Bioinformatics in gene editing
Bioinformatics ensures the precision and accuracy of genome modification techniques such as CRISPR/Cas9, ZFN, TALEN, and DdCBE. Leveraging Next-Generation Sequencing (NGS), bioinformatics provides researchers with the tools to analyse both on-target efficacy and off-target effects, offering a comprehensive view of genomic alterations. This analysis is critical for refining gene editing strategies, ensuring high accuracy while minimising unintended changes in the genome.
On-Target Profiling
One of the core aspects of NGS bioinformatics in gene editing is its ability to perform in-depth analysis of on-target modifications. This includes:
Quantifying the efficiency of gene editing at the intended site.
Characterising the modifications made, such as insertions, deletions, or base substitutions.
With high-throughput sequencing, bioinformatics enables precise measurement of how often and to what extent the desired genetic changes occur at the target loci. This level of detail allows researchers to optimise gene editing protocols and ensure that the intended modifications are achieved accurately.
Off-Target Detection
A major challenge in gene editing is the potential for off-target effects—unintended genetic changes at locations that closely resemble the target sequence. NGS bioinformatics excels in detecting and mapping these off-target events, providing a detailed overview of the genome to:
Assess the specificity of the gene editing tool.
Identify potential risks of unintended genetic modifications that could impact other genes or regulatory elements.
By revealing these off-target sites, bioinformatics helps researchers refine editing strategies and reduce the risk of undesirable consequences, which is essential for both research and therapeutic applications.
Technology-Specific Analysis
Each gene editing technology—CRISPR/Cas9, ZFN, TALEN, and DdCBE—has unique capabilities and limitations. NGS bioinformatics adapts to each tool, offering tailored analysis pipelines that maximise the strengths of the editing technology. For instance:
CRISPR/Cas9: Bioinformatics can identify precise cleavage patterns and modifications at the PAM site, providing insights into its versatility and potential off-target interactions.
ZFN and TALEN: These tools are known for their precision, and bioinformatics can assess the fidelity of editing by pinpointing off-target effects in highly homologous regions.
DdCBE (Deaminase-mediated DNA Base Editors): Bioinformatics helps track the specific base changes introduced, offering a detailed look at the efficiency and accuracy of base editing events.
This technology-specific approach ensures that researchers can continuously improve their gene editing methodologies, driving innovation while maintaining safety and precision.
Bioinformatics has become indispensable in gene editing, offering the insights needed to refine protocols, minimise risks, and enhance the effectiveness of genomic modifications. By combining NGS with cutting-edge bioinformatics analysis, researchers can ensure that gene editing technologies are both precise and efficient, paving the way for more reliable therapeutic applications and groundbreaking genetic research.
A selection of our papers:
Silva-Pinheiro P, et al. (2022) A library of base editors for precise ablation all protein-coding genes in the mouse mitochondrial genome. Nat Biomed Eng doing: 10.1038/s41551-022-00968-1
Silva-Pinheiro P, et al. (2022) In vivo mitochondrial base editing via adeno-associated viral delivery to mouse post-mitotic tissue. Nat commun 18(1):750 dot: 10.1038/s41467-022-28358-w
Gammage PA, et al. (2018) Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo. Nat Med 24(11):1691-1695 doi: 10.1038/s41591-018-0165-9
Gammage PA, et al. (2016) Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs. Nucleic Acids Res 44(16):7804-16 dot 10.1093/nar/gkw676
