Genome Editing Tutorial for Beginners

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Genome editing 

Genome editing is a technique to alter DNA of specific cells or organisms including bacteria, plants and animals. Genome editing has extensive advantages starting from agricultural research to human disease research. Recent advancement of bacterial or engineered nucleases target and or modify genomic DNA regions more accurately and effectively. In the context of agricultural research, genome editing has become a very handy technique. For example, genome editing is often used to edit the genome of the crop plants to increase crop yield or to modify crops to make it stress tolerant (Pixley et al, 2022). Several approaches have been discovered so far to edit the genome. Among them, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas is the most common technique used to edit and modify the genome (Al-Attar et al, 2011). Apart from CRISPR-Cas, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are also utilized to edit the genome (Li et al, 2020).  

  

CRISPR/Cas-based gene editing 

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas nucleases are of two types based on the structural rearrangements (Jinek et al, 2012). Generally, type 1 CRISPR-Cas nucleases formed by multiple effector protein complexes whereas type 2 CRISPR-Cas only have one effector protein. So far, six different CRISPR-Cas types have been discovered and among them, CRISPR-Cas9 (type 2) is the most used system to edit the genome. Cas9 protein was isolated from Streptococcus pyogenes (SpCas9) and it is very specific to target the DNA sequences (Jiang et al, 2013). In brief, CRISPR-Cas9 systems have 2 parts – a single-stranded guide RNA (sgRNA) and a Cas9 endonuclease. The 20 base-pair (bp) sgRNA can be designed to target specific regions of the genome with higher specificity. Just upstream of sgRNA, a short DNA sequence of “NGG” or “NAG” is known as protospacer adjacent motif (PAM) (Sternberg et al, 2014). Cas9 protein identifies and directly binds to the PAM sequences and starts unzipping the downstream DNA sequences. The sgRNA binds to the target region and then the Cas9 cleaves the DNA to make the double-stranded breaks (DSBs). After that, the DNA repair machinery starts repairing the double-stranded breaks (DSBs). Typically, with CRISPR-Cas9, pathways such as nonhomologous end-joining (NHEJ), homology-directed repair (HDR) and microhomology-mediated end-joining (MMEJ) are initiated to repair the genome. During the process of DNA repair, the introduction of small insertions and deletions (INDELs) and targeted DNA base changes can take place. 

On the other hand, ZFNs and TELENs are engineered nucleases which include a non-sequence specific DNA cleavage domain fused to a sequence-specific DNA binding domain to create DSBs. TELENs display higher efficiency and specificity than ZFNs (Li et al, 2020). 

  

Genome editing in agriculture 

Modern agriculture is facing challenges of increasing and diversifying food demand in times of climate change and population growth. With the recent advancement in genome-sequencing and availability of genomic sequences, genome editing has become much easier and more precise nowadays allowing breeders to introduce sequence alterations at a specific locus, facilitating the precise modulation of desired traits in crops.  

It can take plant breeders decades to introduce a new trait into a crop through conventional plant breeding methods, while genome editing has the potential to shorten that timing to a few years. In the context of food, agronomy or abiotic stress tolerance, recent data suggests that more than 40 crops underwent genome editing across 25 countries (Pixley et al, 2022). However, only 6 genome-edited crops (rice, maize, canola, soybean, mushroom and camelina) got approval for commercialization so far (Pixley et al, 2022). 

Because it is simple, cost-effective, and efficient, CRISPR-Cas9-based genome editing has attracted the attention of scientists. On the basis of this principle, several technologies, including base editing and prime editor, are being developed and successfully applied in crop genome editing.  

 

The future of gene editing in plant science 

The future of gene editing in plant science promises transformative advancements. Precision gene-editing technologies hold the potential to revolutionize crop development by enabling the creation of plants with enhanced traits such as disease resistance, climate resilience, and improved nutritional content in addition to assisting the understanding of the development and function of multiple genes that have not been characterized to date. This innovation is expected to contribute to global food security by accelerating crop breeding processes, creating crops tailored to specific environmental conditions, and reducing the environmental impact of agriculture through the development of more sustainable farming practices. However, the future also entails ongoing ethical and regulatory considerations, emphasizing the importance of responsible and transparent use of gene-editing techniques in plant sciences. Global collaboration and interdisciplinary efforts will play a crucial role in navigating these challenges and realizing the full potential of gene editing to address the evolving needs of our growing population and changing climate. 

 

References 

  1. Pixley, K.V., Falck-Zepeda, J.B., Paarlberg, R.L. et al. Genome-edited crops for improved food security of smallholder farmers. Nat Genet 54, 364–367 (2022). https://doi.org/10.1038/s41588-022-01046-7
  2. Al-Attar, Sinan, Westra, Edze R., van der Oost, John and Brouns, Stan J.J.. “Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes” Biological Chemistry, vol. 392, no. 4, 2011, pp. 277-289. https://doi.org/10.1515/bc.2011.042
  3. Li, H., Yang, Y., Hong, W. et al. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Sig Transduct Target Ther 5, 1 (2020). https://doi.org/10.1038/s41392-019-0089-y
  4. Martin Jinek et al. ,A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity.Science337,816-821(2012).DOI:10.1126/science.1225829
  5. Jiang, W., Bikard, D., Cox, D. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31, 233–239 (2013). https://doi.org/10.1038/nbt.2508
  6. Sternberg, S., Redding, S., Jinek, M. et al. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014). https://doi.org/10.1038/nature13011 

 

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About the Authors

Sourav Mukherjee is pursuing PhD in genetics at Monash University, Australia, and a 2023 Plantae Fellow. His study involves understanding the molecular mechanism of temperature responses in Arabidopsis. You can find him on X/Twitter at @SouravBiotech.

Andres Reyes is a biotechnology engineer passionate about plant sciences and scientific communication, and a 2023 Plantae Fellow. He believes that science and knowledge is useless if it is not transmitted to the community. His goal is to become a great researcher and to contribute with new findings to the scientific community. You can find him on X/Twitter at @f_andresreyes.