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 Table of Contents  
Year : 2017  |  Volume : 3  |  Issue : 5  |  Page : 174-180

Recent progress in technological improvement and biomedical applications of the clustered regularly interspaced short palindromic repeats/cas system

1 Translational Medicine Institute, National and Local Joint Engineering Laboratory of High-Through Molecular Diagnostic Technology, The First People's Hospital of Chenzhou, University of South China, Chenzhou; Hunan Province Key Laboratory of Tumor Cellular and Molecular Pathology, Cancer Research Institute, University of South China, Hengyang, Hunan, China
2 Translational Medicine Institute, National and Local Joint Engineering Laboratory of High-Through Molecular Diagnostic Technology, The First People’s Hospital of Chenzhou, University of South China, Chenzhou, Hunan, China
3 Department of Pharmacology, Southern Illinois University, School of Medicine, Springfield, IL, USA
4 Hunan Province Key Laboratory of Tumor Cellular and Molecular Pathology, Cancer Research Institute, University of South China, Hengyang, Hunan, China
5 Division of Stem Cell Regulation and Application, School of Pharmacy, Hunan University of Chinese Medicine, Changsha, Hunan, China
6 Translational Medicine Institute, National and Local Joint Engineering Laboratory of High-Through Molecular Diagnostic Technology, The First People's Hospital of Chenzhou, University of South China; Center for Pathology, The First People's Hosptial of Chenzhou, Southern Medical University, Chenzhou, Hunan, China

Date of Submission21-Jun-2017
Date of Acceptance08-Oct-2017
Date of Web Publication26-Oct-2017

Correspondence Address:
Di-Xian Luo
Center for Pathology, The First People's Hospital of Chenzhou, Southern Medical University, Chenzhou 423000, Hunan
Duanfang Liao
Division of Stem Cell Regulation and Application, School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, Hunan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ctm.ctm_22_17

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The adaptive immune systems of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas) selectively destroy nonnative DNA and defend almost all archaea and about half of the bacteria against infections. In the past years, the system has been genetically engineered to a powerful genome editing tool for a wide variety of organisms. Recently, many progresses have been made in the CRISPR-Cas systems. These improvements include applications in editing multiple genes, correcting mutation genes with one base difference, targeting nondividing cells, reducing off-target, and editing RNAs. The biomedical applications of the technology are to edit not only cells but also embryos in clinical settings. In this review, we briefly introduce the improvements of CRISPR-Cas9 gene editing methods and summarize the recent advances of this technology.

Keywords: Clustered regularly interspaced short palindromic repeats, clustered regularly interspaced short palindromic repeats-Cas, gene editing, progress

How to cite this article:
Li Y, Hu Z, Yin Y, He R, Hu J, Luo W, Li J, Wen G, Xiao L, Li K, Liao D, Luo DX. Recent progress in technological improvement and biomedical applications of the clustered regularly interspaced short palindromic repeats/cas system. Cancer Transl Med 2017;3:174-80

How to cite this URL:
Li Y, Hu Z, Yin Y, He R, Hu J, Luo W, Li J, Wen G, Xiao L, Li K, Liao D, Luo DX. Recent progress in technological improvement and biomedical applications of the clustered regularly interspaced short palindromic repeats/cas system. Cancer Transl Med [serial online] 2017 [cited 2018 Dec 14];3:174-80. Available from: http://www.cancertm.com/text.asp?2017/3/5/174/217259

Yanlan Li, Zheng Hu
These authors contributed equally to this work and are co-first authors.

  Introduction Top

For almost all archaea and about half of bacteria, the adaptive immune systems of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas) selectively destroy nonnative DNA and defend them against infections.[1],[2] To protect microbes from viruses and other invading DNA, the CRISPR-Cas systems act in three steps: (i) Incorporate foreign DNA sequences into CRISPR arrays, (ii) produce mature CRISPR RNAs (crRNAs) containing “protospacer” regions that are complementary to the foreign DNA site, and (iii) guide Cas enzymes by the crRNAs to target and cleave foreign DNAs bearing cognate sequences in the respective invader genomes.[3],[4] All CRISPR-Cas systems characterized to date follow these three steps, although the mechanistic implementation and proteins involved in these processes display diverse extensively.

The CRISPR-Cas9 is a simple system for genome editing and it requires only two CRISPR components, Cas9 and gRNA. The Cas9 enzyme snips through DNA like a pair of molecular scissors along with a single guide RNA (sgRNA or gRNA) by directing the scissors to a specific sequence of DNA. Recently, the system has been developed into a powerful multiplex genome targeting and homologous-directed repair (HDR)-based precise genome editing for a wide variety of organisms. The CRISPR-Cas9 technology has displayed great potentials for treating disease or editing the genes of human embryos and has leaped from laboratory to industry. In the past years, many exciting progresses in the CRISPR-Cas9 system make the field of gene editing to receive worldwide attentions. In this review, we briefly introduce the improvements of CRISPR-Cas9 gene editing methods and summarize the recent advances of this technology.

  Progress of Clustered Regularly Interspaced Short Palindromic Repeats Gene Editing Technology Top

Despite the power of the CRISPR-Cas9 system, it does have some limitations in applications. This has led many scientists to improve the gene editing technique to be more precise.

Editing multiple genomic loci or at one base resolution

Targeting multiple genomic loci with Cas9 is limited by the requirement for multiple or large expression constructs.[5],[6],[7],[8] By contrast, Cpf1, a class II CRISPR endonuclease, has features that are distinct from Cas9, requiring only one Pol III promoter to process several crRNA, and it alone is sufficient for the maturation of crRNA.[9],[10],[11],[12] Taking advantage of the ability of Cpf1, Zetsche et al.[13] used a single customized CRISPR array to edit up to four genes in mammalian cells and three in the mouse brain simultaneously. Zhong et al.[14] also used Cpf1 orthologs to modify multiple genomic targets in a simple way. Ren et al.[15] simultaneously introduced Cas9 RNA and gRNAs targeting TCR, beta-2 microglobulin (B2M), and PD1, using electroporation and Lentivirus, respectively, to generate the CAR T-cells with deficient TCR, HLA class I molecule, and PD1. Liu et al.[16] also used CRISPR-Cas9 system to knock out a pairs of genes (TRAC and B2M) or three genes (TRAC, B2M, and PD-1) simultaneously in the CAR T-cells. Ma et al.[17] described that CRISPR Rainbow can document six chromosomal loci simultaneously. Komor et al.[18] fused CRISPR-dCas9 and a cytidine deaminase enzyme to mediate one base conversion of cytidine to uridine. Nishida et al.[19] combined a sea lamprey-derived “nickase” Cas9, which can create a DNA nick, with the activation-induced cytidine deaminase (AID) to generate a synthetic complex (target-AID), which can edit a single DNA nucleotide and perform highly efficient target-specific mutagenesis. Kim et al.[20],[21] even used the single-base-pair substitutions in mouse embryo cells and confirmed the accuracy of the “base editors.” For human gene therapy, more sophisticated technologies to edit genes with one mismatch are desired, thus requiring either improvement of current systems or identification of new enzymes.

Targeting nondividing cells

The successful application of CRISPR-Cas9 system in gene therapies requires the establishment of appropriate delivery and repair strategies. By taking advantages of endogenous DNA replication mechanisms, the CRISPR-Cas9 system is effective in dividing cells, such as those in skin or gut. However, in non- or slowly-dividing cells such as muscles and neurons, further improvement of CRISPR-Cas9 in efficiency is highly desired. The researchers in the Salk Institute paired nonhomologous end-joining, a DNA-repair cellular pathway that repairs double-strand breaks (DSBs) in DNA without the guidance of a homologous template, with existing gene-editing technology, to successfully knock-in DNA into a precise location in nondividing cells.[22] For the first time, scientists are able to insert a new gene into a precise DNA location in nondividing adult cells, such as those in the eye, brain, pancreas, or heart, thus offering new therapeutic applications in these cells. To understand what happens in our brains as we learn and memorize, Mikuni et al.[23] developed a method called SLENDR (single-cell labeling of endogenous proteins by CRISPR-Cas9-mediated homology-directed repair), which could be used to precisely modify the DNA in neurons in living samples. Together with utero electroporation, this technique allowed to insert DNA into prenatal brain cells that were still in developing and dividing stages through HDR. This method also allows to reliably label two different proteins with distinct colors in the same cell at the same time. Yao et al.[24] devised a homology-mediated end joining-based strategy, which contains guide RNA target sites and 800-bp of homology arms, and yielded a higher knock-in efficiency in neurons.

Reducing off-target and improving functionality

Due to off-target activities of Cas9 proteins, the CRISPR-Cas9 system can induce unwanted off-target mutations.[25] Therefore, extensive research efforts have been focused on reducing the off-target activities of the CRISPR-Cas9 system.[6],[26],[27],[28] Although an enhanced variant of Streptococcus pyogenes Cas9 (SpCas9) exhibits reduced off-target activities and robust on-target activities, its efficiencies are insufficient and it is far from applications.[29] SpCas9-HF1 is a Cas9 mutant and contains shorter amino acid side chains that bind to DNA, thus rendering all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods.[30] Researchers at Western University fused the I-TevI nuclease domain to Cas9 to generate TevCas9 nuclease, which cuts DNA in two sites instead of one, making it much more difficult for the DNA repair to regenerate the cleavage site. TevCas9 nuclease functioned robustly in HEK293 cells and generated 33- to 36-bp deletions at frequencies of up to 40%.[31] Researchers at the University of California, Berkeley, designed a new approach based on the discovery that, after cutting DNA, the Cas9 protein remains attached to the chromosome for up to 6 h, long after it has sliced through the double-stranded DNA, and they achieved an astonishing success rate of 60% when replacing one short stretch of DNA with another.[32] Kim et al.[33] found that off-target effects were completely abrogated using CRISPR-Cpf1 endonucleases. Not like other researches who focused on improving Cas9 nuclease, Doench et al.[34] developed a method to optimize gRNA to reduce off-targets. Some software, such as CRISPETa (the Centre for Genomic Regulation, Barcelona, Spain) and CRISPRdirect (Database Center for Life Science, Chiba, Japan), can scan genome to avoid off-target effects.[35],[36] Two teams even developed methods to identify off-target mutations of CRISPR-Cas9.[37],[38]

Keeping clustered regularly interspaced short palindromic repeats in check with anti-Cas9

Small proteins specifically inhibiting non-Cas9 type I CRISPR-Cas systems were discovered previously, while three bacteriophage-encoded anti-Cas9 genes were recently described.[39] By transfecting human cells in culture with plasmids encoding Cas9, a gene-targeting guide RNA, and the anti-CRISPR protein, researchers found that the new protein prevented Cas9 from binding to its RNA-specified genomic site, thus blocking Cas9 from editing the genome. These anti-Cas9 proteins were able to block the activity of CRISPR-Cas9 in human cells.[40] These findings allow researchers to better understand naturally occurring CRISPR systems and better modulate the activity of CRISPR-based gene-editing tools for research and clinical applications. Another team also found four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages and two of these inhibitors can block the widely used SpCas9.[41] To understand the Cas9 inhibitor proteins clearly, Shin et al.[42] studied the anti-CRISPR protein AcrIIA4 to establish the mechanism of CRISPR-Cas9 inhibition. Whether these anti-Cas9 proteins could substantially decrease off-target effects as claimed still needs further evaluation.

Targeting only RNA

Although DNA serves as the fundamental building blocks of life, RNAs are associated with many diseases including cancer and autism. DNA editing can make the cell genome to produce permanent changes, while the CRISPR RNA-targeted methods allow the researchers to generate temporary changes in RNAs. Abudayyeh et al.[43] found that C2c2 is guided by a single crRNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Compared with the existing methods of RNA interference, C2c2 has gained attentions for its higher specificity as an RNA-targeting tool. East-Seletsky et al.[44] also found that C2c2 has two distinct RNase activities, one is responsible for CRISPR RNA maturation and the other is responsible for RNA-activated single-stranded RNA degradation activity. The system snips and recognizes RNA segments, thus allowing for RNA detection in bacteria and for diagnostic applications.[45] By applying CRISPR-Cas9 to RNA, Nelles et al.[46] altered several features of this system to track RNA movement inside cells. They tracked the RNAs encoding the proteins ACTB, TFRC, and CCNA2 and revealed the movement of RNA into stress granules, a cluster of proteins, and RNAs that form in cytoplasm when cells are under stress. The CRISPR-Cas9 was fused with reverse transcriptases, allowing to recognize and bind target RNAs and convert them into complementary DNAs.[47]

  Clustered Regularly Interspaced Short Palindromic Repeats Editing in Cell Level Top

What CRISPR offers and biologists desire is to disrupt a gene to learn about what it does in the vast expanse of a genome. Researchers worldwide are fast adopting CRISPR-Cas9 to modify the genomes of humans, viruses, and bacteria.

Treating diseases by targeting the genomic DNA

The most promising applications of the CRISPR-Cas9 technology are the direct treatments of deleterious genetic diseases and cancer. In early 2016, three groups used adeno-associated virus to deliver CRISPR-Cas9 into Duchenne muscular dystrophy mice and successfully restored muscle functions by genome editing in vivo.[48],[49],[50] Dever et al.[51] combined Cas9 and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the β-globin (HBB) gene in hematopoietic stem cells (HSCs) to cure β-hemoglobinopathies that were caused by mutations in the HBB gene. Ou et al.[52] attempted to use CRISPR-Cas9-corrected iPSC-derived HSCs to cure this disease. DeWitt et al.[53] replaced the sickle cell disease mutations in human HSCs with CRISPR to develop new treatments. These studies provided a path toward the development of new treatments for hematopoietic diseases. Using gRNA/Cas9 plasmid in combination with electroporation-generated allele-specific disruption of RhoS334, Bakondi et al.[54] prevented retinal degeneration and improved visual functions. Latella et al.[55] edited the human rhodopsin (RHO) gene in a mouse model for autosomal dominant retinitis pigmentosa. Sin et al.[56] restored arginase-1 in Arginase-1 deficiency genetic disorder in humans. Yang et al.[57] reduced the toxicity of polyglutamine to alleviate motor deficits of Huntington's disease and did not affect the mice's viability. Eliminating the entire HIV-1 DNA with CRISPR-Cas9, two teams might find a novel and effective method toward curing AIDS.[58],[59] Tyrosine kinase inhibitors that target abnormal epidermal growth factor receptor pathways cause drug resistance in cancer patients. Tang and Shrager[60] proposed “molecular surgeries” to repair or destroy EFGR mutations on genomic DNA, thus directly targeting the cause of the disease. Wan et al.[61] found that the YEATS domain-containing protein ENL, as a histone acetylation reader, is required for disease maintenance in acute myeloid leukemia. Targeting cancer-causing “fusion genes” may be a specific cancer therapy.[62] Manguso et al.[63] identified new immunotherapy targets for cancer through screening thousands of genes in tumor models using CRIPSR. CRISPR-Cas9 can be used to diagnose and inactivate cancer mutations or rapidly make model of the entire spectrum of cancer of cancer and metastasis possible, thus opening up new possibilities for cancer treatments.[64]

Screening disease-related proteins or genes

Mutating genes with CRISPR-Cas9 have been found in cultured human cells of the gastrointestinal infection Clostridium difficile. The research group led by Tao et al.[65] has identified a cell surface protein called Frizzled after four rounds of screening and sequencing. They found that toxins bind Frizzled to enter cells, and based on this discovery, they developed a potential strategy to protect colons. Using CRISPR-Cas9, Stroud et al.[66] modified a series of cells, of which each cell type lacks a unique gene relating to human mitochondrial Complex I proteins. They not only identified two new genes to bind Complex I, but also uncovered the functional importance of thirty protein components in the complex. Their research shed light into genetic diagnosis of the mitochondrial disease and could be beneficial in identifying potential therapeutic targets for treatments. Noroviruses are the most common viral cause of diarrhea worldwide. However, it is largely unknown about the host factors that mediate norovirus invasion, replication, and pathogenesis, and consequently determine the species tropism of the virus. Orchard et al.[67] performed a genome-wide CRISPR screen for host factors that were required for the infection of mouse cells by murine norovirus (MNoV) and discovered a proteinaceous cellular receptor CD300lf, the primary determinant of MNoV species tropism. Savidis et al.[68] applied the same method and discovered that dengue virus and Zika virus require the host endoplasmic reticulum membrane complex for their early stages of infection. Using CRISPR screen, Spielmann et al.[69] identified ZAK as a key player in mammalian limb patterning. Tzelepis et al.[70] improved and applied CRISPR-Cas9 to identify the genetic vulnerabilities of AML and they found that targeting KAT2A can destroy AML cells in the laboratory while sparing healthy blood cells. The results of these studies have important applications for disease treatments in the near future.

Creating a DNA record to trace the events of cell

The bacterium uses the CRISPR system to snip short DNA elements from virus genomes to integrate into its own genome at the CRISPR locus, thus protecting from virus infection. The RNAs produced from the integrated elements direct the destruction of the corresponding virus. In essence, the bacterium keeps a DNA account of its viral foes, highlighting its potential use in terms of this recording capacity. Shipman et al.[71] identified an Escherichia coli strain that contains a CRISPR DNA locus and a new version of Cas1 and Cas2 that is required for integrating but not destructing the DNA oligomers. They also found that the registered virus sequences at CRISPR loci did accurately reflect the order of specific synthetic DNA sequences that were introduced into cells in a time-dependent manner, thus producing information in living cells like a digitized image.[72] Using a sgRNA-encoding DNA locus to tag a 5'-NGG-3' PAM downstream of the specificity-determining sequence encoding region, Perli et al.[73] developed a cassette, which can take continuous DNA mutagenesis as a function of sgRNA expression. They used this cassette to analyze the temporal sequence evolution dynamics of sgRNAs and create a recording metric. Associating sgRNA or Cas9 expression to specific biological events of interest, such as the treatments of small molecules, exposure to TNFα, or LPS-induced inflammation, the researchers validated Mammalian Synthetic Cellular Recorder Integrating Biological Events (mSCRIBE) as an analog memory device that records information about the duration and/or magnitude of biological events. Moreover, Perli et al.[73] demonstrated that multiple biological events can be simultaneously monitored using independent sgRNA loci. To trace whole-organism lineage, McKenna et al.[74] developed genome editing of synthetic target arrays (GESTALT), a method that generates and accumulates a combinatorial diversity of mutations over many cell divisions within a compact DNA barcode consisting of multiple CRISPR-Cas9 target sites. This method allows to sequence the edit barcodes and analyze the edit patterns to track cell lineages. The team used this approach to trace the lineages of cells in adult Zebrafish organs and found that each adult tissue originated from a small number of founder cells. GESTALT can also be used to track cell lineages in tumors as they grow and metastasize, or to explore the cells that are responsible for regenerating given tissues. A mutation that occurs earlier in an animal's development will appear in many of its cells; however, more recent mutations will appear in a smaller population of cells. Researchers have developed memory by engineered mutagenesis with optical in situ readout, a new method consisting of the seqFISH (sequential single molecule fluorescence in situ hybridization) method and CRISPR editing, to record the life history of animal cells, including their relations with other cells, communication patterns, and the influential events that have shaped them.[75] Bertero et al.[76] developed single-step optimized inducible gene knockdown or knockout (sOPTiKD or sOPTiKO) platforms for the study of human development.

Mapping phenotypic traits to specific loci

During mitosis, DSBs in one arm of a chromosome can be repaired by a mechanism called homologous recombination. It can use the other chromosome in the homologous pair to replace the sequence in the broken arm. Normally, such mitotic recombination happens so rarely as to be impractical for mapping phenotypic traits to specific loci. With CRISPR-Cas9 technology, however, Sadhu et al.[77] used a targeted DSB in one arm of a yeast chromosome labeled with a green fluorescent protein gene to generate a recombined region from the site of the break to the chromosome end using HDR. After this cell divides, each daughter cell will contain a homozygous section known as “loss of heterozygosity.” Sadhu et al.[77] could direct DSBs to any locus along a chromosome of interest, thus controlling the sites of recombination and allowing high-resolution genetic mapping of phenotypic traits in yeast. Using CRISPR-Cas9 blocking mutations along with an inverse relationship of mutation's insert rate with its distance, Paquet et al.[78] achieved a predictable zygosity control and found that the Alzheimer's disease-causing mutations in amyloid precursor protein (APP Swe) and presenilin 1 (PSEN1M146V) are important in human neurons.

  Clustered Regularly Interspaced Short Palindromic Repeats Editing in Gene Engineering Animal Top

Gao et al.[79] used a single Cas9 nickase (Cas9n) to create a modified version of CRISPR-Cas9n to successfully insert a tuberculosis resistance gene, NRAMP1 (natural resistance-associated macrophage protein-1), into the cow genome without off-target effects on the animal genomes. They employed the meticulous approach to identify a best-suited region for gene insertion, which can be targeted with the modified CRISPR to insert new genes and benefit agricultural livestock successfully. To generate pigs that are resistant to Porcine Reproductive and Respiratory Syndrome virus (PRRSV) infection, Burkard et al.[80] used CRISPR-Cas9 to edit CD163, a fusion receptor for PRRSV.

  Clustered Regularly Interspaced Short Palindromic Repeats Editing in Human Embryo Level Top

Chinese researchers reported two cases of gene editing in human embryos that were unable to develop into a baby.[81],[82] Tang et al.[83] even demonstrated that CRISPR-Cas9 can work in normal human (dual pronuclear, 2PN) zygotes. Developmental biologist Fredrik Lanner of the Karolinska Institute in Stockholm is the first researcher to publicly acknowledge editing genes in viable human embryos. Lanner is attempting to edit genes using CRISPR-Cas9 in human embryos to understand genetic regulation during early embryonic development, favoring the treatment and prevention of miscarriages. This will cause permanent alterations on the human genome. However, the public is worried about such types of research, especially those using viable embryos, which causes ethical issues and alters human genome permanently. In February 2016, the United Kingdom granted developmental biologist Kathy Niakan of the Francis Crick Institute in London a license to perform gene editing on early human embryos; however, these experiments have not yet been done.

  Clustered Regularly Interspaced Short Palindromic Repeats Editing in Clinical Level Top

Lu et al.[84] applied the CRISPR-Cas9 technology to knock out gene PDCD1 in the T-cells. PD-1 inhibits immune responses against cancer cells and allows cancer cells to proliferate. The engineered T-cells with PDCD1 knock out were cultured in the laboratory and reintroduced into the patient bloodstream to circulate. In a clinical trial, the modified T-cells without PD-1 were delivered into a patient of aggressive lung cancer to attack cancer cells. To enhance survival of T-cells and improve the immunotherapies for cancer, Eyquem et al.[85] delivered a CAR gene to T-cell receptor alpha chain in T-cell genome using CRISPR-Cas9. Moreover, in a US clinical trial, multiple targets were proposed to edit in participants' cells using CRISPR, aiming to treat different cancers.

  Conclusion and Prospect Top

To date, CRISPR-Cas9 has been quickly optimized and applied to many fields, such as gene therapy, synthetic biology, functional genomic screening, and transcriptional modulation of current model systems. The exciting advances of CRISPR-Cas9 technology in genome editing not only attract close attentions worldwide but also inspire researchers to push forward human genome editing and its implications. Given the potential benefits of genome editing-based therapies, many groups are now striving to make CRISPR-Cas9-based therapies a reality. Further improvement of gene editing technology and wider application of this exciting technology will come in the near future.

Financial support and sponsorship

This work was supported by grants from the National Natural Science Foundation of China (81372825), the China Postdoctoral Science Foundation (2016T90765), the Natural Science Foundation of Hunan Province (2016JJ2014, 2015JJ4094, and 2017JJ2004), the Public Entrepreneurship and Innovation Building Special Grant of Hunan Province ((2016)1069), the Strategic Emerging Industries and New Industrialization Special Grant of Hunan Province ((2015)68), the Natural Science Foundation of Guangxi (No. 2015GXNSFEA139003), the Education Department Project of Hunan Province (13C882), the Health Department Project of Hunan Province (B2012-157, B2015-177, and C2013-009), the Construct Program of the Key Discipline in Hunan Province (Basic Medicine Sciences in University of South China), the Young Natural Science Foundation of Chenzhou (CZ2013063), and the Introducing Foreign Intelligence Project ((2015)116).

Conflicts of interest

There are no conflicts of interest.

  References Top

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