Kanikah Mehndiratta, MSc, University of Glasgow
CRISPR-Cas9 technology is recently being extensively applied in human diseases research in study and effective treatment. Along with its huge therapeutic potential, the technique is also being explored in various other areas where gene editing could improve breeds and promote desirable traits. Traditional approaches to selective breeding in the field of aquaculture poses a lot of limitations.
Majorly, heritability of the desired trait in the progeny is an issue and the average time between the 2 generations of salmon in population lineage is about 3-4 years. This makes breeding an unfavourable approach and a more efficient repair is thus required for obtaining homozygous mutants when using CRISPR-Cas9 for gene editing in aquatic animals. Scientists are recently trying new breeding technologies (NBT) via the CRISPR/Cas9-induced homology directed repair (HDR) method to speed up the genetic improvements in favour of the aquaculture industry.
One such improvement could be disease resistance. A recent study published in the BMC Genomics journal is the first of its kind that uses the method of single nucleotide replacements (SNR) using asymmetrical ODNs and Crispr/Cas9 technology.
CRISPR-Cas9 for salmon gene editing:
It stands for Clustered regularly spaced palindromic repeats, the effector Cas proteins and the technology was developed in 2012 as a cut-and-paste tool for genome editing. The technique activates the non-homologous end joining (NHEJ) pathway by inducing a double-stranded DNA cut in the coding region where it may result in a gene knock-out or a frameshift mutation. This could be applied to study knock-out phenotypes in different fish species. For a precise alteration in the salmon genome, it is essential that an HDR is induced. This could be done by providing a repair template that is homologous to the target site of CRISPR. This would mostly allow changes in single nucleotide polymorphisms (SNPs), insertion of affinity tags for detection of proteins and modification of regulatory elements in order to change expression of the specific gene of interest.
In comparison to traditional breeding, which requires backcrossing and selection, an SNR could simply introduce desirable wild-type alleles for disease resistance into the salmon population in a time saving manner. One important consideration here is to reduce mosaicism, where few cells in the founder salmon genome would have a different genetic makeup than others. Symmetrical ODNs with short homology arms have already been used for knocking in a FLAG element in the fish pigmentation gene slc45a2 with perfect HDR rates. A more efficient rate of HDR using asymmetrical ODNs was the target of the study.
Significant Findings:
The approach successfully did precise SNR and introduced in the primordial germ cell dnd gene of founder salmon fish, a premature stop codon. Using deep sequencing, an efficacy of 59.2% came up for single salmon embryos. The same ODN design also aided in insertion of a FLAG element in the dnd and the slc45a2 regions of the salmon genome with amazing efficacies of about 32% and 36% respectively.
The study concluded that a precise SNR and knock-in approach via the ODN donors is very much possible. HDR efficiency is inversely proportional with the size of the insert and is also dependent on other factors such as the concentration of the template. HDR-induced single nucleotide repair can thus be utilized as an effective NBT by offering efficient introgression of desired alleles in the Atlantic salmon genomes.
Also read: The role of pre-capture multiplexing in exome sequencing
Reference:
- Straume, A. H., Kjærner-Semb, E., Skaftnesmo, K. O., Güralp, H., Lillico, S., Wargelius, A., & Edvardsen, R. B. (2021). Single nucleotide replacement in the Atlantic salmon genome using CRISPR/Cas9 and asymmetrical oligonucleotide donors. BMC Genomics, 22(1), 563. https://doi.org/10.1186/s12864-021-07823-8
2. Edvardsen, R. B., Leininger, S., Kleppe, L., Skaftnesmo, K. O., & Wargelius, A. (2014). Targeted mutagenesis in atlantic salmon (Salmo salar L.) using the crispr/cas9 system induces complete knockout individuals in the f0 generation. PLoS ONE, 9(9), e108622. https://doi.org/10.1371/journal.pone.0108622
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Author info:
Kanikah Mehndiratta is an avid researcher in the field of Genetics with a background in Biotechnology. She is a postgraduate from the University of Glasgow in their Medical Genetics and Genomics program. Currently, based in Chandigarh as a scientific writer, she involves herself mainly in projects related to neurological disorders. Outside of academics, she likes to read novels, travel and is involved in volunteer work mostly.
LinkedIn profile– https://www.linkedin.com/in/kanikah-mehndiratta-301830171
Other publications:
2. https://bioxone.in/news/worldnews/understanding-cancer-pathophysiology-via-isotopic-tracing/
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