CRISPR/CAS9 mediated gene editing for the improvement of beef and dairy cattle

Gene editing;Clustered regularly interspersed short palindromic repeats (CRISPR);Cattle

Polkoff, Kathryn ; Beever, Jonathan, ; ,Hurley, Walter ; Wheeler ; Matthew B.

关键词: Gene editing;    Clustered regularly interspersed short palindromic repeats (CRISPR);    Cattle;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/99109/POLKOFF-THESIS-2017.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

With the growing demands on agricultural production around the world, it is becoming increasingly important to figure out how to feed the growing population. For thousands of years we have been optimizing our agricultural outputs in livestock species through reproductive selection based on favorable traits, many of which are due to genetic variation. Single nucleotide polymorphisms are responsible for a great deal of either beneficial or undesirable mutations. In this study, we examine how we can use the CRISPR/Cas9 system to induce single-base pair substitutions, thereby improving animal genetics through removal of undesirable or insertion of beneficial mutations. In Angus beef cattle (Bos taurus), a mutation in the NHL-repeat containing 2 gene causes a heritable abnormality referred to as Developmental Duplications (DD). Calves homozygous for this mutation are affected with a broad range of neural tube defects and commonly exhibit polymelia, which is the presence of additional limbs. The mutation has been identified as a single-nucleotide polymorphism resulting in a valine to alanine substitution in a highly conserved protein-coding region of the gene. Similarly, in Holstein dairy cattle (Bos taurus) a guanine to adenine substitution mutation in the α-lactalbumin flanking region at (+15) base pairs from the transcriptional start site has been shown to be associated with increased quantity of milk production, likely due to upregulation of the α-lactalbumin protein and increased lactose synthesis. Other breeds of cattle used for dairy production, such as Nelore and other tropically adapted Bos indicus breeds, do not have this mutation unless they are crossed to Holsteins. In this study, the CRISPR/Cas9 system was paired with a guide RNA targeting the aforementioned genes to create a break in the DNA of fibroblast cells from Nelore and Angus cattle. Cells were also supplied with a 90 nucleotide single stranded-oligonucleotide repair template, which allows for homology directed repair to introduce the desired substitution at those loci. Cells from these cattle that are genetically improved by this technique may be used for somatic cell nuclear transfer and resulting embryos will be transferred to recipients. With these genetic improvements, we can preserve high quality beef cattle genetics while eliminating a harmful mutation, and we can increase milk yield of tropically adapted dairy cattle to feed malnourished populations around the world.