Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency
Splice donor site sgRNAs enhance CRISPR/ Cas9-mediated knockout efficiency
Ignacio Garc??a-Tu? o?nID 0 3
Vero? nica Alonso-P e?rezID 0 3
Elena VueltaID 1 3
Sandra Pe? rez- Ramos 1 3
Mar??a HerreroID 1 3
Luc??a Me? ndez 1 3
Jes u?s Mar??a Herna? ndez-S a?nchezID 0 3
Marta Mart??n-Izquierdo 0 3
Raquel Salda?a 3
Julia? n SevillaID 3
Ferm??n Sa? nchez- Guijo 0 3
Jes u?s Mar??a Herna? ndez-Rivas 0 3
Manuel Sa? nchez-Mart??nID 1 2 3
? These authors contributed equally to this work. 3
0 Unidad de Diagno ?stico Molecular y Celular del Ca ?ncer, Centro de Investigacio ?n del Ca ?ncer-IBMCC (USAL- CSIC) , Salamanca, Spain, 2 IBSAL , Instituto de Investigacio ?n Biome ?dica de Salamanca , Salamanca , Spain
1 Servicio de Transge ?nesis, Nucleus, Universidad de Salamanca , Salamanca , Spain , 4 Servicio de Hematolog ??a, Hospital de Especialidades de Jerez , Zacatecas , Spain , 5 Hospital Infantil Universitario Nin?o Jesu ?s , Madrid , Spain , 6 Servicio de Hematolog ??a, Hospital Universitario de Salamanca , Salamanca , Spain
2 Departamento de Medicina, Universidad de Salamanca , Salamanca , Spain
3 Editor: Stefan Maas, National Institutes of Health , UNITED STATES
CRISPR/Cas9 allows the generation of knockout cell lines and null zygotes by inducing sitespecific double-stranded breaks. In most cases the DSB is repaired by non-homologous end joining, resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, these mutations do not produce the desired null result in all cases, but instead generate a similar, functionally active protein. This effect could limit the therapeutic efficiency of gene therapy strategies based on abrogating oncogene expression, and therefore needs to be considered carefully. If there is an acceptable degree of efficiency of CRISPR/Cas9 delivery to cells, the key step for success lies in the effectiveness of a specific sgRNA at knocking out the oncogene, when only one sgRNA can be used. This study shows that the null effect could be increased with an sgRNA targeting the splice donor site (SDS) of the chosen exon. Following this strategy, the generation of null alleles would be facilitated in two independent ways: the probability of producing a frameshift mutation and the probability of interrupting the canonical mechanism of pre-mRNA splicing. In these contexts, we propose to improve the loss-of-function yield driving the CRISPR system at the
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Funding: This work was mainly supported by a
grant from the Fondo de Investigaciones Sanitarias
(FIS) of the Spanish Ministry of Economy and
Competitiveness and the European Regional
Development Fund (ERDF) ?Una manera de hacer
Europa? [grant PI17/01895 to IGT and MSM.];
Junta de Castilla y Leo?n, Fondos FEDER
[SA085U16 to JMHR]; Novartis grant; and by the
SDS of critical exons.
With the recent diversification of genome editing tools, including those involving clustered,
regularly interspaced short palindromic repeats and their nuclease-associated protein Cas9
(CRISPR/Cas9), the landscape of suppression techniques has dramatically changed. Although
CRISPR/Cas9 is similar in action and efficacy to protein-based targeted nucleases, such as zinc
finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)[
ease with which these reagents can be designed and tested through the construction of
singleFundacio?n ?Jabones para Daniel?. JM
Herna?ndezSa?nchez was supported by a research grant from
Fundacio?n Espa?ola de Hematolog??a y Hemoterapia
(FEHH). The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
guide RNAs (sgRNAs) has made gene editing available to a wider variety of users and for a
broader range of applications. Unlike ribozymes, antisense oligodeoxynucleotides (AS-ODNs)
and short interfering RNAs (siRNAs), CRISPR/Cas9 works at the DNA level, where it has the
advantage of providing permanent and full gene knockout, while other methods only silence
]. CRISPR/Cas9 cuts DNA in a sequence-specific manner with the
possibility of interrupting coding sequences, thereby making it possible to turn off cancer drivers in
a way that was not previously feasible in humans[
]. This notable application of permanent
gene disruption is based on the cellular mechanisms involved in double-stranded break (DSB)
repair. Nonhomologous DNA end-joining (NHEJ) is the predominant DSB repair pathway
throughout the cell cycle. Following the creation of a DSB within the coding sequence of a
gene, the predominant and error-prone NHEJ pathway often results in small nucleotide
insertions or deletions (indels)[
]. Its great efficiency at inducing DSB has led to CRISPR/Cas9
technology gaining a reputation as the gold standard for creating null alleles in vivo and in
vitro. These null alleles can arise from NHEJ indels that trigger premature stop codons
(frameshift mutation) and/or non-sense-mediated decay in the target gene, resulting in loss of
function. Currently, CRISPR/Cas9 is extensively used to engineer gene knockouts in most
biological systems, but due to the variable size of the NHEJ-induced indel, it is not always
possible to generate a full KO in one step. When the delivery of Cas9 elements is effective, full KO
generation requires off-frame mutations in both alleles, which is a matter of probability since
the random nature of DNA repair gives rise to considerable heterogeneity within the cell. It
entails dealing with a significant frequency of mutated cells in which the outcome of mutation
could preserve the reading frame (i.e., +3 or -3 mutations)[
]. A possible solution is to use two
or more RNA guides to knock out the gene at several key sites in an attempt to guarantee the
null result. However, a high proportion of off-targets would increase with each new sgRNA
added. Conversely, more sgRNAs at the same time trigger more DSBs, which induces a
stronger p53-mediated DNA damage response[
] and more complex rearrangements[
way, these undesirable effects may be irrelevant in assays in which the knockout cell can be
sequenced, selected and expanded, or the null allele of the animal model can be segregated.
Nevertheless, there are other situations, either in vivo or in vitro, in which cell selection and
clone expansion are not available, and achieving high levels of knockout or gene inactivation
efficiency is crucial[
]. Thus, it is important to study the key exons carefully and, more
importantly, the target areas inside them, before making a selection. Hematological cancer
therapies based on specific oncogenic silencing within primitive pluripotent stem cells may be
the best example of these situations. In this pathological cell context, the highly efficient
interruption of the oncogenic open reading frame (ORF) might be an effective therapeutic option.
It would even be more important for those tumors directed by a single oncogenic event, as is
the case for several leukemias or sarcomas, which are directed by specific fusion oncoproteins
]. A recent study of the BCR/ABL oncogene showed this gene fusion to be an ideal target
for CRISPR/Cas9-mediated gene therapy. A CRISPR-Cas9 application truncated the specific
BCR-ABL fusion (p210) abrogating its oncogenic potential, but to achieve in vivo effectiveness
in a xenograft model, the authors had to select and expand the correctly edited cellular clone
because some of the clones contained in-frame or non-synonymous mutations[
Therefore, in these situations, it is essential to have not only highly efficient Cas9-sgRNA cell
delivery, but also a high capacity for generating null mutations. This is especially critical for cancer
oncogene suppression therapies based on disrupting driver oncogenes. If the efficiency of
CRISPR/Cas9 reagent delivery to the cancer cell is acceptable, the key step to success lies in the
effectiveness with which a specific sgRNA can knock out the oncogene. In this way, for most
knockout studies in which the edited cells or mice can be selected, the sgRNA targets different
positions within the chosen exon, avoiding exon boundaries. In most of these cases, the
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sgRNA design follows only off-target criteria, but for cases in which cellular selection is not an
option and only one sgRNA can be used, the null effect could be strengthened with an sgRNA
that targets splice site consensus sequences or close to them. Following this strategy, the
generation of null alleles would be enhanced in two ways: by producing a frameshift mutation and
by breaking the canonical mechanism of pre-mRNA splicing. In this sense, it has long been
known that mutations in splice-site consensus sequences can affect pre-mRNA splicing
patterns and can lead to the generation of null or deficient alleles[
]. In fact, pioneering genetic
studies indicated that many of the thalassemia mutations in the ?-globin gene affect splice sites
and give rise to aberrant splicing patterns[
]. Recent studies have demonstrated that a
splicing mutation in the STAR gene is a loss-of-function mutation that produces an aberrant
protein. Nonsense-mediated mRNA decay (NMD), a conserved biological mechanism
that degrades transcripts containing premature translation termination codons, could help
secure the null effect when a DSB is induced at splice sites. In addition to transcripts derived
from nonsense alleles, the substrates of the NMD pathway include pre-mRNAs that enter the
cytoplasm with their introns intact[
]. Several mutations of splice donor sites that cause loss
of gene function have recently been identified. A novel mutation at a splice donor site that was
predicted to lead to skipping of exon 10 of the PLA2G6 gene was found in a homozygous state
in infantile neuroaxonal dystrophy patients. This variant has been correlated with loss of
function, providing further evidence of its pathogenicity[
]. Mutations in the ectodysplasin A1
gene (EDA-A1) at the splice donor site have been described in patients with hypohydrotic
ectodermal dysplasia. This novel functional skipping-splicing EDA mutation was the cause of
the pathological phenotype[
]. Studies in a family with premature ovarian failure identified a
variant that alters a splice donor site. This variant resulted in a predicted loss of function of the
MCM9 gene, which is involved in homologous recombination and repair of double-stranded
Taking into account all these findings, we decided to explore the effectiveness of driving
one single sgRNA targeting the splice-donor exon site (SDE-sgRNA) to increase the null allele
yield. To compare the knockout efficiency of SDE-sgRNAs and sgRNAs targeting positions
within the exon (IE-sgRNA) we induced DSB with both guides in critical exons in three genes
(TYR, ATM and ABL), two systems (in vivo and in vitro), and two species (human and mouse).
Finally, we sequenced all mutant alleles generated and analyzed the consequences in silico and
The sgRNA guides targeting splice-donor sites of key exons increase the
generation in vitro and in vivo of null alleles in mouse and human cells
In vitro. Two groups of sgRNAs were created to study the efficiency of SDE-sgRNAs and
IE-sgRNAs at generating null alleles in mouse and human cells (Fig 1). All guides were
designed to target the Tyrosinase, and ATM genes in both species in key exons.
Three individual electroporation assays were performed with each sgRNA in Baf/3 mouse
cells and K562 human cells. Mouse Tyr exon 1, mouse ATM exon 10, human Tyr exon1 and
human ATM exon10 sgRNAs (SDE-sgRNA and IE-sgRNA for each one) were cloned in a
CRISPR-Cas9-GFP mammalian expression vector. An empty CRISPR-Cas9-GFP vector was
used as a control. GFP expression was detectable 24 hours post-electroporation in all cases,
indicating the effective delivery of the CRISPR/Cas9 system and its expression in Baf/3 or
K562 cells (Fig 2A). GFP+ cells were sorted and subjected to Sanger sequencing, which
revealed no variations in the target sequence of control cells. Sanger sequencing identified
indel mutations at the predicted cleavage point in CRISPR/Cas9 assays, while no sequence
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Fig 1. Experimental design of genome edition of TYR, ATM and ABL-1 loci by CRISPR/Cas9 system. (A)
Schematic representation of the mouse and human Tyr loci and the CRISPR/Cas9 experimental design of the two RNA
guides are represented in the exon 1 sequence. SDE-sgRNAs match the splice site between exon 1 and intron 1?2.
IEsgRNAs target a central position at the coding sequence of exon 1. (B) Schematic representation of the mouse and
human ATM loci and the CRISPR/Cas9 experimental design the two RNA guides are represented in the exon 10
sequence. SDE-sgRNAs match the splice site between exon 10 and intron 10?11, and IE-sgRNAs target a coding
sequence of exon 10. (C) Schematic representation of the human ABL-1 locus and the CRISPR/Cas9 experimental
design the two RNA guides. SDE-sgRNAs match the splice site between exon 4 and intron 4?5, and IE-sgRNAs target a
coding sequence of exon 6. Sequences of each SDE-sgRNA are represented (blue line) and its expected cleavage point
(blue arrowhead) at the splice donor sequence (red dotted box). Also, several candidates to SDE-sgRNAs are listed
with its respective scores (red box correspond to selected sgRNAs).
variations were observed in control cells (Fig 2B). Tracking of indels by decomposition (TIDE)
analysis showed similar overall DSB-induced efficiency between SDE-sgRNA and IE-sgRNA
in the Baf/3 or K562 cell lines. In knockout assays with both sgRNAs, the TIDE algorithm of
Baf/3 and K562 mutant cells predicted small deletions (1?7 bp) in most cases (Fig 3).
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Fig 2. In vitro CRISPR/Cas9-mediated edition of Tyr and Atm loci in the Baf/3 mouse cell line, and TYR, ATM
and ABL-1 in the K562 human cell line. (A) Fluorescent microscopy of cells electroporated with empty px480 vector
(controls) and carrying each RNA guides. (B) Sequences of CRISPR/Cas9 edited cells through IE-sgRNA (red box) and
SDE-sgRNA (blue box). Edited cells showed a mixture of sequences around the expected cleavage point for each
To eliminate interference in Cas9 delivery efficiency among assays, we decided to analyze
only the mutant alleles generated by every guide and their consequences for the obviation of
wildtype or well-repaired alleles. In order to gain detailed information about all mutant alleles
for each sgRNA we analyzed the genome of properly electroporated Baf/3 or k562 cells by
next-generation sequencing (NGS) (S1?S4 Tables). Unlike with the Sanger analysis, NGS
revealed a high number of mutated alleles in both groups. Several of detected alleles shown
inframe indels that deleted 1?6 amino acids, thereby preserving the reading frame of the protein
(S1?S4 Tables). However, in silico analysis of the allelic modifications generated by
SDEsgRNA predict the generation of a null allele in all cases, by frameshift mutations or by loss of
canonical splicing sequences, or both simultaneously (Fig 4).
In order to evaluate the functionality of the mutant alleles generated by the CRISPR/Cas9
system in the human ATM gene, protein levels in K562-edited cells were analyzed by western
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Fig 3. TIDE decomposition analysis of edited sequences generated in human and mouse cell lines. TIDE
decomposition algorithm prediction of the overall edition efficacy and most common allele variations generated for
IE-sgRNAs (red panels) and for SDE-sg-RNAs (blue panels).
blot (WB). While IE-hATMsgRNA-transfected cells showed slightly weaker ATM expression
compared with K562 parental cells, low levels of ATM protein were detected in
SDEhATMsgRNA-transfected cells (Fig 5A). Single-cell-derived cell lines from both
IEhATMsgRNA (6 clones) and SDE-hATMsgRNA-SD (6 clones) K562 cells were established and
analyzed by NGS (S5 Table). ATM protein levels of each single-cell-derived clone were
analyzed by WB. Most mutated cell clones (4/6) edited with IE-hATMsgRNA showed ATM
expression (Fig 5B). NGS analysis of all single-cell clones edited with IE-hATMsgRNA had at
least one functional allele, either a wildtype (wt) or with in-frame mutations (S5 Table).
However, several mutated cell clones (5/6) edited with SDE-hATMsgRNA had no levels of ATM
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Fig 4. Next Generation Sequencing (NGS) analysis of TYR and ATM genes in mouse and human edited cells.
Graphic representation of the mutations found in edited cells by IE-sgRNA and SDE-sgRNA, and their predicted
effect. Black and gray circles represent null alleles and functional alleles respectively, while the background indicates
the type of mutation (dark blue: splice donor site in-frame and/or frameshift; light blue: frameshift).
protein that could be detected by WB (Fig 5B). Analyzing them showed splicing mutations
together with in-frame or frameshift mutations in both ATM alleles (S5 Table).
In vivo. One-cell stage embryos from two strains of mice, inbred C57Bl6/J and F2 hybrids
of B6/CBA, were microinjected with Cas9 mRNA and Tyr sgRNAs. No nucleotide
polymorphisms between C57Bl6/J and CBA strains at Tyr exon1/intron1 were found. The
microinjected embryos were divided in two groups, one of which was grown to blast stage and
harvested to obtain the genomic DNA, which was analyzed to detect indels at the sgRNA
cutsites. Embryos of the other group were grown to the two-cell stage and implanted in
pseudopregnant females to visualize the in vivo CRISPR effect on mouse coat color. The microinjected
zygotes grown to blast stage were harvested to obtain their genomic DNA, which was then
analyzed by NGS, revealing a greater abundance of null alleles in the SDE-mTyrsgRNA than in the
IE-mTyrsgRNA embryo group (100% vs. 67.57%) (S6 Table). Briefly, NGS detected seven
mutated alleles at the expected cut-site of IE-mTyrsgRNA. In silico analysis identified three
mutated alleles with in-frame mutations that gave rise to a putative functional protein. NGS in
the group of embryos microinjected with SDE-mTyrsgRNA identified eight mutated alleles, of
which three were in-frame mutations and five were null mutations. However, in this embryo
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Fig 5. Analysis of ATM gene expression by Western blot in K562 edited cells. (A) Western blot analysis of ATM
protein expression in K562-edited cells. A single band of 350 kDa corresponding to ATM was observed in K562 cells
electroporated with empty px458. A lower level of ATM expression was observed in IE-hATMsgRNA-edited cells, and
an even lower level was noted in SDE-hATMsgRNA-edited cells. Vinculin expression of the cells was used as the
loading control. (B) Western blot analysis of ATM expression in single-edited-cell clones. All clones derived from cells
electroporated with empty vector, used as a control, showed a single band corresponding to ATM. Three of six
IEhATMsgRNA edited clones showed no expression of ATM and one of six had a lower level of ATM expression
compared with controls. Only one of six SDE-hATMsgRNA-edited clones expressed ATM, while ATM expression
could not be detected in the other five clones.
group, all alleles (100%) detected were predicted to be null alleles given the splicing site
mutations (Fig 6 and S6 Table).
To confirm the in-silico predictions, one-cell stage embryos from two strains of mice were
microinjected with Cas9 mRNA and both Tyr sgRNAs separately. Embryos microinjected
with SDE-mTyrsgRNA or IE-mTyrsgRNA were implanted in two cell-stage in pseudopregnant
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Fig 6. Next Generation Sequencing (NGS) analysis of CRISPR/Cas9 edited mouse embryos at Tyr locus. Graphic
NGS analysis of CRISPR/Cas9-mediated edition of Tyr locus in mouse blastocysts. Genotyping of embryos
microinjected with sgRNAs targeting Tyr gene, by NGS, revealed that only 67.57% of edited sequences from embryos
microinjected with IE-mTyrsgRNA correspond to null alleles, while 100% SDE-mTyrsgRNA-modified alleles gave rise
to null alleles. Black and gray circles correspond to null and functional alleles, respectively, while the background
indicates the type of mutation (dark blue: splice donor site in-frame and/or frameshift; light blue: frameshift).
females. Full albinos, mosaics, and colored-coat pups were detected in all offspring of each
group of microinjected embryos in both strains (Fig 7). 60 mice per group were analyzed by
Sanger sequencing and a large number of mutant mice with one or two mutant alleles were
detected. To address which sgRNA yielded a higher proportion of null alleles, we excluded all
mice with unmuted alleles. All mice with at least one mutant allele (mosaic mice) were
analyzed in silico. We detected a higher number of albino or mosaic mice in the SDE-mTyrsgRNA
mouse group compared with the IE-mTyrsgRNA group (S7 Table).
Sanger sequencing and TIDE analysis of the SDE-mTyrsgRNA mouse group with any
grade of albinism identified at least two alleles with frameshift mutations and/or splice
mutations. As a representative example we show an offspring where we detected mosaic pups with
three alleles: a wildtype allele, a frameshift null allele and a splicing-site-mutated allele arising
from a point mutation (+1 bp insertion) at the intronic splice-site. We also detected
coat-colored pups in IE-mTyrsgRNA targeted pups exclusively with two mutated alleles: a frameshift
allele and a mutated allele arising from a nonsynonymous mutation (Fig 7 and S7 Table).
The sgRNA guide targeting the exon splice-donor site of BCR/ABL
oncogene increases the efficiency for abrogating cell survival / proliferation
To test the efficiency of SDE-sgRNA and IE-sgRNA guides at switching off oncogenes we
performed similar assays to generate ABL null alleles in the leukemic K562 cell line and to
abrogate the oncogene activity of BCR/ABL oncogene fusion (Fig 1C).
Similarly to TYR and ATM genes, three individual electroporation assays of K562 cells were
performed with each sgRNA directed towards the ABL exon 1 (SDE-hABL-1sgRNA and
IEhABL-1sgRNA) cloned in a CRISPR-Cas9-GFP mammalian expression vector. Sanger
sequencing showed genome edition at expected cleavage point for each sgRNA guide and Tide
analysis predicted a variety of small indels for each guide (Figs 2 and 3). NGS analysis showed
the most frequent allele variations generated in K562 by electroporation with SDE- and
IEhABL-1 sgRNAs (S8 Table). 40% (4/10) of the allelic variations generated by IE-hABL-1
sgRNA gave rise to in-frame mutations. By contrast, SDE-hABL-1 sgRNA gave rise to 100%
(9/9) of knockout sequences, four of which (44.4%) were in-frame mutations, but with an
altered canonical splicing sequence (S8 Table).
To test the ability of SDE sgRNAs to increase the efficiency at knocking out fusion
oncogenes, we compared the proficiency at abrogating the cell survival and proliferation produced
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Fig 7. In vivo Analysis of Tyr-null alleles generation by CRISPR/Cas9-induced mutations. A representative
offspring obtained from SDE-mTyrsgRNA. Most pups of SDE-mTyrsgRNA -edited embryos (4 of 5) showed a mutant
phenotype (1 albino and 3 mosaic). The genetic characterization of the different alleles of the off-spring is showed
by the BCR-ABL oncoprotein through the induction of indels with SDE-sgRNA and
IEsgRNA CRISPR-Cas9 guides. In three independent assays, we electroporated the K562 BCR/
ABL cell line with SDE-hABL-1 and IE-hABL-1sgRNA. After 48 hours, we analyzed the effects
on apoptosis and cell cycle (Fig 8). SDE-hABL-1sgRNA-targeted cells showed a higher level of
apoptosis (86.8%) than noted in IE-hABL-1sgRNA cells (60.1%), while 32.4% of control cells
were apoptotic (Fig 8A). K562 cells electroporated with SDE sgRNA yielded 10% more subG0
DNA content (45.3%) than IE-edited cells (34.5%) (Fig 8B). The quantification of annexin
expression in K562-edited cells with SDE- and IE-hABL-1 sgRNAS showed a higher level of
expression in SDE-hABL-1sgRNA-edited cells (568.2 mfi) compared with
IE-hABL-1sgRNAedited cells (475.5 mfi) and K562 control cells (411.5 mfi) (Fig 8C).
Off-targets analysis showed no differences between sgRNAs designed
against splice-donor site and internal-exon region
To determinate if the predicted off-targets were affected in a major manner by the SDE
sgRNAs we studied the top 5 predicted off-targets of each independent sgRNA (Fig 9). We
tested the ability of each sgRNA to induce genome edition in off-target sequences by the
observation the heteroduplex formed in the edited sequences. The IE-sgRNAs produced genome
edition in 5 of 25 analyzed off-target sequences, and the same proportion of edited off-target
was found in SDE-sgRNAs, producing 4 altered sequences of 25 (Fig 9). Statistical analysis
showed no significant differences between both sgRNAs groups (p value = 0.751).
DSB induced by CRISPR/Cas9 technology is the gold standard for creating null alleles in any
biological system. In most cases, DSBs are typically repaired by NHEJ, resulting in indel
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Fig 8. Functional analysis of BCR-ABL-1 in CRISPR/Cas9 edited K562 cells. (A) Flow cytometry analysis of annexin
V expression and cell cycle of K562-edited cells. SDE-hABL-1sgRNA trigger a higher number of apoptotic cells than
IE-hABL-1sgRNA and control cells after electroporation with the empty vector. (B) The DNA content of the cells
edited with SDE sgRNA gave 10% higher levels than IE-edited cells (45.3% vs. 34.5%). (C) The quantification of
annexin V expression in K562-edited cells with SDE and IE hABL-1 sgRNAs showed a higher level of expression in
SDE-hABL-1sgRNA edited cells (568,2 mfi) than in IE-hABL-1sgRNA-edited cells (475.5 mfi). Graph shows results
from three independent experiments. , p<0.001.
mutations. These mutations can generate knockout alleles when CRISPR/Cas9 is directed at
coding sequences, but due to the variable size of NHEJ-induced indels, generating a full KO in
one step cannot always be achieved at high frequency. This could be especially critical for gene
therapy approaches. If there is an acceptable degree of efficiency of delivery of CRISPR/Cas9
reagents to the target cell, the key step for success lies in the effectiveness of a specific sgRNA
at knocking out the oncogene. In this context, the null effect could be increased by sgRNAs
targeting the exon SD boundaries. Following this strategy, the generation of null alleles could be
increased in two independent ways: by the probabilities of producing a frameshift mutation
and/or breaking the canonical pre-mRNA splicing. In the present work we have demonstrated
that knockout efficiency can be increased using sgRNAs targeting the exon splice donor area.
The study considered the predicted informatic score (most guides with a score of > 75) and
the cut-site of the sgRNAs. It is important to note that for SDE-sgRNAs we chose PAMs to
trigger DSBs inside the coding sequence that were located no further than five nucleotides
from the end of the exon.
We noted that most of the mutant alleles produced in our assays in the Baf3 and k562 cell
lines correspond to small indels, indicating that the DSB is repaired by blunt-end ligation
independently of sequence homology, the classic nonhomologous end joining (C-NHEJ)
]. NGS corroborated the Sanger sequences detected and exposed new mutant alleles
that are likely to be little-represented in the edited cell line. As expected, NGS and Sanger
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Fig 9. Off-targets analysis of sgRNAs. Surveyor analysis of the top 5 predicted off-targets for each sgRNA used. Panel
shows amplified sequence of target and 3 representative off-targets, non-treated and treated with T7 endonuclease I.
Digestion reveals genome edition (red asterisk) when the off-target sequence carries some mutations. Table shows
overall results from all off-targets analyzed. No differences were observed in number of edited off-targets by
IEsgRNAs compared with SDE-sgRNAs (Chi-square test P = 0.751 n.s.).
sequencing highlighted the same alleles in in vivo assays of mouse zygotes, grown to blast or of
mice born from them. In silico analysis of these mutant alleles revealed a full efficiency of the
null effect in SDE-sgRNA compared with IE-sgRNA. When an IE-sgRNA was used, mutant
alleles with mutations preserving the reading frame were detected. To corroborate the in silico
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findings we Sanger-sequenced all mice born in both groups. Excluding unmutated mice, we
detected color mice born from microinjected zygotes with IE-sgRNA with indels in one or
more alleles. It is of particular note that we observed color mice with both alleles mutated, one
of them with a frameshift mutation and the other with a mutation, indicating that some
induced indels are not able to generate a frameshift mutation. By contrast, when we used a Tyr
SDE-sgRNA, we detected albino or mosaic mice featuring one allele with a frameshift
mutation and another with a mutation but a destroyed splice-donor site. This result demonstrates
the higher null efficiency when an SDE-sgRNA is used. To determine whether this effect can
be reproduced in another locus we employed the same assay but targeting the ATM and ABL
loci. A similar result was obtained in both loci in human and mouse cell lines. Western blot
analysis in cell clones from both groups corroborated the NGS and the results of their in silico
analysis. More importantly, this approach can be efficiently used to abrogate oncogene
expression. When a cancer cell is the target, a delivery strategy that can result in the expression of
Cas9 and an oncogene-specific sgRNA in all infected cells is desirable. This is especially critical
for in vitro gene therapy where the expansion processes of a selected edited cell are not available.
Similarly, it is crucial for in vivo approaches in cancer therapies based on disrupting a driver
oncogene. If the efficiency of delivery of CRISPR/Cas9 reagents to the cancer cell is acceptable,
the key step for success lies in the effectiveness of a specific sgRNA at knocking out the oncogene.
In most of these cases, the designs are based solely on off-target criteria. However, for those cases
in which cellular selection is not an option and only one sgRNA can be used, the null effect
could be increased with an sgRNA targeting the exon boundary. Various strategies at different
] have been employed to treat malignant diseases in recent decades, such as
specific drug inhibitors acting at the protein level, gene suppression therapies at the mRNA level,
and genome-editing nucleases at the DNA level. CRISPR/Cas9 works has the advantage of
providing permanent and full gene knockout, and following this strategy, we abrogated p210 (BCR/
ABLp210) oncoprotein expression in the K562 cell line. Using this approach, pools of K562
edited cells electroporated with SDE-sgRNAs or IE-sgRNA were studied. The loss of p210
expression in K562 cells with SDE-sgRNA resulted in a significant increase in apoptosis levels.
Thus, this strategy could be adopted for gene therapy in cases for which cell selection is not an
option and the delivery Cas9 vector only allows the accommodation of one sgRNA.
Genome-editing nucleases, like the popular CRISPR/Cas9, enable knockout cell lines and null
zygotes to be generated by inducing site-specific DSBs within a genome. In most cases, when a
DNA template is not present, the DSB is repaired by non-homologous end joining, resulting
in small nucleotide insertions or deletions that can be used to construct knockout alleles.
However, for several reasons, these mutations do not produce the desired null result in all cases,
giving rise to a similar but functionally active protein. This undesirable effect could limit the
efficiency of gene therapy strategies based on abrogating oncogene expression by CRISPR/
Cas9 and should therefore be borne in mind. The use of an sgRNA-targeting splice donor site
could improve the null result for in vivo gene therapies. This strategy could be adopted to
abrogate in vivo the oncogenic activity involved in tumor maintenance.
Material & methods
This study followed Spanish and European Union guidelines for animal experimentation (RD
1201/05, RD 53/2013 and 86/609/CEE respectively). The study was approved by Bioethics
Committee of the University of Salamanca and Junta de Castilla y Leo?n, Spain (ref.000359).
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Cell lines and culture conditions
Baf/3 is a murine interleukin 3-dependent murine pro-B cell[
]. Baf/3was maintained in
Dulbecco?s Modified Eagle?s Medium (DMEM) (Life Technologies) supplemented with 10% fetal
bovine serum (FBS) and 1% of penicillin/streptomycin (Life Technologies) and 10% of
WEHI3-conditioned medium, as a source of IL-3.
The human CML-derived cell lines K562 were purchased from Deutsche Sammlung von
Mikroorganismen and Zellkulturen (DMSZ). K562 cells were cultured in RPMI 1640 medium
(Life Technologies) supplemented with 10% FBS, and 1% penicillin/streptomycin (Life
Technologies). All cell lines were incubated at 37?C in a 5% CO2 atmosphere. The presence of
mycoplasma was tested frequently in all cell lines with a MycoAlert kit (Lonza), using only
mycoplasma-free cells in all the experiments carried out.
CRISPR/Cas9 system design and sgRNA cloning
pX458 (Addgene plasmid # 48138)[
], which contains the coding sequence of Cas9 nuclease
and GFP, and a cloning site for sgRNA sequence, was digested with BpiI (NEB). To clone the
sgRNAs into the pX458 vector, two complementary oligos were designed for each sgRNA that
included two 4-bp overhang sequences (S9 Table). The sgRNA sequences were designed with
the web tool of the Spanish National Biotechnology Centre (CNB)-CSIC (http://bioinfogp.cnb.
Two sgRNAs were designed for the mouse Tyr locus. One of them, IE-mTyrsgRNA, targets
the exonic sequence in Tyr exon1, and the other, SDE-mTyrsgRNA, targets the
exon1-intron12 junction. Two sgRNAs were designed to target homologous sequences in the human TYR
locus: IE-hTYRsgRNA and SDE-hTYRsgRNA (Fig 1A).
In the same way, two sgRNAs against the mouse Atm locus (IE-mAtmsgRNA and
SDEmAtmsgRNA) and two sgRNAs against the human ATM locus (IE-hATMsgRNA and
SDEhATMsgRNA) were designed, one of each pair in the coding sequence of exon 10 (IE) and the
other against the ATM exon10-intron10-11 splice donor exon (SDE) (Fig 1B).
Finally, two sgRNA against human ABL-1 locus were designed: IE-hABL-1sgRNA, which
targets the exon 6 coding sequence, and SDE-hABL-1sgRNA, which targets the exon 4 splice
donor sequence (Fig 1C).
The two complementary oligos used to conform each sgRNA (S9 Table) were denatured at
95?C for 5 min, ramp-cooled to 25?C over 45 min to allow annealing, and finally ligated with
the linearized px458. 2 ?l of the ligation reaction were used to transform competent cells, and
single colonies were expanded using a QIAprep spin Maxiprep Kit (Qiagen) before plasmid
extraction. The correct insertion of the sgRNA sequences was confirmed by Sanger
In vitro cell electroporation
Mouse Baf/3 and human K562 cells were electroporated with px458 containing sgRNAs
against the Tyr and ATM loci, respectively, using Amaxa Nucleofector II (Lonza). 2 x 106 Baf/3
mouse cells were electroporated with 15 ?g of plasmid in 100 ?l of electroporation buffer (5
mM KCl; 15 mM MgCl2; 120 mM Na2HPO4/NaH2PO4 pH7.2; 25 mM sodium succinate; 25
] using program X001, while 1 x 106 k562 cells were electroporated with 10 ?g
of plasmid using program T016. 24 hours after electroporation, GFP-positive cells were sorted
by fluorescence-activated cell sorting (FACS) using FACS-Aria (BD Bioscience). 72 hours
post-electroporation, the genome editing of the cells was analyzed.
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Sequencing of sgRNA targets sites
Genomic DNA from cells was extracted using the QIAamp DNA Micro Kit (Qiagen) following
the manufacturer?s protocol. To amplify the different target regions of human and mouse TYR
and ATM genes, and human ABL-1, PCR was performed with the oligos described in S10
Table. Genomic DNA from single blastocyst-staged embryo was extracted in 10 ?l of lysis
buffer (50 mM KCL, 10 mM Tris-HCL pH 8.5, 0.1% Triton x-100, and 4 mg/ml of proteinase
K) at 55?C overnight, then heated at 95?C for 10 min. 2 ?l of this DNA solution was used as a
template for two rounds of PCR (30 cycles + 20 cycles) to amplify the target sequences using a
specific primer for each region (S11 Table).
PCR products were purified using a High Pure PCR Product Purification Kit (Roche) and
sequenced by the Sanger method using forward and reverse PCR primers.
The editing efficiency of the sgRNAs and the mutations potentially induced were assessed
using Tracking of Indels by Decomposition (TIDE) software (https://tide-calculator.nki.nl;
Netherlands Cancer Institute), which only required two Sanger sequencing runs from
wildtype cells and mutated cells.
To specifically identify the different generated mutations, Next Generation Sequencing
(NGS) technology was employed with the same Sanger primers with the corresponding
adapters added, to read each edited sequence individually.
The purified amplicons were mixed in equimolar ratios according to the number of
molecules and diluted to a final concentration of 0.2 ng/ul. The indexed paired-end library was
prepared with a Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced using an
Illumina platform (NextSeq or MiSeq, 300 cycles). A median per base coverage of 27,538 reads
(range 2096?88,976) was achieved. To call the sequence variants, an in-house bioinformatics
pipeline was established. Sequencing reads were aligned to the mouse reference sequence
genome (mm9) using bwa-0.7.12 software, and variant calling was performed with VarScan.
v2.4. To visualize read alignment and confirm the variant calls, Integrative Genomics Viewer
version 2.3.26 (IGV, Broad Institute, MA) was used.
Flow cytometry analysis and cell sorting of single-edited cell-derived clone
72 hours after sgRNA electroporation of K562 and Baf/3 cells, GFP-positive cells were selected
by fluorescence-activated cell sorting (FACS) using FACS Aria (BD Biosciences), establishing
the edited K562 and Baf/3 cell pool lines. For K562, single cells were seeded in 96-well plates
by FACS, establishing six random single-cell-derived clones for both ATM sgRNAs, and used
to analyze ATM protein expression. Six clones derived from cells electroporated with empty
vector were used as controls.
ATM protein expression was assessed by SDS-PAGE and western blot using a rabbit anti-ATM
antibody (1:1000; 2873S; Cell Signaling). Horseradish peroxidase-conjugated ?-rabbit antibody
(1:5000; 7074S; Cell Signaling) was used as a secondary antibody. Antibodies were detected
using ECL Western Blotting Detection Reagents (RPN2209, GE Healthcare). The expression of
vinculin (rabbit anti-vinculin; 1:1000; 4650S; Cell Signaling) was used as a loading control.
In vitro transcription of CRISPR/Cas9 system components, animals and
All sgRNA sequences were PCR-amplified from px458-based vector with primers carrying the
T7 RNA polymerase promoter at the 5? ends (S11 Table), and after column purification
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(Roche) the resulting PCR was used as a template for T7 RNA polymerase transcription in
vitro (MEGAshortscript T7 Transcription Kit, Thermo Fisher).
The Cas9 nuclease ORF, including NLS, was also PCR-amplified with primers carrying the
T7 RNA polymerase promoter at the 5? ends (S11 Table). The PCR product was purified and
used as a template for in vitro transcription, 5? capping (mMESSAGE mMACHINE T7
Transcription Kit, Thermo Fisher), and 3? poly(A) tailing (Poly(A) Tailing Kit, Thermo Fisher).
Transcription products were purified with RNeasy Mini Kit (Qiagen) and eluted in
nucleasefree EmbryoMax microinjection buffer (Millipore).
One-cell-staged embryos from superovulated C57BL/6J or B6/CBA hybrid females were
harvested and microinjected with 20 ng/?l of sgRNA and 20 ng/?l of Cas9 mRNA into the
cytoplasm and pronucleus. Embryo donor mice were euthanized by cervical dislocation and
were given humanitarian care in accordance with bioethical committee of University of
Salamanca (ref. 000359) and Spanish and European Union guidelines for animal experimentation.
Apoptosis and cell cycle analysis
Apoptosis was measured by flow cytometry with an annexin V-Dy634 apoptosis detection kit
(ANXVVKDY, Immunostep) following the manufacturer?s instructions. Briefly, 5 ? 105 cells
were collected and washed twice in PBS, and labeled with annexin V-DY-634 and non-vital
dye propidium iodide (PI), allowing the discrimination of living-intact cells (annexin-negative,
PI-negative), early apoptotic cells (annexin-positive, PI-negative) and late apoptotic or
necrotic cells (annexin-positive, PI-positive). In parallel, cell distribution in the cell cycle phase
was also analyzed by measuring DNA content (PI labeling after cell permeabilization). Plots
show results of a representative experiment from three independent replicates.
Predicted top 5 off-targets were analyzed by the T7 endonuclease I (T7EI) mismatch cleavage
assay following manufacturer?s indications (Integrated DNA Technologies) [
]. Target DNA
sequences were amplificated by PCR using specific oligonucleotides (S12 Table). To form the
heteroduplex complexes, PCR products were denatured 95?C for 10 minutes, followed by
temperature ramp (95?85?C, -2?C/sec and 85?25?C, 0.3?C/sec). The heteroduplex products were
incubated with T7E1 1 hour a 37?C and visualized in 2% agarose gel.
Statistical analysis of annexin V expression was performed using GraphPad Prism version 6.00
for Mac OS X, (GraphPad Software, La Jolla California USA, www.graphpad.com).
Experimental results were expressed as median ? standard error (SEM). Nonparametric variables
were analyzed using Kruskal-Wallis followed by Dunn?s multiple comparisons test. Values
with p<0.001 (indicated by three asterisks) were considered to be statistically significant.
Chisquare test was performed to analyze the difference obtained in off-target analysis.
S1 Table. In vitro genome editing of the mouse Tyr locus using sgRNA against exon coding
sequence (IE) and the coding splice-donor exon (SDE) sequence. NGS analysis of allelic
variants induced in Baf/3 mouse cells.
S2 Table. In vitro genomic edition of human TYR locus using sgRNA against exon coding
sequence (IE) and coding SDE sequence. NGS analysis of allelic variants induced in K562
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S3 Table. In vitro genome editing of the human ATM locus using sgRNA against the exon
coding sequence (IE) and the coding SDE sequence. NGS analysis of allelic variants induced
in K562 human cells.
S4 Table. In vitro genome editing of the mouse Atm locus using sgRNA against the exon
coding sequence (IE) and the coding SDE sequence. NGS analysis of allelic variants induced
in Baf/3 mouse cells.
S5 Table. NGS analysis of ATM allelic variants induced in human K562 single-edited
S6 Table. In vivo genome editing of Tyr locus in mouse embryos using sgRNA against the
coding sequence (IE) and the SDE sequence. NGS analysis of allelic variants induced in
microinjected mouse blastocysts.
S7 Table. In vivo genome editing of Tyr locus in mice using sgRNA against the coding
sequence (IE) and the coding SDE sequence. Observed phenotype and Sanger analysis of
allelic variants induced in mice born after CRISPR/Cas9 system microinjection.
S8 Table. In vitro genome editing of the human ABL-1 locus using sgRNA against the exon
coding sequence (IE) and the coding SDE sequence. NGS analysis of allelic variants induced
in K562 cells.
S9 Table. Oligos designed for each sgRNA.
S10 Table. Oligos used for target genome sequence amplification.
S11 Table. Oligos used for in vitro transcription of sgRNA and Cas9 mRNA.
S12 Table. Oligos used for off-target genome sequence amplification.
The authors wish to express their sincere thanks to Dionisio Mart??n Zanca, Alberto Penda?s
(Spanish Research Council, CSIC), for their technical assistance and contribution to our
CRISPR/Cas9 studies; Servicio de Citometr??a, Servicio de Experimentacio?n Animal
(University of Salamanca) and Servicio de Secuenciacio?n (IBMCC) for their technical assistance.
Conceptualization: Ignacio Garc??a-Tu?o?n, Manuel Sa?nchez-Mart??n.
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Formal analysis: Jesu?s Mar??a Herna?ndez-Sa?nchez, Marta Mart??n-Izquierdo.
Funding acquisition: Jesu?s Mar??a Herna?ndez-Rivas.
Investigation: Ignacio Garc??a-Tu?o?n, Vero?nica Alonso-Pe?rez, Elena Vuelta, Sandra
Methodology: Ignacio Garc??a-Tu?o?n, Vero?nica Alonso-Pe?rez, Elena Vuelta, Sandra
Pe?rezRamos, Mar??a Herrero, Luc??a Me?ndez, Jesu?s Mar??a Herna?ndez-Sa?nchez, Marta
Resources: Raquel Salda?a, Julia?n Sevilla, Ferm??n Sa?nchez- Guijo.
Supervision: Manuel Sa?nchez-Mart??n.
Visualization: Manuel Sa?nchez-Mart??n.
Writing ? original draft: Ignacio Garc??a-Tu?o?n, Manuel Sa?nchez-Mart??n. Writing ? review & editing: Ignacio Garc??a-Tu?o?n, Manuel Sa?nchez-Mart??n.
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