Designing of a DNA matrix for transgene integration into the bovine beta-lactoglobulin gene locus using CRISPR/Cas9 technology

E.M Koloskova,K.S Ostrenko,V.A Ezerskiy

doi:10.15421/2020_231

Abstract

Beta-lactoglobulin (BLG) is the main protein in milk serum in almost all mammals, with the exception of rodents and primates. Regulatory regions of the beta-lactoglobulin gene in ruminants (sheep, goats, and cattle) as part of genetic constructs provide tissue - specific expression of recombinant protein in the mammary gland and have been actively used in genetic engineering since the beginning of the era of creating transgenic animals. To work effectively with the CRISPR/Cas9 genomic editing method, it is necessary to know the exact DNA sequence of the target gene: this is necessary both for creating a DNA matrix for homologous recombination and for the targeted accuracy of guide RNAs. A polymorphic variant of the bovine BLG gene was identified, whose sperm was used to fertilize cow oocytes in vitro. The aim of this work was to create a plasmid containing 5’ - and 3’ - arms of homology (ha) to the bovine BLG gene. Based on ??TZ57R/T, the pTZhaBLG plasmid was obtained, which has a unique site for EagI restriction at the junction of the homology arms. A fragment containing a biologically active protein gene can be embedded in the resulting plasmid at this restriction site. We created the pBLGcmvEGFP plasmid containing the green fluorescent protein (EGFP) gene under the cytomegalovirus (cmv) promoter: protein expression can serve as a reliable indicator of successful integration of the transgene into the genome. The resulting plasmids in circular or linearized form are intended for site-specific integration by homologous recombination repair into the BLG gene using CRISPR/Cas9 components.

Highlights

For the constantly growing population of the world, issues of medical, biological and food security will inevitably become more acute in the near future, which makes it urgent to find and develop effective ways to solve them
We presented the scheme for selecting homology arms and fragments for sequencing (Fig. 1)
Since the efficiency of the CRISPR/Cas9 components depends on the quality of the sgRNA target sequences, the possible polymorphism of the targeted gene in the region of potential double-stranded sections (DSS) is of great importance when selecting them

Summary

Introduction

For the constantly growing population of the world, issues of medical, biological and food security will inevitably become more acute in the near future, which makes it urgent to find and develop effective ways to solve them. The application of genetic engineering in animal husbandry has proved challenging, especially for large farm animals with a small number of offspring in the litter. The method of microinjection of zygotes, previously used only for small laboratory animals with a large number of offspring (mice, rats, rabbits), has become available for obtaining transgenic (or other genetically modified) farm animals – goats, pigs, and cattle. The application of endonuclease technologies using the homology directed repair (HDR) mechanism is an effective method for changing the composition of milk by replacing the genes of endogenous milk proteins in farm animals in order to improve the consumer qualities of milk and use the animal's mammary glands as a bioreactor for the production of heterologous proteins (Shepelev et al, 2018).

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