PRIYANKA CHAKRABORTY, AMITY UNIVERSITY, KOLKATA
The CRISPR/ Cas9 system has been rapidly used due to its’ simplicity and adaptability. Genetic disruptions of the CRISPR/Cas9 delivery can be attached either as a targeted disruption looking for recognition of a single gene or in a wider range with the concepts of genetic screening. Lentiviral marking of any cell can extend the functionality of the CRISPR system towards multiplexed perturbations or disruptions. In a greater variety of mammalian cell types, the CRISPR system has proven to be straightforward when passed through a single or one lentiviral vector. But, lentiviral delivery on the CRISPR component of a primary mammalian hematopoietic stem and progenitor cells has proved to be challenging.
But recently, several researchers have shown that ribonucleoproteins and Streptococcus pyogenes Cas9 protein in association with single-guide RNA (sgRNA), could be used to gain effective genome editing in HSPCs when joined by electroporation. It was further seen that a proper sgRNA structure, that improved gene editing efficiency and obtained knockout levels up to 90% for the surface proteins CD45 and CD44 in sgRNA. The combination CRISPR/Cas9 delivery had no negative reaction on CD34 expression in vitro related to non-treated HSPCs. Moreover, gene-edited HSPCs showed integration in- Vivo constitution following transplantation to weaker mice. Taken together, development of a combination of CRISPR/Cas9 delivery that performs efficient gene editing in primary human HSPCs, and is also compatible with high sensitivity both in vitro and in vivo.
The report on the enhancement of a hybrid system, jointly with lentiviral sgRNA delivery and transient delivery system of Cas9 for CRISPR/Cas9 genome editing in primary human HSPCs was analyzed. By delivering Cas9 as mRNA and using a modified sgRNA backbone, it was seen that this approach is highly flexible and compatible, allowing for efficient editing with much lower toxicity, and stem and progenitor cell potential was preserved successfully.
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Source: Yudovich, D., Bäckström, A., Schmiderer, L. et al. Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells. Sci Rep 10, 22393 (2020). https://doi.org/10.1038/s41598-020-79724-x
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