CRISPR Knockouts vs Knockins

The use of target gene editing in preclinical research

Not so long ago, the addition, removal and modification of parts of the genome were only possible in science-fiction movies. In the past years, the CRISPR/Cas9 technology has made these processes a concrete possibility, revolutionizing the field of genome engineering and offering scientists the possibility to perform an endless number of genetic modifications, including knockouts and knockins. In this post, we will go over the available knockout and knockin techniques and how they differ so that you can choose the one that is most appropriate for your experiment.

Differences between knockouts and knockins

Despite the two approaches having only a few different letters in their names, they are completely different from each other: the term knockout refers to a strategy aiming at “removing” a DNA sequence while knockin approaches are meant to do the opposite (“inserting” a sequence).

As mentioned before, the CRISPR/Cas9 technology represents a powerful tool for both procedures: the system acts as molecular scissors and is able to recognize and “cut” specific regions of the DNA to initiate the process of genome editing. Upon DNA damage, the organism puts in place different DNA-repair mechanisms that scientists can take advantage of to produce the intended modification.

The term “knockout” (KO) has been borrowed from combat sports, where it is employed to define a situation in which one of the two opponents is unable to pursue the match as a consequence of an attack suffered. Similarly, in genetics, the term knockout refers to cases in which, as a consequence of the disruption of its sequence, a gene becomes unable to play its biological role (e.g. to produce a protein responsible for a specific function).

“Knockin” (KI) approaches are employed to realize modifications meant to add an exogenous sequence in the targeted locus.

Knockouts: techniques

To generate a knockout, researchers exploit the non-homologous-end joining (NHEJ) pathway, the most common endogenous DNA repair mechanism. Once the CRISPR/Cas9 recognizes and breaks the target DNA region, this repair system tries to restore the original sequence by putting back together the two extremities of the damaged DNA. This mechanism is error-prone and could lead to the insertion or deletion of a few bases (INDELS) that modify the original sequence. When these INDELS are not multiple of 3 can cause frameshift (a change in the reading frame) and introduction of premature STOP codons.

If the target region is located at the beginning of a gene, the generation of INDELS leads to the complete inactivation of the gene impeding the production of the encoded protein. If the target region is placed later, INDELS results in the generation of a modified or truncated protein that is likely unable to perform its biological function.

CRISPR mechanism to induce loss-of-function gene mutation. The Cas9-mediated induction of double-strand break (DSB) activates endogenous mechanisms of DNA repair, aiming to fix the genomic damage produced at the locus of interest. These error-prone mechanisms can induce different outcomes in the target locus.

Knockouts: applications

A knockout model allows us to understand the biological function of a protein by observing what happens if the protein is missing. In this sense, the zebrafish represents an ideal model organism to study the effect of a gene knockout as its small size and transparency offer scientists the possibility to visualize, in real-time, many different biological processes.

An additional application of the knockout approach is the generation of disease models. Many human pathologies are caused by genetic mutations, natural knockouts in which specific proteins are missing or not-functioning. A disease model recapitulates the pathological phenotypes associated with the disease and allows scientists to investigate its causes or to test putative therapies. Also in this case the zebrafish extends a helping hand to scientists as disease modeling in this small organism makes it possible to perform high-throughput screenings to test therapeutic compounds or gene therapies.

Knockins: techniques

Different technologies have been developed to allow the targeted insertion of a genetic sequence. All approaches rely on the provision of a “donor DNA” (containing the exogenous sequence of interest) together with the CRISPR/Cas9 machinery. Two repair mechanisms can be employed to obtain a KI: the already mentioned NHEJ and the homology-directed repair (HDR).

In the first case, the KI is successful if, instead of joining the two extremities of the broken DNA, the NHEJ combines one extremity of the broken DNA with one side of the donor DNA.

In the second option, the HDR employs the donor DNA as a template to synthesize a new DNA chain. To promote HDR, the donor has to contain two “homology arms” (HAs) flanking the exogenous sequence to be inserted into the genome. The HAs need to be identical to the sequences at the sides of the CRISPR/Cas9 genomic target.

Knockins: applications

Knockin approaches can be employed to introduce short or long DNA sequences serving different experimental purposes. Indeed, knockin strategies can be employed to generate single base changes (point mutations) causing small modifications in a protein sequence.

Compared to a knockout, in which the protein encoded by the target gene is missing or nonfunctional, this approach is a better choice to model human diseases in which a point mutation induces an adverse gain of function (e.g. the mutated protein acquires a toxic function). Knockin can be also employed to insert sequences allowing “conditional knockout”, a technique in which the target sequence is selectively removed in specific tissues or at a specific time-point. This approach allows scientists to focus on the role of a protein in a particular cell type or in a precise developmental stage. The knockin of longer DNA sequences makes it possible to label proteins of interest with a tag or a fluorescent reporter, providing researchers with a reliable manner to visualize them. Similarly, selected tissues or cell clusters can be labeled by a fluorescent reporter to generate reporter lines.

Unlike knockouts, whose efficiency has been shown to be very high, knockin approaches are more challenging and require a complex optimization process. Indeed, despite many efforts to increase the success rate of this approach, there is still a poor consensus on which is the optimal strategy to generate the desired knockin. In this context, ZeClinics is strongly committed to establishing the best procedure to modify ad hoc the genome of our favorite model organism: our team is working tirelessly to improve and optimize existing technologies and to develop novel approaches to establish the most efficient pipeline for zebrafish genome modification.

By Flavia de Santis

Flavia obtained her master’s degree in medical biotechnologies from the University of Bari (Italy), then she pursued her scientific education at the Institut Curie (Paris), where she obtained a Ph.D. in neuronews. During her doctorate, she focused her research on vertebrate neural circuit development and, in parallel,  worked on the implementation of innovative CRISPR/Cas9-based genome engineering approaches. Since she joined ZeClinics as a senior scientist, she has been coordinating a wide range of genetics services while working in the development of cutting-edge technologies to achieve a more efficient generation of zebrafish models for #therapeutic target validation.