CRISPR Gene Targeting

Increased Accuracy with CRISPR Gene Targeting and Genome Editing Methods

What Is Gene Targeting?

The gene replacement strategy through the use of homologous recombination is a gene-altering technique that uses genetic recombination to alter a specific endogenous gene. It can be used to delete genes and exons, change individual base pairs through the introduction of point mutations, and introduce specific conditions for the purpose of triggering the modifications it facilitates. The process typically requires the generation of a particular vector for each gene that it targets, and it can be utilized on any gene, regardless of its size, function or transcriptional activity.

Understanding Gene Targeting and Its Practical Applications in Genetics

Scientists and students interested in gene targeting are at the forefront of one of the most advanced and exciting research areas in human history. The ability to alter specific genes and replace them through homologous recombination has recently been perfected, but it is based on studies dating back to Sternberg and Hamilton’s 1981 research. Gene targeting continued to gain worldwide recognition when, in 2007, scientists Mario Capecchi, Martin Evans and Oliver Smithies received the Nobel Prize in Physiology or Medicine using the process to introduce gene modifications in mice with the help of embryonic stem cells.

Targeting Made Easier with CRISPR/Cas9

With the use of CRISPR, gene targeting in experiments involving genome engineering, transgenes and gene knockout has advanced to an impressive extent. While gene targeting was always possible with various other gene editing techniques, such as TALENs and the DSB-inducing of nucleases with customized DNA binding, the advent of CRISPR/Cas9 was a refreshing change brought to the world of genetic engineering. Breakthroughs obtained by scientists like Feng Zhang and George Church have shown that CRISPR/Cas9 can be used in human cell cultures and in many other organisms as well, including plants, fruit flies, nematodes and even mice and primates.

Gene targeting is one of the most significant advantages of the CRISPR/Cas9 system. Not only is CRISPR/Cas9 able to target specific genes with fewer problems and errors, but it also has less difficulty maintaining its accuracy and consistency throughout multiple trials. There are two main components that lead to this superiority, making CRISPR/Cas9 more straightforward and easier to use: reproducible genome cleavage and programmable sgRNA cloning. Even though some aspects that were used to compare Cas9 with other techniques have stayed the same, the differences that set CRISPR/Cas9 apart have led to the possibility of targeted double-strand breaks and improved gene editing overall.

How to Achieve Gene Targeting: Process and Methods

Over the years, there have been several gene targeting methods that are currently widely accepted. A mutational approach proved to be invaluable by examining the roles of gene products in complex biological processes specifically in a mammalian system (Smithies et al., 1985). [1] Gene targeting procedures enable the precise (site-specific) introduction of a mutation to any of the murine genes. Typically, mutations are designed to eliminate gene function, resulting in the generation of ‘‘knockout’’ or ‘‘null mutant’’ mice. The introduction of mutations that produced more subtle alterations in gene function had been achieved. Two major developments made gene targeting experiments feasible: (a) the generation of totipotent embryonic stem (ES) cells, and (b) the elucidation of techniques to achieve homologous recombination in mammalian cells. While this approach has been utilized for several decades to generate mouse models, the most recent technology, CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Regions/ CRISPR associated protein) represents an alternative tool to create gene edited mice or other species. These other species where genes have successfully been targeted and modified on a more consistent basis utilizing CRISPR/CAS9 are rats, rabbits, sheep, cattle swine and a variety of fungi. Even though these methods are remarkable advancements in science, they still have to be perfected, and they are not without their limitations.

There is little evidence of the consequences of off-target genome editing. Indeed, there are only a few assays of genotoxicity based on cells in culture that allow to quantify, stratify and help prevent biological side effects of gene editing in a given target cell population. While many groups have shown improvements in CRISPR gene targeting, the two-donor method continues to lack efficiency, mainly due to the reliability of two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. It has been shown that the methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of mouse models.

Permanent vs. Conditional Targeting

One of the main practical advantages of gene targeting is that it doesn’t have to be permanent. In many cases, permanent targeting solutions are used to obtain changes that are expected to stay in place once they are made. However, with the help of conditional targeting techniques, scientists are able to exercise a greater degree of freedom on the modified genes. They can introduce specific conditions that are accurately targeted to trigger the genetic change at a certain time during the organism’s development, or when a particular tissue-related limitation condition is met.

Gene Trapping and Targeting

The homology regions of gene targeting cassettes require a specific process that adapts them to a particular gene that is aimed for modification. In contrast with this requirement, gene trapping utilizes a different process whereby scientists apply the random insertion of a cassette. While gene trapping is easier to apply to larger projects, targeting is far more precise and effective when it comes to aiming for specific results. Smaller genes are just as easily targeted as larger ones, and the procedure can also be utilized on genes with low transcriptions that would normally be ignored when it comes to gene trapping.

Cas9: The Simplest Method for Genome Targeting

There are many discussions among scientists regarding the relationship between CRISPR and gene targeting. The main answer here is the Cas9 protein, which was named as the component to one of the most accurate systems ever used for genome engineering and gene targeting. The Cas9 system was first used shortly after the development of the initial CRISPR/Cas methods. It was eventually perfected by Jennifer Doudna and Emmanuelle Charpentier, who recombined the two RNAs into a single guide RNA that could more easily target and cut the specified DNA components. Scientists were even able to obtain effective genetic edits in human tripronuclear zygotes in 2015, marking a new stage for human genetic engineering with the help of the CRISPR/Cas9 technique.

Targeting Differences: Cas9 vs Cpf1

In 2015, an important competitor appeared in the CRISPR gene targeting space, which continues to contest the accuracy of gene targeting obtained using Cas9. Cpf1 is a newly discovered nuclease that has successfully been used as a substitute for the Cas9 protein. While results were similar, Cpf1 gene editing resulted in a more “staggered” cut and the need for only a single CRISPR RNA. Moreover, Cpf1 techniques have led to surprisingly positive results when it came to more successful target-specific DNA assembly and superior multiplexed genome editing results.

Practical Gene Mutations and CRISPR’s Role in Genome Engineering Advancements

The targeting process has already led to many important successes in the field of genetics and medicine. It is most often used to study human diseases and the effects that the manipulation of specific genes might have on them. CRISPR continues to raise the bar when it comes to genetic engineering accuracy. The level of genetic targeting that CRISPR/Cas methods are capable of have made them an important research tool pertaining to fields such as biomedicine, gene function research and cancer research. Moreover, with CRISPR, new avenues are open when it comes to successful in vivo genome editing, which was obtained in a number of target organisms such as knockout and knockin mice. Also, the development of specific genetic mutations in humanized mice has led to the creation of accurate human disease models that would have been impossible to create without the use of human test subjects in the past.

Due to these successes and because of the continuing commercial and research backing that it gets, CRISPR gene targeting has a bright future in the years to come. While gene targeting is still in its infancy when we think of its potential in the next few decades, it lays the foundation for what might be possible in the near and far future with advanced genetics.

References

1) Smithies O, Gregg RG, Boggs SS, Koralewski MA, Kucherlapati RS. 1985. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature 317(6034): 230-4.

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