designatedwells of the assay plates using a 12-channel pipette. Note thateach genome-editing (or control) enzyme assay requires threefour-row units of the assay plates for genome-editing assays
with 48 unique gRNAs in triplicates (i.e., a four-row unit of atransfection mix is stamped three times to one and a half
96-well assay plates).
11. Incubate the assay plates for 72 h in a cell culture incubator at
37  C and 0.05% CO2.
3.5 Template
Genomic DNA
Preparation

Here we describe genomic DNA template preparation for theamplicon sequencing procedure. The genome-editing treated cellsamples in the 96-well assay plates are lysed with NaOH andboiling. and neutralized by Tris–HCl. This simple cell lysis is sufficient for the downstream amplicon sequencing library preparation
procedure. Unlike column-based genomic DNA extraction protocols, this protocol processes one assay sample plate (96 samples) in
20 min with minimal hands-on time and also can be implemented
for use with robotic liquid handling systems for further scalability.

214

Soh Ishiguro and Nozomu Yachie

1. Remove cell culture medium (about 250 μL each) from theassay plates using a 12-channel pipette (see Note 9).
2. Put 20 mL of freshly prepared 50 mM NaOH in a disposable
reagent reservoir.
3. Using a 12-channel pipette, transfer 50 μL of the NaOH
solution to every sample well of the assay plates.
4. Using a 12-channel pipette, carefully and gently transfer theentire lysed cell samples to fresh 96-well PCR plates. The celllysate will be sticky. Slowly pipet the samples.
5. Seal the sample PCR plates with optically clear adhesive PCRfilms.
6. Centrifuge the sample PCR plates at 1050  g for 2 min.
7. Incubate the sample PCR plates at 95  C for 15 min, then coolto 4  C using a thermal cycler.
8. Centrifuge the sample PCR plates at 1050  g for 2 min.
9. Prepare 10 mL of 1 M Tris–HCl (pH 8.0) in a disposable
reagent reservoir.
10. Remove the adhesive PCR films and carefully add 5 μL of theTris–HCl solution into every sample well of the sample PCR
plates for neutralization using a 12-channel pipette. The Tris–
HCl volume should be 10% of the input NaOH volume.
11. Seal the sample PCR plates again with optically clear adhesive
PCR films (see Note 10).
12. Centrifuge the sample PCR plates at 1050  g for 2 min to spin
down the remaining cell lysates on well walls and to remove air
bubbles.
13. Proceed to the next step, or the cell lysate plates can be store at20  C for a few months or 80  C for a long time.
3.6 Generation of aMultiplexed Amplicon
Sequencing Library
and Sequencing

In this final section, we describe parallel amplification of genomeediting target sites, pooling and indexing of the PCR products foreach genome-editing (or control) enzyme, and generation of amultiplexed amplicon sequencing library with quality controls forone-shot Illumina sequencing to evaluate hundreds to thousands ofgenome-editing samples. Target genomic regions of genomeedited samples are PCR amplified using the corresponding primers
screened in Subheading 3.1. Then, PCR products of similar sizes
are all pooled for each genome-editing enzyme of each assay replicate and subjected to magnetic bead-based purification. The
pooled PCR samples are reamplified by a second-round PCR to
add Illumina sequencing adapters with unique indices. The indexedsequencing libraries are separately size-selected by agarose gel electrophoresis and quantified by qPCR (Fig. 7). Equal molar quantities of the indexed libraries are further pooled as a single
multiplexed library and analyzed using an Illumina sequencer.

High-Throughput Analysis of CRISPR Genome Editing Outcomes
P5 index

P5

P7 index

PS1.0

Target region

PS2.0

Read1

Lib 1

Lib 2

215

P7
Read2

Lib 3

Lib 4

Lib 5

Lib 6

Lib 7

Lib 8

Lib 9

500bp
300bp

Fig. 7 Preparation of amplicon sequencing libraries. The first-round PCR product pools of different assay
enzymes of different replicates are reamplified by a second-round PCR. The PCR products are then sizeselected by agarose gel electrophoresis. The yellow boxes show the typical area for the size selection

After sequencing, sequencing reads can be demultiplexed into
individual genome-editing assays according to their custom indicesand endogenous target sequences. Sequencing reads of each assay
sample can be used for mutation outcome pattern analyses.
3.6.1 PCR Amplificationof the Target Regions

1. Thaw the curated primer mix plates (48 primer pairs) established in Subheading 3.1 at room temperature and centrifuge
at 1000  g for 2 min.
2. Retrieve the cell lysate plates established in Subheading 3.5(thaw if frozen) and centrifuge at 1000  g for 2 min.
3. To perform the following target amplification PCRs, prepare180 PCR master mix for each genome-editing (or control)
enzyme for a total of triplicate samples (scale the master mix
volume with the number of genome-editing and control
enzymes used).
1 reaction unit
Component

Final concentration

Volume (μL)

5 Phusion HF buffer

1

4

2 mM dNTPs

200 μM2

10 μM primer mix

500 nM each2

DNA template (cell lysate)

2

Phusion DNA polymerase

0.2

Ultrapure distilled water

9.8

Total

20

216

Soh Ishiguro and Nozomu Yachie

180 PCR master mix
Component

Volume (μL)

5 Phusion HF buffer

480

2 mM dNTPs

240

Phusion DNA polymerase

24

Ultrapure distilled water

1764

Total

2508

4. Transfer the PCR reaction mix into a disposable reagent reservoir placed on ice.
5. Prepare the same number of fresh 96-well PCR plates as theassay plates prepared in Subheading 3.3.2. Label the PCR
plates to correspond to the assay plates. Using a 12-channel
pipette, transfer the PCR master mix, 17 μL each, from thereservoir to all the wells of the PCR plates.
6. Using a 12-channel pipette, take 2 μL each from the four-row
unit of the curated primer mix plate (rows A–D) and apply
them to the top four-row unit (rows A–D) of the first PCR
plate. Repeat the same procedure until both of the top andbottom four-row units of the all the PCR plates are filled up.
7. Using a 12-channel pipette, take 2 μL each from the cell lysate
plates and apply them to the corresponding well positions of
the PCR plates. Gently mix the samples by pipetting.
8. Seal the PCR plates with optically clear adhesive PCR films.
9. Centrifuge the PCR plates at 1000  g for 2 min.
10. Perform the first-round target amplification PCR with thefollowing thermal cycle conditions.
Step

Temperature

Time

1

98  C0’30

298  C

0’10

3



60 C

0’10

4



1’00

72 C

Go to 2(29 times)


5

72 C

6



4 C

5’00
1

11. Centrifuge the first-round PCR sample plates at 1000  g for
2 min.

High-Throughput Analysis of CRISPR Genome Editing Outcomes217

12. Proceed to the bead-based DNA purification step, or store the
PCR sample plates at 20  C.
3.6.2 Purificationof the First PCR Products

1. Retrieve AMPure XP beads from a fridge, settle to room temperature, and vortex thoroughly before use (see Note 11).
2. Centrifuge the first-round PCR sample plates at 1000  g for
2 min.
3. Gently remove the plate seals.
4. For each genome-editing (or control) enzyme of each assay
replicate, pool and combine the first-round PCR products,
3 μL each, in a 1.5-mL tube. This yields 144-μL PCR sample
pools each containing amplicon products for 48 gRNA targeting regions.
5. Add 259.2 μL of AMPure XP beads (1.8 volume) to eachfirst-round PCR sample pool and thoroughly mix by pipetting
up and down several times without making bubbles.
6. Incubate the sample tubes at room temperature for 5 min toallow PCR products to bind to the beads.
7. Place the sample tubes on a magnetic stand and wait for 1 min
to allow magnetic separation of beads.
8. Without removing the sample tubes from the magnetic stand,carefully remove all of the clear supernatants.
9. Add 500 μL of 70% EtOH to each sample tube on the magnetic
stand and wait for 30 s.
10. Without removing the sample tubes from the magnetic stand,carefully remove all of the clear supernatants.
11. Repeat steps 9 and 10 one more time. Make sure no EtOH
remains in the sample tubes.
12. Remove the sample tubes from the magnetic stand and airdry
for 1–3 min.
13. Apply 50 μL of ultrapure distilled water and gently mix withthe magnetic beads by pipetting.
14. Incubate the sample tubes at room temperature for 2 min toallow PCR products to elute in the water solvent.
15. Place the sample tubes on the magnetic stand and wait for
2 min to allow magnetic separation of beads.
16. Without removing the samples tubes from the magnetic stand,transfer the clear supernatants each to a fresh 1.5-mL tube.
17. Measure DNA concentrations using a spectrophotometer.
18. Adjust sample concentrations to 10 ng/μL with ultrapure
distilled water.

218

Soh Ishiguro and Nozomu Yachie

19. Proceed with the next pooled indexing PCRs, or store the firstround PCR pool samples at 20  C.
3.6.3 Pooled Indexing
PCR for Multiplexed
Sequencing

1. Vortex the first-round PCR pool samples and spin down wellusing a benchtop centrifuge.
2. To perform the following second-round indexing PCR for eachpooled sample, prepare 1.1 second-round PCR master mix.
Scale up the volume of the master mix with the number of thefirst-round PCR pool samples (the number of genome-editingor control enzymes multiplied by three for assay replicates).
1 reaction unit
Component

Final concentration

Volume (μL)

5 Phusion HF buffer

1

8

2 mM dNTPs

200 μM

4

10 μM P5 index primer

500 nM2

10 μM P7 index primer

500 nM

2

First-round PCR pool

1

Phusion DNA polymerase

0.4

Ultrapure distilled water

22.6

Total

40

1.1 master mix
Component

Volume (μL)

5 Phusion HF buffer

8.8

2 mM dNTPs

4.4

Phusion DNA polymerase

0.44

Ultrapure distilled water

24.86

Total

38.5

3. Aliquot the PCR master mix, 35 μL each, to individually
capped eight-strip PCR tubes.
4. Apply unique combinations of custom Illumina P5 and P7
index primers to the PCR reaction tubes. See Subheading 2.7
for the design of the custom Illumina index primers.
5. Apply the first-round PCR sample pools 1 μL each to the PCR
reaction tubes and mix gently by pipetting. Make sure uniqueindex pairs are assigned to the first-round PCR sample pools.
6. Spin down the PCR reaction tubes using a benchtop
centrifuge.

High-Throughput Analysis of CRISPR Genome Editing Outcomes219

7. Perform PCR with the following thermal cycle conditions.
Step

Temperature

Time

1

98  C

0’30

2



98 C

0’10

3



60 C

0’10

4

72  C

2’00

Go to 2(14 times)


5

72 C

6



4 C

5’00
1

8. Perform gel electrophoresis analysis with 2% agarose gel tocheck the rough yields and size distributions of the reamplified
PCR products.
9. For each reamplified PCR product, cut the gel to obtain the
PCR product in the expected size range (Fig. 7) and purify the
amplicon sequencing libraries using a PCR purification kit (seeNote 12).
10. Elute the size-selected samples into 20 μL of ultrapure distilledwater and collect the eluate in a 1.5-mL LoBind DNA tube.
11. Check the concentration for each amplicon sequencing libraryusing a spectrophotometer (see Note 13).
12. Proceed to the next step below, or store the amplicon sequencing libraries at 20  C.
3.6.4 Multiplexed
Amplicon Sequencing

1. Retrieve the amplicon sequencing libraries (and thaw iffrozen).
2. Vortex the sample tubes well and spin down using a benchtop
centrifuge.
3. Using KAPA Library Quantification Kits (Illumina), quantify
the absolute concentrations of the amplicon sequencing
libraries according to the manufacturer’s instruction (seeNote 14).
4. Pool amplicon sequencing libraries in a 1.5-mL LoBind DNA
tube so that each library is represented equally.
5. Using KAPA Library Quantification Kits (Illumina), quantify
the absolute concentration of the multiplexed library according
to the manufacturer’s instruction.
6. Analyze the multiplexed amplicon sequencing library with 20%
PhiX spike-in using an Illumina sequencer (see Note 15). The
library can be stored long term at 20  C (see Note 16).

220

4

Soh Ishiguro and Nozomu Yachie

Notes
1. We used pSI-356 v2 in our previous studies [4, 35]. pSI-356 v2
encodes the human U6 promoter and 2-kb filler sequence
sandwiched by two BsmBI cloning sites followed by thegRNA scaffold sequence. The filler DNA fragment is replacedwith a target spacer-encoding fragment to obtain a gRNA
expression plasmid.
2. Prepare a fresh NaOH solution immediately before the experiment every time.
3. For better transfection efficiency, use low passage HEK293T
cells that have undergone no more than 10 passages since theirestablishment.
4. PCR efficiency might be affected by the amount of cell lysate.
The template cell lysate concentration may need to be
optimized.
5. To make the gRNA construction easy with a 12-channel
pipette, order forward and reverse ssDNA oligos in the
96-well format. Obtain corresponding forward and reverse
ssDNA oligos in the same positions of two separate plates. We
also recommend obtaining the ssDNA oligos dissolved in
100 μM water or TE instead of a dry shipping option that
requires the additional steps of dissolving and normalizing
the ssDNA oligo samples.
6. Two to three colonies are usually sufficient to obtain a correct
plasmid clone.
7. Before proceeding to the next step, use a microscope to confirm that the seeded cells are uniformly distributed in the welland adhered to the flat well bottom.
8. Midiprep from a 150–200 mL bacterial culture with 12–15 hincubation at 37  C should be sufficient to obtain a genomeediting enzyme expression plasmid for transfection assays with
48 gRNAs in triplicates several times. Note that some genomeediting enzyme expression plasmids are toxic for bacterial
growth and may need further optimization in this step.
9. The experiment can be stopped here. After completely removing the culture medium, the assay plates can be stored at20  C.
10. Cell lysate samples are sticky. PCR films need to be removed
gently to avoid cross-well sample contaminations.
11. AMPure XP beads are sufficient to remove residual primers andprimer dimers from the first-round PCR products. We recommend size-selection using agarose gel electrophoresis whenstrong nonspecific band signals are observed in a gel. The

High-Throughput Analysis of CRISPR Genome Editing Outcomes221

purity of a first-round PCR product greatly affects the result of
the downstream second-round PCR reamplification with custom Illumina index adapters.
12. The second-round PCR products usually show smear bands ona gel in a range of 50 bp from the expected product size.
According to our amplicon sequencing data, we assume thismay be caused by indel mutations induced by genome editing.
We recommend to size-select a wider area of the PCR bands
than what is for usual for molecular cloning.
13. If the measured concentration is more than 10 ng/μL by
spectrophotometry, adjust the sample concentration to
5–10 ng/μL with ultrapure distilled water so that the sample
quantity is within the range of the of standards in thedownstream qPCR.
14. Because the size distribution of the amplicon sequencing
library can be expected by design, we prefer to use qPCR for
library quantification and normalization rather than otherquantification methods, such as TapeStation or BioAnalyzer.
15. The amplicon sequencing library should be sequenced (pairedend) with 20%–30% PhiX spike-in because the library complexity for each sequencing cycle is relatively low, and this is known
to restrict sequencing cluster identification, at least slightly, inan Illumina sequencer. From 20,000 to 50,000 sequencing
reads for each genome-editing assay is usually sufficient for
further data analyses. We usually choose MiSeq Micro (total
output five million reads) for less than 200 genome editing
assays or MiSeq v3 (total output 25 million reads) for larger
assays.
16. DNA binds to the tube wall even if LoBind tubes are used. The
sequencing library should be quantified by qPCR no morethan 2–3 days before the sequencing run.

Acknowledgments
We thank Hideto Mori for optimizing the protocol in concordancewith data analysis pipelines and Mamoru Tanaka for optimizing thedetailed experimental steps. This study was funded by the Japan
Agency for Medical Research and Development (AMED) Platform
Project for Supporting Drug Discovery and Life Science Research(to N.Y.), the New Energy and Industrial Technology Development Organization (NEDO) and AMED PRIME program(17gm6110007) to N.Y., and the Japan Society for the Promotionof Science (JSPS) Grant-in-Aid for Scientific Research (16 J06287)
to S.I.

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Soh Ishiguro and Nozomu Yachie

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