HDR vs NHEJ in CRISPR Gene Editing

Understanding DNA Repair Pathways

The CRISPR/Cas9 system has revolutionized genetic research, offering scientists unprecedented precision in genome editing. However, before delving into genetic modifications, it is crucial to understand the intricate DNA repair pathways that come into play when a DNA sequence is damaged. In this article, we will explore the differences between Homology Directed Repair (HDR) and Non-Homologous End Joining (NHEJ) in the context of CRISPR/cas9-based gene editing and we will discuss when you should, and should not, consider using them.

The Importance of DNA Repair Pathways

Cells rely on DNA as a blueprint for growth and reproduction, making genome protection vital. When DNA damage occurs, various pathways are activated to sense and repair the disrupted sequences. These pathways are essential for maintaining genomic integrity across all organisms. 

Although DNA damage can affect one or both DNA strands, in this article we focus on double-strand breaks (DSBs) repair mechanisms, because this is the type of damage we take advantage of when using the CRISPR/Cas9 system. Although many people believe that the CRISPR/Cas9 machinery is the one performing the desired genetic modification, it actually only performs the cut at a specific location in the genome, and it is the cell's endogenous DNA repair mechanisms that produce genetic editing while joining the two cut ends.

There are many repair mechanisms including:

• Base Excision Repair (BER)
• Nucleotide Excision Repair
• Mismatch repair (MMR)
• Homology directed repair (HDR)
• Non-Homologous End Joining (NHEJ)
• Other minor repair mechanisms

Researchers can leverage these endogenous DNA repair pathways to generate genetically edited organisms that help further the study of human disease, and the development of new therapeutics.

Choosing the Right Pathway

When deciding between HDR and NHEJ for genome editing, researchers must consider the desired outcome of their experiments. HDR is suitable for precise genetic modifications and knockins, while NHEJ is preferred for gene knockout studies due to its ability to induce INDELs effectively. Understanding these pathways allows researchers to tailor their approach based on the specific goals of their research.

Non-Homologous End Joining (NHEJ)

NHEJ is an error-prone DNA repair pathway that rejoins broken DNA ends without requiring a template with homologous sequences. This mechanism often leads to small insertions or deletions (INDELS), making it ideal for gene knockout studies where disrupting gene function is the goal. NHEJ is efficient but lacks the precision of HDR, limiting its use for more complex genetic modifications. However, with the appropriate strategy, NHEJ can also be used for gene knockin generation, as explained here.

Homology Directed Repair (HDR)

HDR is a precise DNA repair mechanism that utilizes homologous sequences (from a sister chromatid, a donor homology plasmid, a single stranded oligodeoxynucleotide (ODN), etc.) to accurately repair DSBs. Unlike NHEJ, which can lead to INDELS, HDR uses homologous regions as templates for error-free repair. 

Researchers can leverage HDR to introduce specific and highly accurate genetic modifications like gene knockins or precise gene edits. To achieve this objective, they need to design a donor template wherein the DNA sequence intended for insertion is flanked by arms homologous to the 5’ an 3’ sites of the DSB. This method is useful for more complex gene editing, such as generation of point mutations or tagged versions of your gene of interest.

Conclusión

In conclusion, HDR and NHEJ are two fundamental DNA repair pathways that play a crucial role in CRISPR/Cas9-based gene editing. By comprehending the differences between these pathways and their implications for genetic modifications, researchers can optimize their strategies for creating genetically modified organisms and advancing scientific discoveries.

By Miriam Martínez

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.