Two CRISPR/Cas9 Gene Knockout Methods

Gene Inactivation vs Deletion of a Protein Functional Domain

Understanding Knockouts in Genetics: CRISPR/Cas9 Methods Explained

The development of CRISPR/Cas9 editing has revolutionized the ability to create precise gene knockouts (KO). While CRISPR/Cas9 KO methods have garnered significant attention, understanding the nuances between different applications of these techniques is crucial. This article delves into the two primary methods of generating knockout models using the CRISPR/Cas9 technology: disrupting a gene or deleting specific regions of a protein.

What does knockout mean in genetics?

The study of biology often involves disrupting systems to better comprehend their function. This disruptive approach is widely utilized across various fields, from brain lesion studies to gene knockout investigations. 

Knockout studies serve as a valuable tool in a scientist's arsenal, with advancements in technology enabling more precise gene disruption. Notably, the CRISPR/Cas9 system empowers researchers to target specific genes (or parts of them) for genomic removal or genomic modification leading to different possibilities to impact gene function.

INDELs vs large deletions

We previously saw how DNA can be edited with the CRISPR/Cas9 technology and the components this system requires, the nuclease Cas9 and a single guide RNA (sgRNA). By playing with the number of guides employed, it is possible to drive the Cas9 to induce small mutations or large genomic deletions.

If only one sgRNA is used, in response to Cas9-mediated double-stranded breaks, the cell activates the Non-Homologous End Joining (NHEJ) pathway, an error-prone DNA repair mechanism that might result in small insertions or deletions (INDELs).

INDEL method for gene knockout
Figure 1. Example of CRISPR/Cas9 INDEL approach with one sgRNA to disrupt a gene.

If two guides are employed, large genomic deletions can be obtained instead of small INDELs. 

In this case, the two guides direct the Cas9 toward each side of the genomic region slated for deletion, resulting in the generation of two double-stranded breaks. Repairing these breaks might result in the joining of the remaining ends, allowing researchers to delete substantial portions of a gene or even its entire sequence.

exon deletion for gene knockout
Figure 2. Example of CRISPR/Cas9 approach with two sgRNA to delete a specific region of a gene.

Inactivating a gene with CRISPR/Cas9

When the goal of a researcher is to completely abolish protein expression and function, the sgRNAs can be designed to target either the early coding sequence of a gene or its regulatory region.

In the first case, if only one guide is employed, it might be possible that the INDELs carried by the mutated sequence give rise to a mutated protein that maintains its functionality. However, when these alterations are not multiple of 3, INDELS can effectively inactivate the gene by causing a frameshift (a change in the reading frame). 

In this case, the entire DNA sequence that follows the mutation will be incorrectly read, leading to different possible outcomes:

  • the addition of wrong amino acids to the protein (non-functional protein) and/or
  • the creation of a premature STOP codon (truncated protein)

In any case, the gene product is likely unable to perform its biological function, resulting in the complete loss of function of the gene. 

More than one guide could be employed to maximize the probability of generating early frameshifts in the coding sequence (e.g. if two guides are employed, a frameshift can be introduced after each guide’s cutting site or after a large deletion).

An alternative approach to completely disrupt gene expression is to generate large deletions within the regulatory region of a gene. The removal of the promoter will impede the transcription of the gene and, as a consequence, will prevent the translation of the protein. 

Both strategies result in the total switch-off of a gene, an approach extremely useful to explore gene function.

Deleting specific regions of a protein with CRISPR/Cas9

If the goal of a scientist is to remove a specific region of a protein without abolishing its expression, large genomic deletions have to be induced. In this case, the sgRNAs should be directed against sequences located before and after the genomic region encoding the target protein domain. It is crucial to avoid altering the transcript's reading frame to preserve the wild-type sequence of the remaining peptide. A possible strategy to achieve this is the selective removal of a specific exon and the consequent elimination of a particular set of amino acids.

This approach might be powerful  for deleting a critical part of a gene, however the challenge becomes more pronounced as the size of the sequence targeted for deletion increases (making it difficult, for example, to remove the entire sequence of a gene). This strategy enables researchers to study the role of functional domains of a protein without interfering with its overall expression.


CRISPR/Cas9 has revolutionized genetic research by providing precise and versatile tools for gene knockout studies. This article highlights the nuances between two primary CRISPR/Cas9 knockout approaches: disrupting a gene and deleting specific protein domains. Both methods offer unique advantages and are tailored to different research goals. While l total gene inactivation provides insight into the function of a protein in toto, the targeted deletion approach offers greater precision in dissecting the functions of specific domains 

By understanding and applying these distinct CRISPR/Cas9 strategies, researchers can model prevalent human disorders such as genetic conditions caused by loss-of-function mutations in genes. Additionally, reverse genetic approaches utilizing knockouts have been instrumental in advancing our knowledge of biological processes. With an increasing number of researchers harnessing CRISPR technology, we anticipate exciting new insights and discoveries arising from our expanding ability to manipulate the genome.

CRISPR/Cas9 Gene Knockout

Get the edit you want along with the support you need to start your knockout project.

Miriam-Martinez-ZeClinics By Miriam Martínez Navarro

Miriam is a Human Biologist expert in neuropharmacology. After a master’s degree in Pharmaceutical and Biotech Industry, she obtained her PhD in Biomedicine from Pompeu Fabra University (Barcelona). During her doctorate, she focused her research on the behavioral analysis of animal models for neurophenotypical characterization. Since then, she has been working in the healthcare marketing and publicity sector, where she has contributed to developing marketing campaigns for several pharmaceutical brands. In 2021, she joined ZeClinics with a branding and marketing strategy focus.

CRISPR/Cas9Disease modelingDisease modelsGene-editinggenetic modelsknock-out