ESTABLISHMENT of BETA-LACTOGLOBULIN KNOCKOUT BUFFALO CELL LINES using CRISPR-RNP COMPLEXES and INDIGENOUS LAB-MADE TRANSFECTION BUFFER
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Date
2023
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ICAR-NDRI, KARNAL
Abstract
Beta-Lactoglobulin (BLG) is a major milk allergen present in farm animals; but it is
absent in human milk. Engineered endonucleases, including CRISPR, enable targeted genetic
modification in livestock. In India, the importance of buffaloes in the dairy industry is
pronounced, accounting for approximately 45% of the country's milk production. This
highlights the dominant position of buffaloes in fulfilling the milk requirements of the Indian
population. The production of -lactoglobulin knockout buffaloes could have significant
implications, including the availability of hypoallergenic milk and increased demand for
buffalo milk among individuals with milk allergies. By considering this in mind, the present
thesis study was designed to establish -lactoglobulin knockout cell clones, and the
production of -lactoglobulin-gene edited embryos. To achieve targets of this study, five
major experiments were performed: 1) Designing of CRISPR guides against BLG gene; 2)
Determination of editing efficiencies of designed guides using different methods such as T7E
assay, TIDE, and ICE analysis; 3) Generation of single cell clones from electroporated cells;
4) Validation of mono-allelic or bi-allelic mutations in single-cell colonies; 5) Production of
cloned blastocyst stage embryos from edited BLG-gene edited cells and assessment of their
quality. In summary, a total three sgRNAs were designed, of which two sgRNAs were
efficient to edit the BLG locus with indigenous lab-made buffer. With the best optimized
condition, in the present study, the seven BLG-gene edited single cell clones were established
that are 1) C1 has bi-allelic heterozygous mutation, one allele with -107 bp deletion and the
other allele with - 9bp deletion with C/T conversion; 2) C2 has bi-allelic homozygous
mutation, both alleles have -32 bp deletion; 3) C4 has mono-allelic mutation, in which one
allele has -151 bp deletion; 4) C8 has the mono-allelic mutation, in which one allele is with -8
bp deletion; 5) C10 has bi-allelic heterozygous mutation, one allele with -150 bp deletion and
the other allele with one bp T insertion; 6) C11 has mono-allelic mutation, in which one allele
is with -14 bp deletion; and 7) C13 has bi-allelic heterozygous mutation, one allele with -27
bp deletion and the other allele with - 26 bp deletion. Following the use of some edited single
cells clones as nuclear donor for SCNT applications, the embryonic developmental rates were
different among the cell type; however, the total cell number and apoptotic index were
similar among edited and non-edited (wild type) donor cells. Thus, SCNT outcome indicated
that genetic modification using CRISPR did not affect the cloned embryonic developmental
competence. The results of this study demonstrated the successful establishment of a pipeline
for CRISPR-based manipulation of the -lactoglobulin locus in buffaloes and the production
of BLG-gene-edited cloned embryos. The generated -lactoglobulin knockout cell clones will
provide a valuable resource for the production of live genome-edited buffaloes.