Establishment of Biallelic Knockout Technique in Nile Tilapia(Oreochromis niloticus)Based on CRISPR/Cas9 System:A Case Study of SLC24A5 Gene
Currently,the clustered regularly interspaced short palindromic repeat(CRISPR)-associated protein 9(Cas9)system(CRISPR/Cas9)stands out as a primary technology for enhancing genome editing efficiency in eukaryotes.However,for species with longer reproductive cycles,such as the Nile tilapia,the application of CRISPR/Cas9 technology faces challenges due to its low homo-zygous efficiency,especially in large-scale genetic screening studies.To solve this problem,a highly efficient CRISPR/Cas9 method was developed using SLC24A5 gene as an example in tilapia,which can directly achieve F0 generation biallelic knockout with a rela-tively stable probability in injected embryos.Specifically,two highly effective guide RNAs(gRNAs)were used for mixing,the con-centration of Cas9 protein was 800 ng·µL-1,the mass ratio of Cas9 protein to gRNA was 4∶1,and the injection dose was controlled at 1 nL,that is,800 pg Cas9 protein and 200 pg gRNA.This knockout technique enabled the direct production of individuals with a sig-nificant phenotype expressivity(Lv.1,Lv.2,Lv.3,and Lv.4)of 71%in F0 generation embryos of the new GIFT Nile tilapia,with a significantly phenotypic penetrance(Lv.1 and Lv.2)of 17%.This breakthrough technology provided a convenient and efficient means for genetic screening in Nile tilapia.
New Gift Nile tilapiabiallelic knockoutknockout efficiencyCRISPR/Cas9SLC24A5
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