An Overview of Gene Editing Techniques
What is genome or gene editing?
In brief, it is the alteration of the genetic code with a set of tools, techniques or technologies
Gene editing describes the process of modifying an organism’s genetic code. Genome engineering relies on two aspects, the cutting phenomenon - performed artificially, and the repair mechanism triggered by the cutting, which is realized naturally. Gene editing makes a smart use of these to achieve a specific objective. Various gene editing techniques exist. But regardless of the specific technique used, all are based on the use of enzymes called "nucleases" that directly interact with the DNA in a cell. Nucleases can be repurposed to specifically target a defined site within the genome. Once the site directed nuclease recognizes, binds to the target site, and generates a DNA-strand, several editing approaches can be realized.
What is gene editing used for?
One of the goals of gene editing is to understand the function of a specific gene, gene regulatory element, or single-nucleotide variation(s) by means of several technologies and approaches. In fact, about half of the existing pathogenic genetic variants are due to single-nucleotide variants. Consequently, there is a clear need to develop methods and tools capable of correcting or introducing single-nucleotide variants with high efficiency to improve the clinical interpretation and the understanding of how human genetic variations impact health. To gain such knowledge, a genome editing technique must demonstrate high on-target efficiency and a reduction of off-target edits. In addition to those key properties, each gene editing technique must provide a range of approaches to alter the genome at a set location.
What are the different gene editing approaches?
Two main gene editing approaches can be performed: namely, gene knock-out and gene knock-in. These approaches rely on the generation of a DNA-strand break by the nuclease. As a response, the cell tries to repair the break via either: a non-homologous end joining repair (NHEJ) or a homology-directed repair (HDR). The repair response to the breakage will define the resulting gene editing approach.
Gene Knock-out (KO)
To understand the function of a gene, the first gene editing approach that can be performed is simply to render the gene non-functional and to study the consequences. This approach is referred to as “knock-out”. The gene knock-out relies on a non-homologous end joining repair (NHEJ): It is performed by direct ligation of the break ends without the use of a template. The NHEJ is prone to errors and leads to insertions and deletions of base pairs, collectively called indels, potentially resulting in a Knock-out (KO) of the targeted gene. These indels can result in frame shift mutation and/or premature stop codons resulting in a non-functional mRNA. Alternatively, a specific region of a gene can be targeted by a two-guide-RNA approach. Here, instead of relying on the formation of indels at the targeted region, two individual gRNA are designed such that a specific portion of the target gene is deleted.
Gene Knock-In (KI)
The second gene editing approach is called gene "knock-in”. There are several types of knock-in approaches serving different purposes:
• The insertion of a transgene into a defined locus.
• The insertion of a small or large tag into the coding sequence of a gene.
• The generation of a point mutation by exchanging base pairs.
These three approaches can be employed to achieve different goals. The first approach can be used to add a gene to the targeted cell and its function or effect on the phenotype can be explored. The second approach is used to detect and localize a gene product within a cell (e.g.: fluorescent tag) or add a sequence to facilitate the detection or the purification of the gene product for biochemical assays. The latter approach is mainly used to model and eventually treat the effect of point mutations in the context of genetic diseases. The knock-in is based on a homology-directed repair (HDR). This mechanism originates from the use of a template-based homology-directed repair pathway to precisely edit the targeted region according to the respective template. Here, the DNA sequence to be inserted is flanked by regions with sequence homology to the targeted region of the DNA. This DNA sequence is used as a template for HDR of the double-stranded breaks.
Both knock-out and knock-in gene editing approaches can be performed with a broad range of gene editing techniques. The following section details the evolution of gene editing techniques.
Evolution of Gene Editing Techniques
Seven decades ago, the complex evolution of genome engineering started with the discovery of the DNA double helix. Throughout the years, researchers uncovered different techniques to perform gene editing.
1970s - Restriction Enzymes
Restriction enzymes are the precursors of modern genome editing. For the first time, it became possible to recognize and cut at specific patterns of nucleotide sequences, and to further insert new DNA material at a targeted location. Yet, some cutting limitations remained due to the necessity to operate at predetermined site. 
1980s - Zinc Finger Nucleases (ZFNs)
Precise genome editing was first demonstrated with the use of zinc finger nucleases (ZFN). ZFNs contain a nuclease domain and specific zinc finger DNA-binding domains that recognize three-base pair sites on DNA. Multiple ZFNs can combine to form dimers. The dimerization process permits to recognize longer nucleotide sequences, amplify the specificity, and improve the adaptation to the target.
2011 - Transcription activator-like effector nucleases (TALENs)
While ZFNs had paved the way for precision genome editing, transcription activator-like effector nucleases (TALENs) brought the recognition down to the single-nucleotide resolution. TALENs also contain a nuclease fused to a DNA-binding domain sequence but they are capable of single-nucleotide recognition, thereby increasing targeting capabilities and specificity compared to ZFNs. One of the main drawbacks of platforms such as ZFNs and TALENs is the requirement of designing and validating a new zinc-finger nuclease or TALEN protein for each new target editing site. 
2013 - CRISPR-Cas9
An important step for genome engineering was achieved with the discovery of the Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR technology.  The technology is based on a guide RNA and the Cas9 endonuclease; the guide RNA recognizes target sites and the Cas9 endonuclease cuts and triggers the repair mechanism. Both CRISPR and TALENs can perform gene editing with a single-nucleotide resolution, but CRISPR is less time-consuming and cheaper, making it a more attractive alternative.
2017 - Base Editing
Unlike previous techniques, genomics and transcriptomics with base editing avoids cleavage of nucleic acid backbones and instead, directly modifies target nucleobases.  Based on a ‘catalytically dead’ Cas9 (dCas9) fused to bacterial enzymes called DNA deaminases, scientists can perform single nucleotide substitutions by providing a single guide RNA (sgRNA) to direct it to the target sequence. 
2019 - Prime Editing
Prime editing, compared to base editing, is more versatile because it can precisely perform targeted small insertions, deletions, and base exchanges while limiting negative effects.
What sets those tools apart from the CRISPR-cas9 technology is the ability to make targeted edits without breaking the double strand.
The prime editing technology is built with a Cas9 nickase, which induces single-stranded breaks in DNA, fused to a reverse transcriptase enzyme. It uses a single engineered construct known as a prime editing guide RNA (pegRNA), which is made up of the primer binding site (PBS) sequence and a sequence containing the desired edit. 
The revolution: CRISPR-cas9 Genome Editing
If we are to tell concisely the story of gene editing techniques, a specific chapter should be dedicated to CRISPR-Cas9, the system that transformed genome editing.
Where does CRISPR come from?
Originally, CRISPR systems were known as a natural protection for bacteria against invading bacteriophages. Nowadays, in modern biology, CRISPR systems are repurposed for gene editing by inducing double-stranded DNA breaks or RNA cleavage at user-defined loci in living cells and organisms. [3,7,8] This discovery has revolutionized life sciences, as recognized by the 2020 awarded Nobel Prize in Chemistry.  Before proceeding to examine further the reasons behind CRISPR-Cas9 success, it is important to understand the working principle of the CRISPR-Cas9 system.
How does CRISPR work?
The CRISPR-Cas9 system relies on a ribonucleoprotein complex, the so-called CRISPR-complex, which includes two components:
• The Cas (CRISPR associated) protein, most often Cas9, that carries the endonuclease enzymatic activity.
• The guide RNA (gRNA), that drives the complex to the genomic locus of interest through the sequence homology of the gRNA with the genomic DNA. Upon binding, an RNA-DNA duplex is formed.
To sum-up, the Cas9 protein exerts its endonuclease activity and induces a double-stranded break (DSB) in the targeted DNA. As illustrated in the figure below, the double-stranded break activates one of the DNA repair pathways in mammalian cells: either non-homologous end joining (NHEJ) or homology-directed repair (HDR) introduced previously. Bear in mind that there are alternative repair pathways, such as microhomology mediated end joining (MMEJ) that can repair DNA damage.
Schematic representation of CRISPR-Cas9 Gene editing method
Now that you have understood the working principle behind the term "CRISPR-Cas9", you might still wonder what such system brought to research?
What has CRISPR gene editing brought to research?
Compared to previous gene editing methods, CRISPR enables a simplified, more versatile and direct genome editing. The ability to insert a gene at a specific locus is a tremendously useful method, notably to study gene function and to dissect disease mechanisms. Hence, it is with no surprise that this broadly applicable and versatile technology has experienced an enormous adoption in nearly all molecular life science disciplines, ranging from fundamental and pharmaceutical research to applications in agriculture and gene and cell therapy . CRISPR-Cas9 gene editing method has now certainly outstripped its predecessors in terms of ease of use and editing efficiency. As an example, CRISPR with its very high specificity and efficiency is recommended for applications where multiple simultaneous edits are required within the same cell line or organism.
Nonetheless, CRISPR is not the solution to all the existing genome engineering problematics. Each researcher needs to select the right genome editing method and supporting tools to find the right comprise between time, budget and safety. Furthermore, some challenges remain for CRISPR-Cas9 technology. Despite its high specificity, other parameters such as the efficiency of the selected cell transfection method, strongly impacts the success of a gene editing experiment. Another recurring challenge for the technique is the reduction of off-target editing that remain crucial, notably for clinical application.
To overcome the delivery limitations of standard transfection methods, Cytosurge employed its proprietary patented technology - the FluidFM - to offer unprecedented genome editing capabilities with a unique in-vitro solution to perform direct intra-nuclear delivery. This solution allows to improve drastically the efficiency and applicability of CRISPR across a variety of cell types and for cell line development.
 Smith, Hamilton O., and K. W. Welcox. "A restriction enzyme from Hemophilus influenzae: I. Purification and general properties." Journal of molecular biology 51.2 (1970): 379-391.
 Komor, Alexis C., Ahmed H. Badran, and David R. Liu. "CRISPR-based technologies for the manipulation of eukaryotic genomes." Cell 168.1-2 (2017): 20-36.
 Jinek, Martin, et al. "A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity." science 337.6096 (2012): 816-821.
 Porto, Elizabeth M., et al. "Base editing: advances and therapeutic opportunities." Nature Reviews Drug Discovery 19.12 (2020): 839-859.
 NICOLE, M. GAUDELLI. "Programmable base editing of AT to GC in genomic DNA without DNA cleavage (Includes Methods)." vol 551: 464-471.
 Anzalone, A.V., Randolph, P.B., Davis, J.R. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019). https://doi.org/10.1038/s41586-019-1711-4
 Gasiunas, Giedrius, et al. "Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria." Proceedings of the National Academy of Sciences 109.39 (2012): E2579-E2586.
 Mali, Prashant, et al. "RNA-guided human genome engineering via Cas9." Science 339.6121 (2013): 823-826.
 Adli, M. The CRISPR tool kit for genome editing and beyond. Nat. Commun. 9, 1911 (2018).