Molecular Biotechnology (2021) 63:1–12

https://doi.org/10.1007/s12033-020-00280-w

ORIGINAL PAPER

A Novel White‑to‑Blue Colony Formation Assay to Select for Optimized

sgRNAs
Chaogang Wei1 · Tong Chen1 · Yueyue Zhang1 · Yanfeng Wang1 · Dai Shi1 · Zhen Jiang1 · Kai Li2 · Li Xiao2 ·
Junkang Shen1 

Accepted: 7 October 2020 / Published online: 12 October 2020

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
CRISPR/Cas9-mediated genome editing technology consists of a single-guide RNA (sgRNA), and the Cas9 endonuclease
has the potential to treat genetic diseases in most tissues and organisms. In this system, the Cas9 protein can be directed to
target genomic DNA sequences as “molecular scissors” with the guidance of sgRNAs. However, the target-specific activities
of different sgRNAs are highly variable; thus, it is crucial to search for a simple, quick and economical method to screen
for optimized sgRNAs with high target specificity. We have adopted and verified a newly developed white-to-blue colony
formation assay to quickly screen for sgRNAs optimized for the EphA2 gene, which is highly expressed in hormone-resistant
prostate cancer (PC-3) cells. This assay promises to screen for optimized sgRNAs more simply, rapidly, and efficiently.
Our results suggest that the white-to-blue colony formation assay might be a useful screening strategy to quickly select for
optimized sgRNAs.

Keywords  CRISPR/Cas9 · Blue/white · Optimized sgRNA · Gene editing · EphA2

Introduction the CRISPR-associated (Cas) protein is necessary in this

The clustered regularly interspaced short palindromic repeat
(CRISPR)/Cas-mediated genome editing system is a micro-
bial immune system that applies RNA-guided nucleases to
cleave exogenous DNA. Three types of CRISPR systems
have been identified from bacterial and archaeal hosts, and
Chaogang Wei and Tong Chen have contributed equally to the
work.
Li Xiao and Junkang Shen are co-corresponding authors.
system. The type II CRISPR/Cas system, consisting of a
single-guide RNA (sgRNA) and Cas9 protein, has been
widely used in biomedical research [1–3]. SgRNA, which is
fused together by CRISPR RNA (crRNA) and transactivat-
ing crRNA (tra-crRNA), can direct the Cas9 endonuclease
to cleave specific target DNA sites [4]. The Cas9 protein
can be guided to modify and cleave target DNA sequences
by mainly relying upon sgRNA sequences with base-pair
complementarity to the DNA sequences, and the target
specificity is mainly determined by sgRNA sequences [5].

* Li Xiao Dai Shi

lixiao0626@sina.com shidai318@163.com
* Junkang Shen Zhen Jiang
shenjunkang@suda.edu.cn j102@163.com
Chaogang Wei Kai Li
weichaogang1122@163.com kaili34@yahoo.com
Tong Chen 1
Department of Radiology, The Second Affiliated
chentong1120@163.com
Hospital of Soochow University, 1055 Sanxiang Road,
Yueyue Zhang Suzhou 215004, Jiangsu, People’s Republic of China
yxyxzyy@126.com 2
Center of Laboratory, The Second Affiliated Hospital
Yanfeng Wang of Soochow University, 1055 Sanxiang Road,
826780491@qq.com Suzhou 215004, Jiangsu, People’s Republic of China

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2 Molecular Biotechnology (2021) 63:1–12
Notably, target DNA sites must contain related sequences
complementary to the 5′-end of the sgRNA and PAM (proto-
spacer adjacent motif) sequences, which are essential for
the modification of the CRISPR/Cas9 system. Without the
PAM sequence at the 3′-end of the target DNA sequence,
Cas9 will not perform its cleavage function, although there
is a complete match between the target DNA sequence and
the sgRNA sequence [6]. Previous studies have confirmed
that the genomic editing efficiency of the 5′-NGG-3′ PAM
sequence is higher than that of the 5′-NAG-3′ or 5′-NGA-3′
PAM sequence [7].
Although sgRNA is easily prepared, and PAM has a high
incidence in genomes, the CRISPR/Cas9 system can have
severe off-target effects, whereby almost all genetic loci
can be readily modified, producing gene editing effects on
nontargets. The target-specific activity of different designed
sgRNAs is highly variable, which means that the editing
efficiency of the CRISPR/Cas9 system is not stable [8].
Optimization of sgRNA is a feasible solution to address
this problem. At present, many technologies and methods
have been applied to assess the target specificity of sgRNAs,
including HTS (high-throughput sequencing), GUIDE-seq
(genome-wide unbiased identification of DSBs enabled by
sequencing), Digenome-Seq (digested genome sequencing),
T7E1 endonuclease assays, Surveyor endonuclease assays,
Luciferase reporter assays and fluorescent polymerase chain
reaction (PCR)-based methods [9–14]. However, these
approaches may be complicated, costly, time consuming
and difficult to manipulate due to the involved equipment
and technologies. In the present study, we provide a novel
screening assay based on white-blue screening in E. coli
to screen an optimized sgRNA more simply, rapidly and
efficiently.
Prostate cancer is one of the most common malignant
tumours in elderly men in developed countries. After a
period of endocrine therapy, hormone-sensitive prostate
cancer becomes hormone-resistant, for which there is no
effective treatment [15]. Eph (erythropoietin-producing
hepatocellular) receptors are the largest subfamily of recep-
tor tyrosine kinases, which are classified into two subfami-
lies, EphA and EphB, according to their sequence homolo-
gies and binding affinity for their ligands, Ephrins [16, 17].
Studies have demonstrated that Eph receptor A2 (EphA2)
is highly expressed in human hormone-resistant prostate
cancer cells [18]. Overexpression of EphA2 is involved in
tumour progression as well as the correlation between patho-
logical grading and clinical staging [19]. Previous studies
have confirmed that the level of EphA2 mRNA and protein
increases significantly in human PC-3 and DU-145 cells
(hormone-resistant prostate cancer cells) compared with that
in human LNCaP cells (hormone-sensitive prostate cancer
cells), suggesting that EphA2 participates in the change in
prostate cancer from hormone sensitive to hormone resistant
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[20]. Hence, regulation of the EphA2 gene may reveal poten-
tial options for the treatment of hormone-resistant prostate
cancer. In the present study, we successfully designed five
relative sgRNAs according to the EphA2 gene and screened
for the optimized sgRNA among them for further research
in the CRISPR/Cas9 system. In addition, we used the track-
ing of indels by decomposition method (TIDE, https​://tide.
deskge​ n.com/) to quantify the editing efficiency and identify
the predominant types of insertions and deletions (indels)
in the DNA of PC-3 cells [21]. This method enables direct
comparison of the effectiveness of sgRNAs.
Materials and Methods
Design of Relative sgRNA Sequences
Relative sgRNA sequences were intelligently designed
with online design software (Optimized CRISPR Design
(https​://crisp​r.mit.edu) and DNA2.0 gRNA Design Tool
(https​://www.dna20​.com)) according to the EphA2 gene
DNA sequences (GenBank: NC_000001.11). Five sgRNA
sequences were successfully selected based on the above
software, as shown in Table 1. Each oligonucleotide was
biologically synthesized according to the sgRNA sequences.
Reagents
The pCas9 and pMD-19T plasmids were used in the
present study. The pCas9 plasmid was purchased from
Addgene (#42876), while the pMD-19T plasmid was
purchased from TaKaRa Biotechnology (Dalian, China).
An RFP (red fluorescent protein)-marked pX330 plasmid
was supplied by Professor Xiao Li (Center of Laboratory,
The Second Affiliated Hospital of Soochow University).
As shown in Tables 2 and 3, the oligonucleotides were
synthesized by Sangon Biotechnology (Shanghai, China).
BsaI, BbsI, KpnI and HindIII were all purchased from
New England Biolabs (Massachusetts, USA). T4 DNA
ligase was provided by Thermo Scientific Inc. (Waltham,
USA). The Universal DNA Purification Kit, TIANprep
Mini Plasmid Kit and TIANamp Genomic DNA Kit were
Table 1  Five selected gRNA sequences in the EphA2 gene
EphA2 Gene Sites Corresponding sgRNA Sequences PAM
EphA2-1 GCG​CCT​GCT​TCG​CCC​TGC​TG TGG​
EphA2-2 GCC​TGC​TTC​GCC​CTG​CTG​TG GGG​
EphA2-3 CTC​CTT​GAC​GCG​CGC​AAC​TT TGG​
EphA2-4 TTC​CAA​AGT​TGC​GCG​CGT​CA AGG​
EphA2-5 CTT​TCC​CCC​TGC​ATC​CCA​TG GGG​
Bold bases in each sequence represent PAM sequences
Molecular Biotechnology (2021) 63:1–12 3
Table 2  The sequences of the oligos used for the pCas9 plasmid
Oligos Corresponding Sequences (5′–3′)
gRNA-1F AAA CGC GCC TGC TTC GCC CTG CTG G
gRNA-1R AAA ACC AGC AGG GCG AAG CAG GCG C
gRNA-2F AAA CGC CTG CTT CGC CCT GCT GTG G
gRNA-2R AAA ACC ACA GCA GGG CGA AGC AGG C
gRNA-3F AAA CCT CCT TGA CGC GCG CAA CTT G
gRNA-3R AAA ACA AGT TGC GCG CGT CAA GGA G
gRNA-4F AAA CTT CCA AAG TTG CGC GCG TCA G
gRNA-4R AAA ACT GAC GCG CGC AAC TTT GGA A
gRNA-5F AAA CCT TTC CCC CTG CAT CCC ATG G
gRNA-5R AAA ACC ATG GGA TGC AGG GGG AAA G
purchased from TIANGEN BIOTECH (Beijing, China).
DH5α competent cells were also provided by TIANGEN
BIOTECH (Beijing, China).
Establishment of Recombinant pCas9‑sgRNA
Plasmids
pCas9 was enzymatically digested by the BsaI restriction
endonuclease, and a relative sticky end was generated fol-
lowing enzyme digestion. Additionally, five synthesized
oligonucleotides of sgRNA were annealed according to
the following conditions: the samples were incubated for
5 min at 95 °C and 10 min at 70 °C, and the temperature
was slowly decreased to 37 °C. The ligation product was
formed by connecting the pCas9 plasmid and the anneal-
ing product of sgRNA under the catalysis of T4 DNA
ligase. Ten microlitres of ligation mixture was added to
50 μL of DH5α competent cells for transformation on an
LB plate with chloromycetin for selection; the plates were
incubated for 12–16 h at 37 ℃. Following the selection
of a single clone, the recombinant pCas9-sgRNA plasmid
was confirmed by DNA sequencing analysis.
Table 3  Five target sequences Oligos
of the oligos used for the PMD-
19T plasmid EphA2-1F
EphA2-1R
EphA2-2F
EphA2-2R
EphA2-3F
EphA2-3R
EphA2-4F
EphA2-4R
EphA2-5F
EphA2-5R
Construction of the pMD‑19T‑Modified Plasmid
with the EphA2 Gene
The pMD-19T plasmid was enzymatically digested by the
KpnI and HindIII endonuclease enzymes. Meanwhile, five
oligonucleotides of the EphA2 gene were annealed accord-
ing to the above conditions, similar to the construction of the
pCas9 plasmid. Annealed target sequences were connected
to the purified plasmid and further transformed on LB plates
with ampicillin for selection for 12–16 h. After selecting a
single clone and extracting the plasmid, the modified pMD-
19T plasmid was verified by DNA sequencing.
Cotransformation of the Two Plasmids and Colony
Analysis
After successful identification by DNA sequencing, equal
quantities of the two plasmids were cotransformed into
DH5α competent cells, and they were further selected on
LB plates containing ampicillin-chloramphenicol-Xgal-
IPTG at 37 °C overnight. Following the cotransformation
of the pCas9 plasmid with sgRNA sequences and the pMD-
19T plasmid with EphA2 target sequences on LB plates, the
on-target editing efficiency of sgRNAs could be evaluated
by the proportion of blue colonies to total colonies. Because
the EphA2 target sequences identified by sgRNAs and dou-
ble-repeat fragments are inserted into the LacZ region of
the pMD-19T plasmid, causing a frame-shift mutation, the
colony appears white. Meanwhile, after the CRISPR/Cas9
system cuts the target sequences, the double-repeat frag-
ments recombine and recover the coding frame, and the
white colony turns to a blue colony. The on-target effect of
sgRNA was evaluated by the proportion of blue colonies to
total colonies. In our study, EphA2-2 and EphA2-5 with the
highest on-target efficiency were selected after blue colony
sequencing verification. EphA2-2 and EphA2-5 sequences
were then recombined into pX330 plasmids (sgRNA-Cas9-
EphA2-2 and sgRNA-Cas9-EphA2-5) for subsequent cell
experiments.
Corresponding Sequences (5′–3′)
CCC ACA GCA GGG CGA AGC AGG CGC GA
AGC TTC GCG CCT GCT TCG CCC TGC TGT GGG GTA C
CCC CCA CAG CAG GGC GAA GCA GGC GA
AGC TTC GCC TGC TTC GCC CTG CTG TGG GGG GTA C
CCC AAA GTT GCG CGC GTC AAG GAG CA
AGC TTG CTC CTT GAC GCG CGC AAC TTT GGG GTA C
CCC TTG ACG CGC GCA ACT TTG GAA CA
AGC TTG TTC CAA AGT TGC GCG CGT CAA GGG GTA C
CCC CCA TGG GAT GCA GGG GGA AAG GA
AGC TTC CTT TCC CCC TGC ATC CCA TGG GGG GTA C
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4 Molecular Biotechnology (2021) 63:1–12
Cell Culture
The human hormone-resistant prostate cancer (PC-3) cell
line was purchased from the Type Culture Collection of the
Chinese Academy of Sciences (Shanghai, China) and pre-
served in the Laboratory of the Second Affiliated Hospital
of Soochow University. Cells were grown in high-glucose
Dulbecco’s modified Eagle’s medium (DMEM) (HyClone,
GE Healthcare Life Sciences, Logan, USA) containing 10%
fetal bovine serum (Gibco, Thermo Fisher Scientific Inc.,
Waltham, USA). Cells were grown in humidified incubators
with 5% ­CO2 and 95% air at 37 °C.
Construction of sgRNA‑Cas9‑EphA2 Cell Lines
Cells in good growth conditions were selected for
transfection after cell thawing and subculture. The
­lipofectamineTM3000 reagent (Thermo Fisher Scientific
Inc., Waltham, USA) was used, and different proportions
of the sgRNA-Cas9-EphA2 recombinant plasmid DNA
(μg) and liposome 3000 (μL) in the range of 1:1.5 to 1:3
were selected for transfection and observed by fluorescence
microscopy (Zeiss Observer A1, Germany) to ensure the
appropriate transfection proportion in subsequent formal
experiments. After determination of the optimal transfection
ratio between the sgRNA-Cas9-EphA2 recombinant plasmid
DNA and liposome 3000, the sgRNA-Cas9-EphA2-2 and
sgRNA-Cas9-EphA2-5 recombinant plasmids were trans-
fected into PC-3 cells. Recombinant plasmids in the control
group (sgRNA-con-EphA2-2 and sgRNA-con-EphA2-5)
were also transfected into PC-3 cells under the same condi-
tions. The transfected PC-3 cells (2.5 × 105 cells/mL) were
then seeded at an initial density in 6-well plates. Forty-eight
to 72 h after cell transfection, the transfection efficiency was
further evaluated by fluorescence microscopy according to
the number of cells with positive RFP expression.
Detection of the Mutation by DNA Sequencing
PC-3 cells carrying the sgRNA-Cas9-EphA2 plasmid were
collected to detect specific double-strand DNA mutations
with a Knockout and Mutation Detection Kit (Shanghai
GeneChem Co., Ltd., Shanghai, China). After 72 h of cell
transfection, genomic DNA was extracted from the sam-
ples using a TIANamp Genomic DNA Kit. The specifically
designed primer sequences were as follows: sgRNA-2 sense,
5′-GGC CCC TTT AAA GAC ATT CC-3′; sgRNA-2 anti-
sense, 5′-GAC ACC AGG TAG GTT CCA AA-3′; sgRNA-5
sense, 5′-CAG ATT CAG TGG CTG TTG ATG A-3′; and
sgRNA-5 antisense, 5′-GAG AGA CAG CTG GAA TCA
CTA T-3′. PCR was carried out according to the manufac-
turer’s protocol using the following cycling conditions: 30
cycles of degradation at 94 °C for 30 s, annealing at 58 °C
13
for 30 s and extension at 72 °C for 30 s. PCR products were
then detected by DNA sequencing to verify the on-target
editing effect.
TIDE Methods
TIDE is web software that can rapidly and reliably assess the
genome editing efficiency of a target locus by CRISPR-Cas9.
It only needs sequence trace data from two standard capillary
(Sanger) sequencing reactions: one control and one edited
sample. We set the indel size = 10 and P threshold ≤ 0.001.
Statistics
All data are presented as the means ± standard deviation.
The statistical analyses were performed using SPSS 19.0
software (IBM Corp., USA). Significant differences between
the groups were analysed using independent sample t-tests.
P < 0.05 was considered to indicate a statistically significant
difference.
Results
Results of the White‑to‑Blue Colony Formation
Assay
pCas9 plasmids independently containing five sgRNA
sequences were successfully constructed (Fig. 1). Three
parallel and independent white-to-blue colony formation
experiments were performed, and the screening results of
the five sgRNAs are shown in Fig. 2. Through three par-
allel and independent experiments, the average proportion
of blue colonies among the five designed sgRNAs (sgRNA
1–5) is listed in Table 4. According to the histogram results
(Fig. 3), the average proportion of blue colonies in sgRNA-2
was the highest and accounted for nearly 34% of the total
colonies, followed by sgRNA-5, with an average percent-
age of approximately 24%. The average proportion of blue
colonies for the remaining three sgRNAs was approximately
14% (sgRNA-3), 15% (sgRNA-1), and 7% (sgRNA-4). The
results show that sgRNA-2 had the highest average propor-
tion of blue colonies, and this proportion was significantly
different from those of the other four sgRNAs (P < 0.05).
Our results indicate that the editing efficiency of the five
sgRNAs is highly variable in the EphA2 gene. The results
confirm that sgRNA-2 and sgRNA-5 can be selected as opti-
mized sgRNAs with high on-target effects. It was further
illustrated that the white-to-blue colony formation assay is
useful for the selection of effective sgRNAs.
Molecular Biotechnology (2021) 63:1–12 5

Fig. 1  DNA sequencing dia-

grams of recombinant pCas9
plasmids independently contain-
ing sgRNA-1 (a), sgRNA-2 (b),
sgRNA-3 (c), sgRNA-4 (d) and
sgRNA-5 (e). The results of
the five sgRNA sequences are
correct after comparison with
the reference sequences. The
red underlined sections refer
to sequences of corresponding
sgRNAs (Colour figure online)

Selection of the Optimized sgRNA with a High Construction of Cell Lines Carrying

on‑Target Effect
DNA sequencing of the white and blue colonies with
sgRNA-2 and sgRNA-5 was performed before subsequent
experiments, as shown in Fig. 4. The white colony was
selected to extract plasmid DNA for sequencing, and the
results are shown in Fig. 4a, c. After the CRISPR/Cas9
system cut the target sequences, the double-repeat frag-
ments recombine and recover the coding frame, turning
the white colony blue. The sequencing results of the blue
colony are shown in Fig. 4b, d. The sequencing results show
that the recombination was successful, suggesting that the
CRISPR/Cas9 system has a target site cleavage effect.
the sgRNA‑Cas9‑EphA2 Plasmid and Fluorescent
Transfection Effect
According to the manufacturer’s protocol, sgRNA-Cas9-
EphA2-2 and sgRNA-Cas9-EphA2-5 recombinant plasmids
were transfected into PC-3 cells with the help of liposome
3000. Appropriate transfection conditions were as follows:
the optimized proportion of the sgRNA-Cas9-EphA2 recom-
binant plasmid DNA (μg) and liposome 3000 (μL) for trans-
fection was 1:1.8 (2.5 μg plasmid DNA and 4.5 μL lipo-
some 3000). The fluorescent results showed that RFP was
expressed in PC-3 cells, which indicates that the sgRNA-
Cas9-EphA2-2 and sgRNA-Cas9-EphA2-5 recombinant

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6 Molecular Biotechnology (2021) 63:1–12

Fig. 2  The screening results of

five sgRNAs by a novel white-
to-blue colony formation assay.
Serial numbers 1–5 represent
sgRNA1-5. The proportion of
blue colonies can be calculated
by the ratio between the number
of blue colonies and the number
of total colonies. (The scale
in the lower right corner of
the image represents the size
relative to a cell culture dish.)
(Colour figure online)

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Molecular Biotechnology (2021) 63:1–12 7

Table 4  The results of blue colonies among the five designed sgR- was 22% (with 8.7% of + 1 insertion and 13.3% of -1 dele-
NAs tion spectra), and the frequency of mutations of sgRNA-
sgRNAs The proportion of blue colo- The average proportion Cas9-EphA2-5 was only 4.5% (+ 5 insertion), as shown
nies (%) of blue ­colonies* (%) in Fig.  7. At the eukaryotic level, the editing efficiency
First Second Third of sgRNA-Cas9-EphA2-2 was nearly quadruple that of
sgRNA-Cas9-EphA2-5.
sgRNA-1 14 13 18 15.00 ± 2.65
sgRNA-2 32 38 33 34.33 ± 3.21
sgRNA-3 15 14 12 13.67 ± 1.53 Discussion
sgRNA-4 10 7 5 7.33 ± 2.52
sgRNA-5 26 21 24 23.67 ± 2.51 In our research, the on-target effect of sgRNA was evaluated
*Mean ± standard deviation by a novel white-to-blue colony formation assay, depend-
ing upon the proportion of blue colonies to total colonies.
The assay is formed by cotransferring the target site and
plasmids transfected with liposome 3000 successfully the CRISPR vector containing the corresponding sgRNA,
entered the cells, as shown in Fig. 5. observing the colour change in the colonies and quantita-
tively analysing the efficiency of the CRISPR/Cas9 system.
Detection of Mutations by DNA Sequencing To reflect the efficiency of the system, vectors containing
and TIDE Methods HBV double-repeat fragments were constructed, and target
sequences were then inserted between the repeated frag-
The sequencing results of genomic DNA of PC-3 cells trans- ments; both tested target sequences and double-repeat frag-
fected with EphA2-2 and EphA2-5 recombinant plasmids ments are located in the LacZ region, causing a frameshift
are shown in Fig. 6. In the experimental group, sgRNA- mutation that makes the colony appear white. However,
Cas9-EphA2-2(A) and sgRNA-Cas9-EphA2-5(C) showed after the CRISPR/Cas9 system cut the target sequences, the
small miscellaneous peaks with irregularity in addition to double-repeat fragments recombine and recover the coding
a single peak, indicating that EphA2-2 and EphA2-5 on- frame, turning the white colony blue. The on-target editing
target site cleavage was induced by the CRISPR system (as effect of the CRISPR/Cas9 system can be verified through
marked in red underline). In the control group, both sgRNA- the colour change in white-to-blue colonies combined with
con-EphA2-2 (B) and sgRNA-con-EphA2-5(D) showed DNA sequencing technology [22–24]. The assay is visual-
a single peak, which proves that no gene editing effect ized and efficient at selecting an optimized sgRNA, largely
occurred. Furthermore, with quantitative analysis of TIDE, due to its incomparable advantages of directly assessing
the total frequency of mutations of sgRNA-Cas9-EphA2-2 changes in the colour of the colonies. By calculating the

Fig. 3  Histogram of the average sgRNA-1

proportion of blue colonies to 40
total colonies among the five sgRNA-2
sgRNAs (Colour figure online)
35 sgRNA-3
The average proportion of blue

sgRNA-4
30 sgRNA-5

25
colonies %

20

15

10

0
sgRNA-1 sgRNA-2 sgRNA-3 sgRNA-4 sgRNA-5

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8 Molecular Biotechnology (2021) 63:1–12
Fig. 4  DNA sequencing diagrams of white-to-blue colonies in
sgRNA-2 (a, b) and sgRNA-5 (c, d). The red underlined sections
refer to sequences of sgRNA-2 and sgRNA-5. In a and c, the white
colony was selected to extract plasmid DNA for sequencing. Homolo-
gous sequences are marked in yellow. After the CRISPR/Cas9 system
number of blue colonies out of the total colonies, the editing
effect of the CRISPR/Cas9 system on the EphA2 gene can
be directly reflected.
In our study, we successfully designed five sgRNAs
associated with the EphA2 gene and utilized a novel
white-to-blue colony formation assay to screen for opti-
mized sgRNAs. This assay confirmed that the editing
efficiencies of the five sgRNAs were highly variable.
The editing efficiency was the highest for sgRNA-2
(34%), and the efficiency was nearly 5 times higher for
sgRNA-2 than for sgRNA-4 (7%), which had the lowest
editing efficiency. This assay suggested that the editing
efficiency of sgRNA is not stable, in accordance with
Lee’s results [25]. Possible reasons for this phenomenon
include the nucleotide composition, genomic context of
the target protospacer DNA sites and sgRNA structure
[26, 27]. In addition, the TIDE method was used in this
13
cuts the target sequences and successfully cleaves the site, the white
colony turns blue, which means that the homologous sequences have
recombined. The sequencing results of the blue colony are shown in b
and d (Colour figure online)
study to quantitatively test and analyse the on-target effi-
ciencies of sgRNA-EphA2-2 and sgRNA-EphA2-5 in
eukaryotic cells. With quantitative analysis of TIDE, the
total frequency of mutations of sgRNA-Cas9-EphA2-2
was 22% (with 8.7% of + 1 insertion and 13.3% of -1 dele-
tion spectra), and the frequency of mutations of sgRNA-
Cas9-EphA2-5 was only 4.5% (+ 5 insertion). Through
the above experiments, the comparison of the gene
editing efficiency of the two targets was basically the
same in bacterial and human cells; that is, the efficiency
of sgRNA-EphA2-2 was higher than that of sgRNA-
EphA2-5. Under these circumstances, it is particularly
important to seek a quick and efficient method for detect-
ing on-target effects of sgRNAs. Currently, screening
optimized sgRNAs mainly involves HTS, GUIDE-seq,
Digenome-Seq, T7E1 endonuclease assays, Surveyor
endonuclease assays, and fluorescent reporter assays
Molecular Biotechnology (2021) 63:1–12 9
Fig. 5  Red fluorescence expression in PC-3-sgRNA-Cas9-EphA2-2
(4A) and PC-3-sgRNA-Cas9-EphA2-5 (4B) cells observed by
fluorescence microscopy. PC-3-sgRNA-Cas9-EphA2-2 and PC-
[9–13]. However, these technologies and methods are
complicated and time consuming. Compared with other
detection methods, the white-to-blue colony formation
assay is easier and requires only two steps, including con-
structing the two types of plasmids and cotransforming
them into cells on an ampicillin-chloramphenicol-Xgal-
IPTG plate to calculate the proportion of blue colonies
out of the total colonies. It has incomparable advantages
with strong operability, shortened experiment cycles, and
direct observation. More importantly, optimized sgRNAs
selected from the assay are verified to be accurate and
reliable by routine detection-mutation DNA sequencing.
Hence, the white-to-blue colony formation assay is a rec-
ommended approach to quickly, conveniently, efficiently
and reliably screen optimized sgRNAs.
In general, the white-to-blue colony formation assay
takes advantage of the colonies’ blue or white colour
changes to intuitively calculate the editing efficiency of
sgRNAs, depending on the proportion of blue colonies to
total colonies. This assay has the potential to be a useful
method for sgRNA screening due to its speed, simplic-
ity and reliability. However, the experiment still presents
some deficiencies. First, we mainly evaluated the advan-
tages and values of the white-to-blue colony formation
assay in terms of the on-target effects of the sgRNA, but
3-sgRNA-Cas9-EphA2-5 cells indicated that the sgRNA-Cas9-
EphA2-2 and sgRNA-Cas9-EphA2-5 recombinant plasmid trans-
fected PC-3 cells (Colour figure online)
the meaningfulness of the assay in the off-target effects
of the sgRNA has not been studied, and these effects
should be explored in further studies. Second, the assay
is only used for screening optimized sgRNAs and can-
not improve the specificity of sgRNAs. In future studies,
we will investigate the possibility of using this assay to
enhance the specificity of sgRNAs.
Conclusion
A novel white-to-blue colony formation assay was used to
screen optimized sgRNAs in the present study. Depend-
ing upon this assay, the on-target effect of sgRNAs was
assessed by the proportion of blue colonies to total col-
onies, and the accuracy was further verified by detect-
ing mutations with DNA sequencing. Compared to other
assays, this assay has many incomparable advantages due
to the visual, sensitive, simple, low-cost and rapid nature
of the screening. The selection of optimized sgRNAs by
the white-to-blue colony formation assay provides great
convenience for further functional cell experiments,
which could be a promising and desirable approach for
the application of the CRISPR-Cas9 system in gene edit-
ing research.
13

10 Molecular Biotechnology (2021) 63:1–12

Fig. 6  Genomic DNA sequencing map after recombinant plasmid and EphA2-5 on-target site cleavage was induced by the CRISPR sys-
transfection. In the experimental group, sgRNA-Cas9-EphA2-2 (a) tem (as marked in red underline). In the control group, both sgRNA-
and sgRNA-Cas9-EphA2-5 (c) showed small miscellaneous peaks con-EphA2-2 (b) and sgRNA-con-EphA2-5 (d) showed a single peak
with irregularity in addition to a single peak, indicating that EphA2-2 (Colour figure online)

13
Molecular Biotechnology (2021) 63:1–12 11

Fig. 7  With quantitative analysis of TIDE, the total frequency of mutations of sgRNA-Cas9-EphA2-2 was 22% (with 8.7% of + 1 insertion and
13.3% of − 1 deletion spectra), and the frequency of mutations of sgRNA-Cas9-EphA2-5 was only 4.5% (+ 5 insertion)

Acknowledgements  The authors were financially supported by the References

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