Genome engineering and synthetic genomics (designing artificial genomes) are currently among the most promising technologies in terms of applied biological research and industrial innovation. Genome engineering refers to the strategies and techniques developed for the targeted, specific modification of the genetic information or genome of living organisms. Early technologies developed to insert a gene into a living cell, such as transgenes, are limited by the random nature of the insertion of the new sequence into the genome. It is also having a risk of inducing cancer or other genetic diseases. The major advantage of genome engineering, is that it enables a specific area of the DNA or deoxyribonucleic acid - the hereditary material in humans and almost all other organisms - to be modified, thereby increasing the precision of the correction or insertion, preventing any cell toxicity and perfect reproducibility. Initial genome engineering technologies focused on modifying genetic sequences using only homologous recombination.  It was possible to induce homologous recombination between a cellular DNA strand and an exogenous DNA strand inserted using a vector such as the modified genome of a retrovirus.

Modern methods of gene editing currently fall into two broad categories. The first relies exclusively on homologous recombination, a natural DNA-repair mechanism, to perform endogenous DNA alterations and is best exemplified by the use of recombinant AAV (rAAV) as a gene editing tool. The second category functions through the stimulation of locus specific DNA repair events as a consequence of introducing double strand DNA breaks and is best exemplified by zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Meganucleases.

Advances in genomes technologies developments could enhance the rate of homologous recombination in somatic cell types. They are endonucleases (restriction enzymes), such as zinc finger nucleases (ZFNs), mega nucleases and transcription activator like effector nucleases (TALENs).  The second method is recombinant adeno-associated virus (rAAV) mediated genome engineering which induces high frequencies of homologous recombination.

Procedures in genome engineering insertion involves introducing a gene into a chromosome to obtain a new function (for example to obtain a better drought-resistant plant) or to compensate for a defective gene, particularly by making it possible to manufacture a functional protein if the protein produced by the patient is defective (such as factor VIII in hemophilia A). Inactivation, or “knock-out”, is mainly used in fundamental research to shed light on the function of a gene by observing the abnormalities that occur as a result of its inactivation. It can also have other applications, for example to remove a persistent viral sequence from infected cells, or in agriculture to eliminate the irritant or allergenic properties of a plant. Gene correction aims to remove and replace a defective gene sequence with a functional sequence. Gene correction can be performed on a very short sequence, sometimes just a few nucleotides, such as in the case of sickle cell anemia.

The zinc finger nucleases motifs occur in several transcription factors. The zinc ion is found in 8 per cent of all human proteins and zinc plays an important role in the organization of their three-dimensional structure of proteins. In transcription factors, it is most often located at the protein-DNA interaction sites, where it stabilizes the motif. The C-terminal part of each finger is responsible for the specific recognition of the DNA sequence. The method generally adopted for this involves associating two proteins – each containing 3 to 6 specifically chosen zinc fingers – with the catalytic domain of the endonuclease which can be dimerized and then cut the DNA molecule.

Meganucleases are endodeoxyribonucleases characterized by a large recognition site with double-stranded DNA sequences of 12 to 40 base pair. Meganucleases are therefore considered to be the most specific naturally occurring restriction enzymes. Among Meganucleases, the LAGLIDADG family has become a valuable tool for the study of genomes and genome engineering. Meganucleases are "molecular DNA scissors" that can be used to replace, eliminate or modify sequences in a highly targeted way. Meganucleases are used to modify all genome types, whether bacterial, plant or animal. They have opened up wide avenues for innovation, particularly in the field of human health, for example the elimination of viral genetic material or the "repair" of damaged genes using gene therapy.

Transcription Activator-Like Effector Nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). Transcription Activator-Like Effectors (TALEs) can be engineered to bind practically any desired DNA sequence, so when combined with a nuclease, deoxyribonucleic acid can be cut at specific locations. The restriction enzymes can be introduced into cells, for use in gene editing or for genome editing in situ, a technique known as genome editing with engineered nucleases. Alongside zinc finger nucleases and CRISPR/Cas9, TALEN is a prominent tool in the field of genome editing.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are segments of prokaryotic DNA containing short repetitions of base sequences. The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages, and provides a form of acquired immunity. CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. CRISPR/Cas genome editing techniques have many potential applications, including altering the germline of humans, animals, and food crops. The use of CRISPR Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015.

The CRISPR/Cas several inherent advantages like simple to adopt as it requires just two key components to use. The Cas9 nuclease comprises of the HNH (Histadine and Aspriginine residue) and RuvC like nucleases domain which cleaves the two deoxyribonucleic acid strands there by forming a Double Strand Break (DSB). The second component is sgRNA which is called CRISPR. sgRNA is designed to be complementary to the DNA sequence around modification site, which is engineered from the bacterial system. This requires both Trans activating RNA and CRISPAR RNA. Successful targeting of the Cas9 and CRISPR is dependent on the target deoxyribonucleic acid itself, a short sequences called as proto spacer adjacent motif (PAM). This separation of targeting and nuclease elements is a key feature of the CRISPR/Cas9 system. The relative ease of generating CRISPR molecules rather than reengineering proteins is time and cost saving. After DSB, repair can occur through non homologous end joining (NHEJ) pathways. Alternatively repair by homologous recombination (HR) pathways enables the introduction of specific changes within the specific sequence. The CRISPR/Cas9 methodology allows gene knock-outs, knock-ins, introduction of specific mutations and insertions. The most significant changes are about modifications to the endogenous gene, which allows study of the proteins at the native level.

It is very important in drug discovery to identify a novel validated targets whose pharmacological modulation may yield the therapeutic lead. Due to inefficient technologies, it was not possible to explore fully the druggable genome, for example siRNA. The siRNA libraries has limitations like incomplete inactivation of true function loss. The CRISPR/Cas9 offers an improved ability to modulate the endogenous gene specifically and result in complete genetic knock-out with minimum off target effects. The ability to specifically modulate the endogenous gene with CRISPR/Cas9 and introduce a complete genetic knockout, while minimizing off target effects, offers an improved approach to target identification. One of the special advantage is to develop a generation of genome wide CRISPR libraries, coupled with lentiviral delivery methods enabling a high throughput loss-function screens. For example The Sanger Institute of the Wellcome Trust in Cambridge/United Kingdom has established a library of approximately 88000 sgRNAs comprising 19000 genes that modulate specific endpoints. Further benefit of the CRISPR/Cas9 technology is the speed and relative ease with which cellular models can be generated.

A crucial step in attempting to identify better lead molecules and reducing candidate drug attrition is the development of more physiologically relevant assays. The CRISPR modified cells allows one to assays which allows to monitor both the native and modified target at the endogenous levels. The incorporation of fluorescent tag, GFP offers direct visualization of the target in both native and also in real time leading to high content imaging assays a possibility. This allows one to locate, movement and behaviour of the protein with in the cell. This visualization offers the advantage of target identification in presence of over expression of tagged proteins. Recently, SunTag approach with CRISPR/Cas9 was successfully used to tag a single protein with 24 copies of fluorescent tag.

CRISPR technology offers drug discovery teams the potential to respond to decipher not only preclinical but clinical data. For e. g., a key challenge with in cancer is the capability of a tumor to acquire drug resistance. This is mediated by mutations with change in protein expression. The efficient CRISPR targeting allows one to observe clinically and characterize the mutation. This gives a precise clue for drug discovery team to design the next generation of therapeutic molecules. CRISPR Technologies can be extended to generate isogenicaly paired cell lines to use in assay to identify the sensitivity of drugs in particular diseases. Isogenic approach will be highly valuable with need to assign lead molecules to a specific clinical patient segment without much delay.

Applications of CRISPR is not restricted to small molecule drug discovery, but can be applied to therapeutic solutions which is novel gene therapy. The correction of tyrosinemia disease phenotype through the direct hydrodynamic delivery of CRISPR/Cas9 has raised the hope of resolving all the genetic diseases which were considered impossible to cure. CRISPR-Cas9 is a unique technology that enables geneticists and medical researchers to edit parts of the genome by removing, adding or altering sections of the DNA sequence. It is currently the simplest, most versatile and precise method of genetic manipulation and is therefore causing a buzz in the science across the world. Attempts to knock out CCR5 of HIV DNA from HIV patients by using ZFN using CRISPR/Cas9 technologies can offer a cure from HIV.

CRISPR-Cas9 technologies are one of the exciting field of research with many promises in drug discovery and gene therapy. Lots of investments and efforts are done in this genome engineering research.