RNA Biology

ISSN: 1547-6286 (Print) 1555-8584 (Online) Journal homepage: https://www.tandfonline.com/loi/krnb20

CRISPR/Cas9-mediated noncoding RNA editing in

human cancers

Jie Yang, Xiaodan Meng, Jinchang Pan, Nan Jiang, Chengwei Zhou, Zhenhua
Wu & Zhaohui Gong

To cite this article: Jie Yang, Xiaodan Meng, Jinchang Pan, Nan Jiang, Chengwei Zhou, Zhenhua
Wu & Zhaohui Gong (2018) CRISPR/Cas9-mediated noncoding RNA editing in human cancers,
RNA Biology, 15:1, 35-43, DOI: 10.1080/15476286.2017.1391443

To link to this article: https://doi.org/10.1080/15476286.2017.1391443

Published online: 09 Nov 2017.

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RNA BIOLOGY
2018, VOL. 15, NO. 1, 35–43
https://doi.org/10.1080/15476286.2017.1391443

REVIEW

CRISPR/Cas9-mediated noncoding RNA editing in human cancers

Jie Yanga,b,†, Xiaodan Menga,b,†, Jinchang Pana,b, Nan Jiang a,b
, Chengwei Zhouc, Zhenhua Wud, and Zhaohui Gonga,b
a
Department of Biochemistry and Molecular Biology, Medical School of Ningbo University, Ningbo, Zhejiang, China; bZhejiang Provincial Key
Laboratory of Pathophysiology, Medical School of Ningbo University, Ningbo, Zhejiang, China; cDepartment of Thoracic Surgery, The Affiliated Hospital
of Medical School of Ningbo University, Ningbo, Zhejiang, China; dDepartment of Otolaryngology Head and Neck Surgery, The Affiliated Ningbo
Medical Center Lihuili Eastern Hospital of Medical School of Ningbo University, Ningbo, Zhejiang, China

ABSTRACT ARTICLE HISTORY

Cancer is characterized by multiple genetic and epigenetic alterations, including a higher prevalence of Received 18 July 2017
mutations of oncogenes and/or tumor suppressors. Mounting evidences have shown that noncoding Revised 26 September 2017
RNAs (ncRNAs) are involved in the epigenetic regulation of cancer genes and their associated pathways. Accepted 8 October 2017
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (CRISPR/ KEYWORDS
Cas9) system, a revolutionary genome-editing technology, has shed light on ncRNA-based cancer therapy. CRISPR-Cas9; genome
Here, we briefly introduce the classifications and mechanisms of CRISPR/Cas9 system. Importantly, we editing; microRNA; long
mainly focused on the applications of CRISPR/Cas9 system as a molecular tool for ncRNA (microRNA, long noncoding RNA; circular RNA;
noncoding RNA and circular RNA, etc.) editing in human cancers, and the novel techniques that are based RNA editing; cancer
on CRISPR/Cas9 system. Additionally, the off-target effects and the corresponding solutions as well as the
challenges toward CRISPR/Cas9 were also evaluated and discussed. Long- and short-ncRNAs have been
employed as targets in precision oncology, and CRISPR/Cas9-mediated ncRNA editing may provide an
excellent way to cure cancer.

Introduction mutations in the cancer cell genome is an appealing way to

conquer cancer.9 A wealth of scientific evidence has shown that
Currently, cancer is one of the leading causes of death and is a
CRISPR/Cas9 system can correct mutations that cause cancer
refractory disease around the world which challenges the life
and have potential to be harnessed as a promising therapeutic
and health of human beings.1 Although a multitude of exciting
technique to protect patients at the genetic level.10 Accumulat-
achievements have been made in the area of cancer therapy,
ing data have shown that CRISPR/Cas9-based system can tar-
including surgery, chemotherapy, radiation therapy, targeted
get not only protein-coding genome but also the noncoding
biological therapy and new integrated combination therapy,
RNAs (ncRNAs) in human beings.10-12 In this review, we sum-
the high possibility of relapse and chemo-/radiation- resistance
marized the recent applications of CRISPR/Cas9 system in
and the detrimental side effects and toxicity are hurdles to
ncRNA-related genome editing, the off-target effects and
ensure the quality of life.2 Therefore, further technical advances
CRISPR/Cas9-based novel techniques as well as the limitations
and novel therapeutic approaches for cancer therapy are
and hurdles.
urgently needed. Fortunately, the advent of the clustered regu-
larly interspaced short palindromic repeats (CRISPR)-associ-
ated nuclease 9 (CRISPR/Cas9) system has shed light on cancer
therapy.
CRISPR system and mechanism
To date, CRISPR/Cas9 system is known as a molecular scis-
sor and is widely used in various kinds of studies, including CRISPR was firstly discovered in Escherichia coli by Ishino and
cancer research, drug discovery, treatment of mental disease, colleagues in late 1987.13 They found that the clustered repeat
applications in plants, etc. CRISPR is a heritable and adaptive segments were interrupted by a series of spacer sequences and
antiviral immune system of prokaryote, which primarily works this phenomenon was later termed as CRISPR.14 Till now,
against infectious invading viruses and phages and can be used researchers have discovered more than a dozen of different
to specifically cleave DNA in vitro.3-6 It is of importance and CRISPR/Cas systems that are categorized into three major
well established that cancer is a genetic disease characterized by groups (type I, II and III) and many subtypes according to their
mutations of multiple DNA and RNA in the cellular genome, diverse mechanisms.15,16 CRISPR/Cas9 is one of the type II
and these characteristics of cancer confer cancer cells some dis- CRISPR systems in Streptococcus pyogenes (SpyCas9), and is
tinct biological capabilities, including progressive growth, inva- the most globally used system in mammalian due to its high
siveness, chemoresistance, and so forth.7,8 Correcting the efficiency and accuracy.

CONTACT Zhaohui Gong [email protected] Department of Biochemistry and Molecular Biology, Medical School of Ningbo University, 818 Fenghua Road,
Ningbo, ZJ 315211, China.
y
Equal contribution
© 2018 Taylor & Francis Group, LLC
36 J. YANG ET AL.

be directed by one or more guide RNAs (gRNAs) to up-regulate

the expression of some specific genes in human cells, like,
VEGFA and NTF3.28 Except from its use in human beings,
CRISPRa is also used in other species. In another study, Cheng
et al. used a nuclease-deficient dCas9, dCas9VP48, which can
efficiently activate not only some exogenous reporter genes
both in human and mouse cells, but also a number of endoge-
nous genes, including IL1RN, SOX2, and OCT4.29
RNA interference (RNAi) is a universally used technique to
suppress the expression of genes at the RNA level. Since dCas9
loses the endonuclease activity and acts as a DNA recognition
protein-RNA complex when co-expressed with the sgRNA, this
RNA-guided DNA recognition complex adds a new tool for
researchers to control the expression of interested genes. Lei S
and colleagues successfully demonstrated that CRISPRi can
selectively and efficaciously repress transcription of target genes
through silencing the transcription initiation and elongation.
Figure 1. Schematic illustration of double strand breaks (DSBs) created by CRISPR- Interestingly, the off-target effects, which are commonly found
Cas9. Combined with crRNA and tracrRNA, Cas9 can specifically cut DNA double
strands and cause DSBs.
when using CRISPR KI and CRISPR KO, are rarely detected.25
In another study, researchers used CRISPRi system to stably
The CRISPR/Cas9 system is also the first engineered CRISPR/ and effectively suppress the endogenous genes, like, CD71 and
Cas system for genome editing, partly because it contains an associ- CXCR4, in HeLa cells.24
ated easily programmable single guide RNA (sgRNA) with a short
recognition sequence which is only 20 nucleotides in length.17-19
CRISPR-X
The sgRNA consists of both CRISPR RNA (crRNA), which has a
sequence complementary to the targeted sites, and trans-activating Recently, a powerful approach was based on dCas9 and was intro-
crRNA (tracrRNA), which is separately transcribed and partly duced to engineer and study the protein functions. Gaelen T et al.
complementary to the crRNA.19-21 Likewise, to exert the genomic employed a system, named CRISPR-X, which includes catalytically
editing function, the CRISPR/Cas9 system also needs a key enzyme inactive dCas9 and cytidine deaminase (AID) recruited by dCas9,
component, Cas9 nuclease,3 which is closely related to these two to create specific point mutations in situ with limited off-target
RNA components. These RNAs are required to guide the Cas9 pro- effects in the mammalian genome. Furthermore, the researchers
tein to the targeted sites and to activate the Cas9 nuclease. sgRNA mutated PSMB5 that was targeted by the therapeutic cancer drug
combines with Cas9 protein to form a complex and strictly recog- bortezomib, and found CRISPR-X could identify the mutations
nize the targeted complementary DNA sequence flanked at its 30 that are closely related to bortezomib resistance.30 In short, this
end near the protospacer adjacent motif (PAM), which is com- neoteric strategy can be used to investigate and improve the func-
monly composed of NGG or NAG (N D A, T, G or C), and then tions of proteins, and can be also applied to reveal the unknown
initiate the DNA double strand breaks (DSBs) (Fig. 1).22 mechanisms of drug-resistance.

Base-editing
dCas9, CRISPRa, CRISPRi, CRISPR-X and base-editing in
cancer The base-editing technology, which is based on the combina-
tion of diverse cytidine deaminases with dCas9, is another
dCas9, CRISPRa and CRISPRi
novel application of CRISPR/Cas9 system.31,32 Paired this engi-
Apart from CRISPR knock in (CRISPR KI) and knock out neered fusion with a gRNA, this complex can find cytosine
(CRISPR KO), the CRISPR-Cas9 system has other diverse within a targeted desire range of five nucleotides (nt) and then
applications in cancer research area (Fig. 2). The catalytically transform it into uracil, finally, facilitating a base substitution
inactive/dead Cas9 mutant (dCas9) is engineered to act as the of C-to-T or G-to-A without introducing DSBs.31 This technol-
transcriptional activator or suppressor of the target gene by ogy was firstly used by Komor and colleagues to mediate direct
blocking both the enzymatic activities of RuvC and HNH transition from cytidine to uridine thus generating single-base-
domains of the Cas9 nuclease, without altering the pair substitutions in human and murine cell lines.31 With the
sequence.23-25 Many studies have indicated that these special help of electroporation and microinjection, this technology was
reconstructive Cas9 can be fused to different domains to exert used by Kim et al. to edit mouse embryos on a single base pair
its stimulative or suppressive function of genetic transcrip- level efficiently and precisely.33 Aside from using in mamma-
tion, and these strategies are now named as CRISPR activa- lian cells and embryos, CRISPR/Cas9 derived base-editing is
tion (CRISPRa) and CRISPR interference (CRISPRi), likewise used in yeast and plants,34-36 indicating the huge
respectively.24-27 potential of this novel technology in cancer research and other
CRISPRa now is generally used to activate the expression of areas in the coming future.
endogenous human genes. In one study, for example, Morgan In summary, CRISPR KI and CRISPR KO are globally and
L et al. demonstrated that a variant of dCas9, dCas9-VP64, can effectively used for creating mutations both in vivo and in vitro
 RNA BIOLOGY 37

Figure 2. Techniques based on CRISPR-Cas9 system. (A) CRISPR-KI. Guided by the sgRNA, Cas9 can specifically knock the donor DNA into the targeted sequence. (B)
CRISPR-KO. Guided by the sgRNA, Cas9 can specifically knock out the gene of interest. (C) CRISPRa. Guided by one or more sgRNAs, dCas9 can significantly activate the
expression of endogenous genes. (D) CRISPRi. Co-expressed with the sgRNA, dCas9 can selectively and efficaciously repress transcription of targeted genes. (E) CRISPR-X.
Combined with cytidine deaminase (AID), catalytically inactive dCas9 can be used to investigate and improve the functions of protein. (F) Base editing. Combined with
diverse AIDs, dCas9 can mediate direct transition from C to T or from G to A without causing DSBs.

while CRISPRa and CRISPRi are employed to modulate tran- of cancer and other genetic diseases as long as we efficiently
scriptional expression without causing any mutations. As a handle them and overcome limitations brought by them.
neoteric technology, CRISPR-X paves a path to investigate pro-
tein-protein interaction and provides a new skill for scientists
to uncover the mystery of resistance to drugs. Another brand-
CRISPR/Cas9 as a tool for ncRNA editing
new technology, base-editing, can result in specific point muta-
tion without causing the cleavage of targeted sequence, which Prior to the emergence of CRISPR/Case9, RNAi was widely used to
is different from CRISPR KI and CRISPR KO. These technolo- suppress the expression of protein coding genes.37 However, RNAi
gies may open various bright therapeutic avenues for the cure has been proven to be an inefficient approach when using for the
38 J. YANG ET AL.

interference of ncRNAs.38 ncRNAs, primarily including micro- CRISPR/Cas9 for lncRNA editing in cancer
RNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular-
In addition to miRNAs, lncRNAs have been reported to be suc-
RNAs (circRNAs), have been proven to play a critical role in
cessfully edited/regulated by CRISPR/Cas9 system. UCA1 (uro-
cancers (reviewed in39,40). As the majority of (about 99%) human
thelial carcinoma-associated 1), an upregulated lncRNA in
genome, ncRNA-related region lacks protein-coding capability,41,42
bladder cancer, could be targeted by specifically designed
and most of the genetic alterations occur in this vast region, target-
gRNAs of CRISPR/Cas9, and efficaciously inhibited both in
ing non-coding area with CRISPR/Cas9 system, therefore, maybe a
vivo and in vitro,53 suggesting that CRISPR/Cas9 system can be
feasible approach toward cancer therapy. Here, we mainly discuss
used to modulate the expression of lncRNAs and further
the applications of CRISPR/Cas9 system in the areas of miRNAs
employed as a therapeutic approach towards clinical cancer
and lncRNAs.
therapy.
One of the limitations to apply CRISPR/Cas9 system to non-
CRISPR/Cas9 for miRNA editing in cancer coding genes is that tiny indels may not necessarily generate
functional loss of a certain noncoding gene. To overcome this
So far, many studies have indicated that CRISPR/Cas9 system is
obstacle, Ho and colleagues adopted a specific selection system,
an excellent option for ncRNA-related genome editing/regulat-
HR (homologous recombination), to integrate marker genes
ing (Summarized in Table 1). As one of the primary ncRNAs
into the genome, and with the help of CRISPR-Cas9 system,
discussed in this review, miRNAs have been widely investigated
UCA1 and lncRNA-21A as well as AK023948 were successfully
in CRISPR/Cas9-related area. Cloning CRISPR/Cas9 constructs
knockout in HCT-116 and MCF-7 cells, respectively.54 Addi-
with sgRNAs which specifically target the biogenesis processing
tionally, Shechner and colleague developed CRISPR-Display
sites of interested miRNAs, Chang et al. demonstrated that
(CRISP-Disp), a targeted localization method that uses Cas9 to
alterations generated by CRISPR/Cas9 on the structure of pri-
deploy large RNA cargos to DNA loci. They found that
mary miRNAs can lead to the downregulation of these mature
functional RNA domains up to at least 4.8 kb long could be
miRNAs both in vivo and in vitro.43 Moreover, this study also
inserted in CRISPR gRNA at multiple points, allowing the
demonstrated that properly designed the sgRNAs, CRISPR/
construction of Cas9 complexes with natural lncRNAs.55 This
Cas9 can obviously minimize the off-target effects for the miR-
method may open an avenue for lncRNA study in the field of
NAs with highly conserved sequences across the same family.
cancer research.
In another study, Zhou and colleagues used CRISPR/Cas9 sys-
tem to successfully knock out miR-3188 in hepatocellular carci-
noma (HCC) cell lines, and found that miR-3188 KO effectively
CRISPR/Cas9 for lncRNA editing in other diseases
suppressed cell growth, invasion and migration, and inhibited
xenografts tumor growth in nude mice.44 Recently, Yue’s group Except for the application in lncRNA-related cancer research,
reported that lentiviral CRISPR/Cas9 vectors were highly effi- CRISPR/Cas9 is also used for studying other diseases. LncRNA
cient in introducing indels (insertions and deletions) into the PEAT (Pax1 enhancer antisense transcript) is located near the
precursor miRNA sequences. Using this method, they success- upstream of the Pax1 gene and lacks an ORF and consensus
fully disrupted the expression of miR-21 and found that disrup- TSS (transcription start site). In order to delete its transcribed
tion of pre-miR-21 sequences results in decreased cell unit completely via CRISPR/Cas9, researchers employed the
proliferation, migration and invasion in ovarian cancer cells.45 existing annotation (mm10) and mapped RNA-seq reads to
Except for editing/regulating these miRNAs discussed forecast the transcribed region in a mouse line.56 They also
above, CRISPR/Cas9 has also shown vast applications toward found that the deletion of PEAT mildly increased the expres-
other miRNAs, like, miR-137, miR-93, miR-309, miR-126a/b sion of bone morphogenetic protein target gene as well as a
etc., in various cancer cells or organisms.46-52 number of ribosomal protein mRNAs. Another group showed

Table 1. Applications of CRISPR-Cas9 in ncRNA study.

ncRNA Object Effect Efficiency Reference

miR-21 HEK-293 cells Knockout >1000-fold 54

54
miR-29a HEK-293 cells Knockout High
miR-17 In vitro and in vivo Knockdown 96% 43

miR-200c In vitro and in vivo Knockdown 96% 43

miR-141 In vitro and in vivo Knockdown 96% 43

43
miR-3188 HCC cells Knockout High
45
miR-21 SKOV3 and OVCAR3 cells Disruption High
46
miR-137 A2780 cells Disruption High
48
miR-309 Mosquito embryos Disruption 51.5%
47
miR-93 HeLa cells Disruption 16%
49
miR-126a Zebrafish embryos Knockdown High
49
miR-126b Zebrafish embryos Knockdown High
50,52
Several miRNAs Zebrafish/MV4-11 cells Disruption 90%
Rian In vivo Knockout 33.3% 93
54
UCA1 HCT-116 cells Knockout High
54
lncRNA-21A HCT-116 cells Knockout High
54
AK023948 MCF-7 cells Knockout High
 RNA BIOLOGY 39

that combining CRISPR/Cas9 with siRNAs or GapmeRs to Besides, there are many investigations about non-coding
delete or silence MANTIS (lncRNA n342419), a downregulated genome which may not be transcribed into ncRNAs to expand
lncRNA in patients with idiopathic pulmonary arterial hyper- the application of CRISPR/Cas9 in non-coding area. Zhang’s
tension (IPAH), can efficiently inhibit the angiogenic sprouting group,63 for example, developed a high-throughput strategy
of endothelial cells.57 using pooled CRISPR/Cas9 sgRNA system targeting 715 kb of
sequence which surrounds three different genes NF1, NF2, and
CUL3. Using this unbiased mutagenesis method, they showed
CRISPR/Cas9-based screening
how a Cas9-mediated systematic separation of non-coding sites
To facilitate the applications of CRISPR/Cas9 in both miRNA can identify functional elements that are involved in cancer
and lncRNA-related studies, several CRISPR/Cas9 based drug resistance and gene regulation.
screening technologies have been invented. Studies about ncRNA-related pathways also attract scien-
With the help of CRISPR/Cas9 technology, Wallace et al. tists’ interest. Lately, Golden et al. used CRISPR/Cas9 genome-
performed an unbiased global LOF screening to examine wide loss-of-function (LOF) screening combined with a fluo-
miRNAs which are involved in MV4-11 cell lines. Using this rescent reporter of miRNA activity to identify new regulators
approach, they found that a subset of (27/197) evolutionarily that are involved in miRNA pathways in human cells.64 This
conserved miRNAs can promote or inhibit the cellular prolifer- strategy expands the use of CRISPR/Cas9 in the area of non-
ation and survival.52 This approach has the potential to identify coding genome and provides a novel insight into the investiga-
functional non-coding components in mammalian genomes tion of ncRNAs-related genome.
and provides a resource to guide coming work. In another ear- The emergence of CRISPR/Cas9 is opening a new way for
lier study,58 researchers focused their concentration on a func- scientists to edit genome. With the development CRISPR/Cas9
tional screening to identify the cis-regions that control the technology, ncRNA-based gene editing will become more effi-
processing of a certain miRNA, miR-142. Using a CRISPR/ cient and the significant roles of ncRNAs in diseases will be
Cas9 based molecular chipper technology, they carried out a fully investigated in the near future.
functional screen to identify cis-regulatory elements that con-
trol the biogenesis of miR-142 and verified it as an economical
and easily customizable way, compared with microarray-based Off-target effect and solutions
oligonucleotide synthesis, for sgRNA library construction.
Despite its limitations, this Molecular Chipper technology Off-target effect
can be used not only for identifying functional non-coding While the CRISPR/Cas9 system has many advantages over
regions in mammalian genomes but also for performing gene- other genome editing technologies, like, ZFNs and TALENs,
expression-based screening of protein-coding gene regulation there are still some knotty problems that need to be solved,
in the future. such as off-target effect. Sometimes, Cas9 can improperly bind
So far, to our knowledge, more screenings for lncRNAs have to targets and generate mutations within the sequence which is
been developed compared to miRNAs. In a recent study, Mo’s outside the targeted sites.10
group used a CRISPR/Cas9-based synergistic activation media- As off-target sites are determined by the nuclease and the
tor (SAM) system to identify the underlying lncRNAs that have sequence of sgRNA, therefore, some algorithms and methods
the ability to regulate AKT activity, and determined lncRNA including Genome Engineering, CCTop, Cas-OFFinder, E-
AK023948 as a positive regulator for AKT in breast cancer CRISP, CRISPOR and CHOPCHOP v2 have been exploited to
cells,59 indicating that CRISPR/Cas9-based SAM library screen- predict and assess the off-target sites and the efficiency of
ing is a promising strategy for lncRNA investigation. Using a sgRNA22,23,65-78 (for detailed information, see Table 2). Off-
lentivirally delivered paired-guide RNA (pgRNA) CRISPR/ target effect can generate some unnecessary mutations outside
Cas9 library, Zhu et al. invented a high-throughput genome-
wide screening for human lncRNAs. With this method, they
found more than fifty lncRNAs that can either positively or Table 2. Common tools for off-target prediction and assessment.
negatively regulate human cancer cell growth.60
Tool name Application Classification Reference
CRISPRi, an important CRISPR/Cas9 based technology, is
also developed to screen lncRNAs. Since CRISPRi exerts its Genome Engineering Prediction Web-based 22
65
function only within a small range (1 kb) around the targeted CCTop Prediction Web-based
66
Cas-OFFinder Prediction Web-based
desire TSS,26 and dCas9 blocks only 23 bp of the targeted E-CRISP Prediction Web-based 67

sequence,61 CRISPRi can generate precise interference of any CRISPOR Prediction Web-based 68
69
lncRNA gene. Enlightened by this idea, Liu et al. developed a CHOPCHOP v2 Prediction Web-based
74
CROP-IT Prediction Web-based
CRISPRi based platform for large-scale systematic screening.62 GUIDE-Seq Detection Web-based 78

Targeting 16,401 lncRNA loci with CRISPRi libraries in diverse WGS Assessment/Detection Assay 70,73,77
71,72
cell lines, including transformed cell lines and iPSCs (human SURVEYOR Assessment/Detection Assay
23,75,76
ChIP-Seq Assessment/Detection Assay
induced pluripotent stem cells), they identified 499 lncRNAs Deep-targeted sequencing Assessment/Detection in silico 73

which are needed for robust cell growth.62 This study indicated
that the CRISPRi based platform may help to delineate the bio- CCTop, CRISPR/Cas9 target online predictor; CROP-IT, CRISPR/Cas9 off-target pre-
diction and identification tool; GUIDE-seq, Genome-wide unbiased identification
logical characteristics of lncRNA genome, and add a skill for of DSBs enabled by sequencing; WGS, Whole-genome sequencing; ChIP-seq,
large-scale screening of functional lncRNAs. chromatin immunoprecipitation sequencing.
40 J. YANG ET AL.

the desired target site and may cause some serious consequen- Challenges and perspectives
ces.70-73 However, Schaefer et al. found that two CRISPR-
Given the easy operability and economical nature of CRISPR/
treated mice (F03 and F05) shared the identical 117 indels and
Cas9 system, scientists gain a better choice in their tool kit to
1397 SNVs (single-nucleotide variants) by using whole-genome
easily and economically edit the genome of interest. Although
sequencing (WGS). The unexpectedly high number of muta-
techniques based on CRISPR/Cas9 system have many advan-
tions were induced after CRISPR/Cas9 editing in vivo.77 This
tages over ZFNs, TALENs and RNAi in gene editing and in
work manifested that off-target effect is an extensively existed
altering gene expression at the transcriptional level, there are
phenomenon in contrast to former studies. However, there are
still several limitations for scientists to overcome. Here, we
controversies about Schaefer et al’s standpoint. Lareau and
mainly discussed the limitations in ncRNA area, off-target
colleagues argued that Schaefer et al’s experimental design
effect, immune response and the ethical issues.
lacked control, and the alleged “unexpected mutations” were
Although the CRISPR/Cas9 system has been widely used to
merely due to the mice used in their experiment, which shared
edit protein-coding genes or noncoding genes in human and
the common SNPs and indels prior to the Cas9-induced
other creatures, many limitations exist, especially when it is
mutations. Therefore, data presented in Schaefer et al’s paper
applied in non-coding RNA area. For one thing, due to the lack
were insufficient to support their conclusions.79 In addition to
of distinct open reading frame (ORF), small indels generated
the viewpoint from Lareau et al., there are some other research-
by CRISPR/Cas9 system may fail to cause functional knockout
ers questioning Schaefer et al’s point of view. Kim et al.80 and
of a given non-coding gene. For another, since many lncRNAs
Wilson et al.,81 for instance, both think that the observed
are derived from sense/antisense genes or bidirectional pro-
variants in Schaefer et al’s experiments are much more proper
moters, there is a possibility to affect the overlapping or adja-
to be explained by the differences in the genetic background of
cent genes in loci that harbor multiple genes when CRISPR/
the mice rather than mutations caused by Cas9. Thus, this issue
Cas9 system is applied in lncRNA editing. This phenomenon
is currently under drastic controversy and more efforts are
occurred to the lncRNAs NOP14-AS1, LOC389641, MNX1-AS1
needed to clarify it.
etc when they were edited by CRISPR/Cas9 system.91 And this
problem with lncRNA editing by CRISPR/Cas9 system needs
to be solved prior to the use of CRISPR/Cas9 systems for edit-
Solutions toward off-target
ing lncRNAs on a genome wide scale.
To further reduce and finally, if possible, eliminate off-target The off-target effect, which could result in undesired genetic
effect, several approaches have been implemented. First, in one alterations that may lead to cancer or other knotty problems, is
study, Ran et al. described an approach, which used the mutant the main bottleneck of CRISPR/Cas9 technology. As discussed
variant of Cas9 with paired guide RNAs to cause DSBs at the above, although many efforts have been invested the improve-
desired target site, leading to dramatically minimized off-target ment of predicting and detecting the off-target effect, it is far
activity.82 Additionally, this strategy has also been generally from enough. To more precisely and effectively edit the tar-
applied by many other laboratories.83-86 Second, Slaymaker and geted sequences, researchers should exploit more techniques
colleagues showed that enhanced specificity SpCas9 (eSpCas9) and algorithms besides the approaches aforementioned.
variants can essentially reduce off-target effects and retain pow- Previous studies70-73 hold the view that off-target effect is an
erful on-target cleavage.87 Another high-fidelity variant, named uncommon phenomenon, however, Schaefer and colleagues
SpCas9-HF1, was designed to retain on-target activities and raised doubts about this conclusion. They believe that the off-
reduce non-specific DNA contacts. Due to its exceptional preci- target effect extensively exists.77 Although their viewpoint chal-
sion, SpCas9-HF1 can avoid most of the off-target mutations lenges the former conclusion, this is not a crisis for the use of
that are generally induced by wild-type SpCas9.88 Third, trun- CRISPR/Cas9, it is, on the contrary, a “pushing hand” for
cating sgRNA by 2–3nt was demonstrated to minimize unde- researchers to carefully detect the off-target effect when they
sired mutagenesis at off-target sites.86 Fourth, the delivery use this novel technology. And their work will undoubtedly
system is also an important factor which can affect the off-target drive this technology toward a better orientation.
phenomenon. Ramakrishna et al. showed that conjugating the Another obstacle is the unfavorable immune response,
Cas9 protein to cell-penetrating peptide (CPP), and then comb- which is brought by bacterial Cas9. Generally, bacterial Cas9 is
ing the gRNA to form nanoparticles as a delivery system resulted delivered by viral vectors, especially by adenoviral vectors,
in efficient genome editing with distinctly reduced off-target which are well-known to cause an immune response. In a pre-
effect compared to plasmid transfections.89 Finally, combining vious study,92 for example, researchers delivered Streptococcus
Cas9 with other nuclease may be a good choice. A study by Tsai pyogenes–derived Cas9 (SpCas9) into mice by the adenoviral
et al. identified that fusing Cas9 together with RNA-guided FokI vector to edit Pten. They found remarkable increase of antibod-
nucleases (RFNs) immensely improved the on-target specificity ies, including IgG1, IgG2a/b, as well as IL-2, in the mice which
compared to the monomeric CRISPR-Cas.90 had received SpCas9. These results raised some questions: How
Briefly, in addition to taking off-target effect into consider- can we reduce the immune response? What can we do to man-
ation when we use this powerful tool, we should keep in mind age immune response in the patients when using CRISPR/Cas9
that CRISPR/Cas9 system is not originally present in mamma- in the clinic in the future? Fortunately, with the huge number
lian genome, and thus it may cause immune response and and variety of bacteria, we have enough time to find and engi-
weaken treatment effectiveness when we push this system into neer both new and less immunogenic variants of Cas9 before
clinical application. putting CRISPR/Cas9 technology into the clinical area.
 RNA BIOLOGY 41

Ethical issues are problems that researchers cannot bypass bacterial immunity. Science. 2012;337:816-21. doi:10.1126/
before using CRISPR/Cas9-related technique in real applica- science.1225829.
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No potential conflicts of interest are disclosed. tide sequence of the iap gene, responsible for alkaline phosphatase iso-
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Funding
16. Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Hor-
This work was supported by research grants from the Natural Science vath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF, et al. Evolution
Foundation of Zhejiang (LY15C060003, LQ18H200001), the Non-profit and classification of the CRISPR-Cas systems. Nat Rev Microbiol.
Technology Research Program of Zhejiang (LGF18H160006), the Natural 2011;9:467-77. doi:10.1038/nrmicro2577.
Science Foundation of Ningbo (2017A610247), the Scientific Innovation 17. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X,
Team Project of Ningbo (2017C110019), the National Undergraduate Jiang W, Marraffini LA, et al. Multiplex genome engineering using
Training Program for Innovation and Entrepreneurship (201711646018) CRISPR/Cas systems. Science. 2013;339:819-23. doi:10.1126/
and the K.C.Wong Magna Fund at Ningbo University. science.1231143.
18. Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. RNA-programmed
genome editing in human cells. Elife. 2013;2:e00471. doi:10.7554/
ORCID eLife.00471.
19. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE,
Nan Jiang http://orcid.org/0000-0002-8423-7675 Church GM. RNA-guided human genome engineering via Cas9. Sci-
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20. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada
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