CRIPSR/Cas9 and Biosafety Issues
Mohammad Arif Ashraf and Abidur Rahman
The United Graduated School of Agricultural Sciences, Iwate University, Morioka, Japan
In last few decades, discovery and extensive use of three molecular tools; restriction
enzymes, plasmid, and ligase had a significant impact on genetic engineering. It allowed us to
cut the genetic material from one source and introduce it to another organism to get the desired
traits or results. However, genome editing in precise position was still elusive. Bacteria
originated CRISPR/Cas9 system, for the first time provided the endless opportunity to edit the
gene precisely. Specifically, with this system, one can knock-out or knock-in desired genes,
enhance or repress gene expression, create a single point mutation or even repair a mutation in
the gene. Undoubtedly the precision and convenience of system will make it the most desired
technique to manipulate the genes in both mammalian and plant species for next few decades.
One of the major issues with gene editing lies with the biosafety regulation, which literally
blocked the distribution of lots of successful genetically modified crops to the consumer market.
The question remains whether the mediated gene editing will also be under the same regulation
or can skip the conventional biosafety regulations. In the present article, we tried to discuss the
issue to increase the public awareness.
Introduction
CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) and CRISPRassociated (Cas) genes have revolutionized molecular biology. It was initially identified as an
immune system of bacteria, which helps bacteria to make its database from previous exposure to
virus or bacteriophage. Interestingly, these enigmatic repeats had been observed more than 20
years ago, and scientists had no clue about those spacer sequences. Lately, it had been found that
these spacer sequences have higher homology with bacteriophage DNA. Barrangou and
colleagues in 2007 (Barrangou et al., 2007) identified the functional significance of those
incorporated bacteriophage sequences and demonstrated that these integrated sequences help the
bacteria to become resistant to particular bacteriophage.
Mechanism of CRISPR/Cas9
CRISPR/Cas9 has two components. The first one is an enzyme, Cas9 and it functions as an
endonuclease to cut DNA. Naturally, bacteria use it to destroy the genetic code of invading
viruses. The other component is an RNA guide that leads the Cas9 to the precise nucleotides.
Upon infection, CRISPR loci are transcribed and translated. At the same time, it also produces
Cas9 protein and CRISPR RNA (crRNA). This crRNA is complementary to known or previously
invaded bacteriophage sequences. It makes the job easier for bacteria to eliminate it before the
advent of infection. In addition, this system also has the capability to incorporate unknown or
newly invader bacteriophage sequences.
The overview of mediated editing system
This bacteria originated technique has opened a new field of genetic engineering with
unlimited opportunities. All we need to do is designing guide RNA (crRNA and tracer RNA)
targeting the gene of interest. By designing guide RNA (gRNA), one can target literally any gene
regardless of organisms. gRNA contains one portion, crRNA, to target the sequence and another
component, tracer RNA, which helps to anchor with Cas9. Because of the sequence specificity,
this system is more accurate compared to other available gene editing techniques. The most
interesting part is with this technique one can introduce point mutation in the desired gene or can
repair the existing mutation in the desired gene, which is almost impossible to achieve with the
existing genome editing techniques.
The delivery system has remarkable differences considering mammalian cells and plants.
DNA delivery into mammalian cell lines is quite broad and includes several different methods.
Three major recommended delivery methods for mammalian cell lines are Transfection (lipidmediated, cationic polymers, calcium phosphate), Electroporation (nucelofection), and Viral
delivery (lentivirus, retrovirus, adenovirus, adeno-associated virus). In case of plants, for getting
stable genetically edited lines, the only option is Agrobacterium tumefaciens mediated
transformation.
Application of CRISPR/Cas9
In crops, obtaining a variety with new traits largely relies on breeding technique. The
cross-breeding is a time consuming and economically costly method. On average, to release a
new bred variety, it takes around 10 years. Hence, the scientists and farmers are in search of
techniques which may speed up the process and make it more economical and acceptable to
consumers. CRISPR system is possibly the long-sought technique which offers the mechanism to
alter the code of life very precisely. This technique will bring a new revolution in the agricultural
sector.
In mammalian system, CRISPR has also the potential application in treating inborn error
genetic diseases and helping in sustaining ecological balance. CRISPR/Cas9 may be potentially
useful in case of genetic disorder such as Cystic fibrosis (CF), where patient has shortened lives
due to chronic pulmonary infections. If both parents are carriers of defective genes, it indicates
that their children have 25% chance of having Cystic fibrosis. Genetically it is known as
autosomal recessive disease. It occurs because of mutations in the cystic fibrosis transmembrane
conductance regulator (CFTR) gene, encodes for a chloride ion channel regulated by cAMPdependent phosphorylation (Colemeadow et al., 2016). Correcting the mutated nucleotide
through CRISPR will ensure longer life span of the patients.
In case of human embryo, Junjiu Huang and his team at Sun Yat-sen University in
Guangzhou used the CRISPR/Cas9 in 'non-viable' embryos. They edited the gene called HBB,
which encodes the human β-globin protein. Mutations in this particular gene are responsible for
β-thalassaemia. This was the first example of using CRISPR/Cas9 technique in human embryos
(Liang et al., 2015). Another Chinese team at Guangzhou Medical University in China used
CRISPR/Cas9 genome editing to introduce a mutation in a gene called CCR5 into patients’
embryos. Some human individuals naturally carry this mutation which is known as CCR5Δ32.
CCR5Δ32 containing individuals are resistant to HIV. This mutation modifies the CCR5 protein
in such a way that prevents the virus to infect that individual. Last year Fan’s team claimed that
CRIPSR/Cas9 mediated mutation in CCR5 may help to cure HIV infection (Kang et al., 2016).
CRISPR/Cas9 can also be used to alter the ecological landscape. James and his team at
University of California, Irvine published a paper in PNAS two years ago explaining how
CRISPR will help to engineer a version of Anopheles mosquitoes that makes them incapable of
spreading the malaria parasite. They added a small portion of genes that will not hamper the
normal functions of mosquitoes but will prevent them to transmit parasites. This CRISPRmediated change will eventually make mosquitoes sterile and inept to infect humans (Gantz et al.,
2015).
The finest example of the advantage of using CRISPR/Cas9 system for basic plant
biologists was published in 2015. ABP1 (Auxin Binding Protein 1) was considered as auxin
receptor for more than two decades. Newly published research in PNAS used CRISPR/Cas9
system to knock out this particular gene and showed that there is no developmental defect and
change in auxin response (Gao et al., 2015). This research highlights the problem of off-target
gene silencing by other genome editing technique and at the same time provided the evidence of
importance of precise editing. .
In agriculture, successful stories using CRISPR/Cas9 are emerging. Scientists have used
this system to insert mutation in the tomato genome. One research group in Japan (Ito et al.,
2015) targeted the tomato RIN gene, which encodes MADS-box transcription factor regulating
fruit ripening. Three regions within the gene were targeted and by using CRISPR-cas9 either
insertion or deletion was induced in the targeted regions. RIN mutated tomato has incomplete
ripening property compared to regular tomatoes (Ito, Nishizawa-Yokoi, Endo, Mikami, & Toki,
2015). Another example came from Caixia Gao and her team at the Chinese Academy of
Sciences in Beijing. They have created a wheat strain that is resistant to powdery mildew by
introducing mutation in TaMLO-A1 (Wang et al., 2014).
Effect of mediated late ripening phenotypes in tomatoes (Ito et al., 2015)
Ailsa Craig is used as wild type and LA3754 shows mutational phenotype for rin. G1, G2, and
G3 are rin mutants obtained by CRISPR-Cas9 technique.
Biosafety issues of CRISPR/Cas9
This new genome editing tool has enormous power to solve our food security issues. But,
one of the major hindrances is the approval of transgenic crops. Conventional genetically
modified crops have encountered the same issue, and most of them have failed to reach the
market as commercial variety in most part of the globe because of the fervent protests of the antiGMO activists.
Anti-GMO activists opposition to genetically modified crops largely centered on two
issues. 1) Insertion of foreign DNA (DNA from other organisms) to plants may have deleterious
effect to human health in the long run, and 2) Insertion of T-DNA along with antibiotic
resistance genes. For instance, Golden Rice is a GMO engineered to contain genes necessary to
produce vitamin A in the edible part of the grain, something that doesn’t happen naturally in rice
plants. In Golden rice, two genes were introduced. One is phytoene synthase (psy) originating
from daffodil (Narcissus pseudonarcissus) under the control of the endosperm-specific glutelin
(Gt1) and another one is a bacterial phytoene desaturase (crt1) originating from Erwinia
uredovora placed under the constitutive CaMV (cauliflower mosaic virus) 35S promoter (Ye et
al., 2000). Combination of these two enzymes - phytoene synthase and phytoene desaturase –
helps to produce beta-carotene, a precursor of vitamin A, in the edible part of the rice. Each year
up to half a million children in developing world go blind for lack of vitamin A, and this golden
rice could easily solve the problem. But anti-GMO activists have interfered with research and
prevented any commercial production of Golden rice. Transferring genes from viral or bacterial
source is highlighted as one of the major concerns by anti-GMO group. This particular problem
of inserting foreign gene is avoided in CRISPR/Cas9 system as through this technique only the
endogenous genes are edited. No foreign genes would be introduced. Moreover, using
CRISPR/Cas9, it is possible to introduce point mutation in desired genes, which is impossible to
achieve using the existing gene editing techniques. The option of introducing point mutation by
CRISPR/Cas9 will have a profound impact in agriculture as it will ultimately result in insertion
of SNPs (Single Nucleotide Polymorphism) in the targeted crop genomes. In the breeding
process, farmers cross among varieties to get desired traits and deep down genetic mechanism is
incorporation of SNPs. CRISPR-mediated technique will serve the purpose of inserting SNPs in
a more precise manner and shorter the time compared to traditional breeding.
The second issue of the anti-GMO activists is T-DNA and antibiotic resistance gene
insertions in plant system. However, what they are missing is T-DNA is a natural DNA present
in Agrobacterium tumefaciens, a soil borne bacteria used for gene transformation. A recent study
showed that among available 291 accessions of sweet potato, all of them contain one or more
transfer DNA (T-DNA) sequences. These sequences are analyzed in one accession known as
“Huachano” and suggested that an Agrobacterium infection occurred in evolutionary times
(Kyndt et al., 2015). The study is a profound example of natural insertion of T-DNA in crops and
indicates that Agrobacterium and T-DNAs are natural component in our edible food stuffs and
pose no threat for health. The last issue is antibiotic resistance genes used for selection of
transformed plants. Actually, it is possible to get transgene-free genetically edited crops in 13:3
ratio (Khatodia et al., 2016) in a very short period of time. With a proper selection process, the
CRISPR edited plants can produce transgene free offspring, which should have no issues for
health safety, should satisfy anti-GMO activists and bypass the current biosafety regulation.
Modified white button mushroom to reduce browning by (Agapito-Tenfen, 2016)
The above conclusion of bypassing the biosafety reguation of CRISPR edited crops is
eleganly supported by the recent work of Yinong Yang group at Chinese Academy of Science.
They engineered common white button mushroom (Agricus bisporus) by CRISPR method to
prevent browning by deleted few base pairs in the mushroom’s genome which encodes one of six
polyphenol oxidase (PPO) enzyme, responsible for browning, and reduced the enzyme activity
up to 30%. US Department of Agriculture (USDA) indicated that they will not regulate this
engineered mushroom modified through CRISPR-cas9 tool (Agapito-Tenfen, 2016) as no
foreign DNA was inserted.
In summary, the CRISPR/Cas9 technology, which is arguably the safest genome editing
technology, has an enormous potential to bring another revolution in agriculture and solve the
burning issue of future food security. For now, the most important task for the plant scientist’s
community would be to convey the message of advantage of using CRISPR-cas9, explain why it
should be under minimal biosafety regulation, and how it can improve our food production to the
government body as well as the common people. Building a strong public opinion and awareness
would be crucial for using this wonderful technique in our country.
References
Agapito-Tenfen, S. Z. (2016). Biosafety Briefing. Nature. Volume 532, Issue 7599.
Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., . . . Horvath, P.
(2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science,
315(5819), 1709-1712.
Colemeadow, J., Joyce, H., & Turcanu, V. (2016). Precise treatment of cystic fibrosis–current
treatments and perspectives for using CRISPR. Expert Review of Precision Medicine and
Drug Development, 1(2), 169-180.
Gantz, V. M., Jasinskiene, N., Tatarenkova, O., Fazekas, A., Macias, V. M., Bier, E., & James, A.
A. (2015). Highly efficient Cas9-mediated gene drive for population modification of the
malaria vector mosquito Anopheles stephensi. Proceedings of the National Academy of
Sciences, 112(49), E6736-E6743.
Gao, Y., Zhang, Y., Zhang, D., Dai, X., Estelle, M., & Zhao, Y. (2015). Auxin binding protein 1
(ABP1) is not required for either auxin signaling or Arabidopsis development.
Proceedings of the National Academy of Sciences, 112(7), 2275-2280.
Ito, Y., Nishizawa-Yokoi, A., Endo, M., Mikami, M., & Toki, S. (2015). -mediated mutagenesis
of the RIN locus that regulates tomato fruit ripening. Biochemical and biophysical
research communications, 467(1), 76-82.
Kang, X., He, W., Huang, Y., Yu, Q., Chen, Y., Gao, X., . . . Fan, Y. (2016). Introducing precise
genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome
editing. Journal of assisted reproduction and genetics, 33(5), 581-588.
Khatodia, S., Bhatotia, K., Passricha, N., Khurana, S., & Tuteja, N. (2016). The CRISPR/Cas
genome-editing tool: application in improvement of crops. Frontiers in plant science, 7.
Kyndt, T., Quispe, D., Zhai, H., Jarret, R., Ghislain, M., Liu, Q., . . . Kreuze, J. F. (2015). The
genome of cultivated sweet potato contains Agrobacterium T-DNAs with expressed
genes: an example of a naturally transgenic food crop. Proceedings of the National
Academy of Sciences, 112(18), 5844-5849.
Liang, P., Xu, Y., Zhang, X., Ding, C., Huang, R., Zhang, Z., . . . Li, Y. (2015). -mediated gene
editing in human tripronuclear zygotes. Protein & cell, 6(5), 363-372.
Wang, Y., Cheng, X., Shan, Q., Zhang, Y., Liu, J., Gao, C., & Qiu, J.-L. (2014). Simultaneous
editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to
powdery mildew. Nature biotechnology, 32(9), 947-951.
Ye, X., Al-Babili, S., Klöti, A., Zhang, J., Lucca, P., Beyer, P., & Potrykus, I. (2000).
Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free)
rice endosperm. Science, 287(5451), 303-305.