Efficient generation of gene-modified pigs via injection of zygote with Cas9/sgRNA

Abstract

Co-injection of zygotes with Cas9 mRNA and sgRNA has been proven to be an efficient gene-editing strategy for genome modification of different species. Genetic engineering in pigs holds a great promise in biomedical research. By co-injection of one-cell stage embryos with Cas9 mRNA and Npc1l1 sgRNA, we achieved precise Npc1l1 targeting in Chinese Bama miniature pigs at the efficiency as high as 100%. Meanwhile, we carefully analyzed the Npc1l1 sgRNA:Cas9-mediated on- and off-target mutations in various somatic tissues and ovaries, and demonstrated that injection of zygotes with Cas9 mRNA and sgRNA is an efficient and reliable approach for generation of gene-modified pigs.

Germline genome-editing research and its socioethical implications

“…In addition, Cas9 treatment apparently induced off-target mutations. These results suggest that there is room to optimize the protocol for germline genome editing in terms of (i) the methodology used to introduce the nucleases (the dose and the form of the enzymes -plasmid, mRNA, or protein [42,43]; cytoplasmic or pronuclear injection), (ii) mosaicism [14,19,20,24,25,28,[30][31][32]34,37], and (iii) possible off-target mutations [21][22][23][24]27,30,31] (Table 1). The risk of offtarget mutations is one of key technical issues.…”

“…Embryo microinjection -which is outwardly similar to a common ART technique, intracytoplasmic sperm injection (ICSI) [13] -demonstrated efficient generation of genetically modified animals including mice [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29], rats [30][31][32], pigs [33][34][35], sheep [36], cattle [33,36], and non-human primates [2,3,37], via NHEJ or the HDR pathway (Table 1). Moreover, germline gene modifications are also attainable via genome editing of spermatogonial stem cells (SSCs), potentially preventing mosaicism in offspring by avoiding the totipotent states that accompany embryogenesis.…”

Generation of human organs in pigs via interspecies blastocyst complementation

“…Recently, rapid evolving field of genome editing has also embraced pig (Carlson et al, 2012;Hai, Teng, Guo, Li, & Zhou, 2014;Hauschild et al, 2011;Wang et al, 2015;Whitworth et al, 2014;Whyte & Prather, 2012). By combining SCNT and genome editing, a number of useful pig models for human diseases have been created, such as cystic fibrosis (Rogers et al, 2008), diabetes (Renner et al, 2010;Umeyama et al, 2009), Alzheimer's disease (Kragh et al, 2009), retinitis pigmentosa (Petters et al, 1997;Ross et al, 2012) and spinal muscular atrophy (Lorson et al, 2011).…”

Optimizing Crispr Cas9 Genome Editing System:A Review

“…The CRISPR/Cas9 technology has been proved to be effective in large mammalian animal models, such as pigs and non-human primates. Gene modified pigs have been generated successfully by the co-injection of Cas9 mRNA and target gene sgRNA into one-cell stage embryo 37 . Recently Yang L demonstrated that pigs are almost perfect alternative for engineering human 39 .…”