Protocol for assessment of the efficiency of CRISPR/Cas RNP delivery to different types of target cells

Abstract

Background Delivery of CRISPR/Cas RNPs to target cells still remains the biggest bottleneck to genome editing. Many efforts are made to develop efficient CRISPR/Cas RNP delivery methods that will not affect viability of target cell dramatically. Popular current methods and protocols of CRISPR/Cas RNP delivery include lipofection and electroporation, transduction by osmocytosis and reversible permeabilization and erythrocyte-based methods. Methods In this study we will assess the efficiency and optimize current CRISPR/Cas RNP delivery protocols to target cells. We will conduct our work using molecular cloning, protein expression and purification, cell culture, flow cytometry (immunocytochemistry) and cellular imaging techniques. Discussion This will be the first extensive comparative study of popular current methods and protocols of CRISPR/Cas RNP delivery to human cell lines and primary cells. All protocols will be optimized and characterized using the following criteria i) protein delivery and genome editing efficacy; ii) viability of target cells after delivery (post-transduction recovery); iii) scalability of delivery process; iv) cost-effectiveness of the delivery process and v) intellectual property rights. Some methods will be considered ‘research-use only’, others will be recommended for scaling and application in the development of cell-based therapies.

Production of Genetically Modified Porcine Embryos via Lipofection of Zona-Pellucida-Intact Oocytes Using the CRISPR/Cas9 System

“…For this reason, the use of lipofectamine in oocytes and embryos is starting to be applied and optimized [28]. Lipofection is an efficient method that has been widely used in many kinds of somatic cells to introduce foreign molecules, including the CRISPR/Cas9 system [39,40].…”

HAP1, a new revolutionary cell model for gene editing using CRISPR-Cas9

“…The DNA template is required to be homologous to the region immediately located around the DSB, and to be delivered simultaneously as either DNA or with the gRNA complexed with the Cas9, called ribonucleoprotein complexes (RNP). Therefore, coupled with the high specificity of CRISPR-Cas, DSB can be directed to a specific genomic site and the DNA donor sequence delivered used as template to precisely make various types of genome modifications, from single nucleotide substitutions to insertions of longer DNA sequences via HDR (Tyumentseva et al, 2021).…”

Plant biomacromolecule delivery methods in the 21st century

“…In addition to this immediate activity, directly delivered RNPs produce fewer off target effects since they are typically degraded a few hours after transfection, reducing the exposure time and the chances of off target cleavage (Gaj et al, 2012;Liang Z. et al, 2017;Yamagishi et al, 2019;Nicolia et al, 2020;Yang et al, 2021), behaving in a transient manner while still yielding permanent edits. The use of geneediting RNPs also allows for greater versatility and rapid screening of guide RNAs (Kim et al, 2014;Liang et al, 2015;Tyumentseva et al, 2021), eliminating the need to generate new plasmids for each target loci. Finally, direct delivery of RNPs also avoids integration into nuclear DNA (Kim et al, 2014;Liang Z. et al, 2017;DeWitt, Corn and Carroll, 2017), which can bypass regulatory hurdles and alleviate public concern over transgenic organisms (Woo et al, 2015).…”

“…For example, not all gene-editing RNPs function equally well due to unknown reasons when they are not expressed from plasmids (Vakulskas et al, 2018). Low cell viability has also been reported as a complication of direct delivery (Yamagishi et al, 2019;Tyumentseva et al, 2021). Direct delivery of RNPs, may also in some cases, require repeated transfections and large amounts of RNPs to be effective (Liang et al, 2015;Tyumentseva et al, 2021).…”

“…Low cell viability has also been reported as a complication of direct delivery (Yamagishi et al, 2019;Tyumentseva et al, 2021). Direct delivery of RNPs, may also in some cases, require repeated transfections and large amounts of RNPs to be effective (Liang et al, 2015;Tyumentseva et al, 2021). Despite these challenges, direct delivery of RNPs holds great promise for quick, versatile, and accurate genome editing.…”