The Peculiarities Of Genome Engineering

Abstract

Genome editing has recently emerged as an important tool for biomedical research and provides hope for correcting inherited diseases. New developments in genome editing have allowed scientists to manipulate a specific gene in a variety of species and tissues, including cells grown in vitro, and animal organs. This article describes the basic principles of genome editing, particularly the CRISPR/Cas9 system, as well as the potential application of genome editing tools in the treatment of various human diseases.

Introduction

Genome editing, or genome engineering, allows scientist to precisely and efficiently introduce a variety of genetic alterations in the DNA of living organisms, such as gene insertions or deletions, modification of single nucleotide variants and deletion of chromosomal regions. Older genetic engineering methods randomly insert DNA into a host genome, however genome editing targets the DNA modifications to site specific locations [1].

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Genome editing requires the use of highly specific, engineered nucleases. The nucleases create site-specific double-strand breaks (DSBs) at targeted sites in the genome. The induced double-strand breaks are repaired by a DNA repair mechanism know as homology directed repair (HDR), resulting in the desired change being inserted at the site of the DSB. Routinely used restriction enzymes normally recognize and cut at multiple sites in DNA. To create site-specific DSB, different types of nucleases have been found and engineered: The zinc finger nucleases (ZFNs), meganucleases, transcription-activator like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) system, are all examples of programmed nucleases currently being used [2].

Genome editing is now a routine experimental strategy in many research laboratories, and is used for a range of experimental systems from plants to animals. For examples, the generation of transgenic animals, studying gene function with stem cells, endogenous gene labelling and targeted gene mutation is now routinely carried out in biomedical research laboratories [3,4].

CRISPER/Cas9 Genome Editing

Compared to other existing genome editing methods, the CRISPR/Cas9 system is faster, cheaper, more accurate and more efficient. This method has been used in a range of models, for example transgenic animals, cell lines, plants and even human clinical trials [4,5]. The CRISPR/Cas9 technology was adapted from a naturally occurring genome editing defense system of certain bacteria against viruses and plasmids. The CRISPR/Cas9 method involves directing the Cas9 nuclease to create a sitedirected doublestrand DNA break using a small RNA molecule as a guide. Permanent modification of the gene target sequence is thus enabled by this technique, resulting in the specific repair of the damaged DNA [6].

Disease treatment and prevention

Genome editing, specifically CRISPR/Cas9, has immense therapeutic potential for treating and prevention of various human diseases in which the genetic cause of dysfunction is known. Through the creation of cell or animal models, many diseases can be studied in-depth,and with greater ease and efficiency than ever before.

An example of how gene editing has been used for correcting a disease mutation in stem cells is demonstrated by a recently published study on cystic fibrosis [7]. Intestinal stem cells of cystic fibrosis patients were obtained and the homozygous ∆508 mutation in the cystic fibrosis trans-membrane conductance regulator (CFTR) gene was corrected using CRISPR/Cas9 in vitro. Sickle-cell disease (SCD) and β-thalassemia are β-hemoglobinopathies, which are caused by mutations in the β-globin (HBB) gene and affect a significant percentage of the human population. Preclinical studies have outlined a CRISPR-based methodology for targeting hematopoietic stem cells (HSCs) and induced pluripotent stem cells (iPSCs) by homologous recombination at the HBB gene locus [8].

Genome editing also holds promise for the treatment and prevention of more complex diseases, such as cancer, neuropsychiatric disorders, human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) and cardiovascular disease [9,10,11,12].Manipulation of normal and cancer cell genomes is vital for disease modelling, and for studying genes involved in cancer initiation, progression and therapy [9].Experiments using CRISPR/Cas9 technology have identified genes NANOG and NANOGP8, which contribute to the high malignant potential of prostate cancer. Knockouts of these two genes in human prostatecell lines significantly decreased the malignant potential, compared with that of parental prostate cell lines [13].BCR/ABL fusion, which is responsible for Chronic Myeloid Leukemia pathogenesis, is an ideal target for gene therapy. The CRISPR/Cas9 genomic editing system has been used to modify the BCR/ABL fusion gene successfully preventing its possible oncogenic effects. The study shows a CRISPR-Cas9 application for truncating the specific BCR-ABL fusion (p210) at the genetic level in a cellular model [14]. In HIV/AIDS treatment and prevention, CRISPR/Cas9 was used both in vitro in human patient cells and in vivo in animal models. Gene editing by this method can likely result in fully curing HIV/AIDS by eliminating or disrupting HIV-integrated genomes or HIV-infected cells from multiple HIV reservoirs [10].

Gene editing has also been used for the study of gene function and for gene drive to suppress mosquito population levels, and thereby eradicate vector-transmitted diseases, such as dengue, chikungunya, yellow fever and malaria [4]. CRISPR-Cas9 endonuclease constructs that function as gene-drive systems in Anopheles gambiae, the main vector for malaria, were constructed that confer a recessive female sterility phenotype upon disruption [15].

Genome editingto alter human genomesraises anumber of ethical concerns [16]. The majority of changes introduced with genome editing are confined to somatic cells. These changes affect only certain tissues and are not passed on to the next generation. However, germ-line cells and embryo genome editing give rise to many ethical challenges, including whether it would be permissible to use this technology to enhance normal human traits (For example, height or intelligence). Concerns about potential accidental DNA changes (off-target effects) - particularly with regards to potentially oncogenic mutations- that might arise from CRISPR gene editing have emerged [17].

Conclusion

The rapid developments in the field of genome-editing technology has provided investigators the opportunity to manipulate virtually any gene or genome. Experiments as varied as forward genetic screens to correction of pathogenic mutations in induced pluripotent cells (iPSC)-derived human cells can be carried out with high efficiency, precision and cost effectiveness. While the journey towards the treatment of human disease is long, the pace of innovation of this technology over the past decade anticipates a promising future ahead.

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