Rapid and efficient analysis of gene function using CRISPR‐Cas9 in Xenopus tropicalis founders

Abstract

Recent advances in genome editing using programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, have facilitated reverse genetics in Xenopus tropicalis. To establish a practical workflow for analyzing genes of interest using CRISPR-Cas9, we examined various experimental procedures and conditions. We first compared the efficiency of gene disruption between Cas9 protein and mRNA injection by analyzing genotype and phenotype frequency, and toxicity. Injection of X. tropicalis embryos with Cas9 mRNA resulted in high gene-disrupting efficiency comparable with that produced by Cas9 protein injection. To exactly evaluate the somatic mutation rates of on-target sites, amplicon sequencing and restriction fragment length polymorphism analysis using a restriction enzyme or recombinant Cas9 were performed. Mutation rates of two target genes (slc45a2 and ltk) required for pigmentation were estimated to be over 90% by both methods in animals exhibiting severe phenotypes, suggesting that targeted somatic mutations were biallelically introduced in almost all somatic cells of founder animals. Using a heteroduplex mobility assay, we also showed that off-target mutations were induced at a low rate. Based on our results, we propose a CRISPR-Cas9-mediated gene disruption workflow for a rapid and efficient analysis of gene function using X. tropicalis founders.

Targeted gene disruption by use of CRISPR/Cas9 ribonucleoprotein complexes in the water flea Daphnia pulex

“…In addition, the risk of nonspecific insertion is the lowest among these methods because neither DNA nor Cas9 mRNA is used (Kim, Kim, Cho, Kim, & Kim, ; Liang et al., ). Ever since delivery of Cas9 RNPs was first reported in the nematode Caenorhabditis elegans (Cho, Lee, Carroll, Kim, & Lee, ), it has been used as a rapid and highly efficient tool for the analysis of gene function in many organisms such as zebrafish Danio rerio (Sung et al., ), tropical clawed frog Xenopus tropicalis (Shigeta et al., ), fruit‐fly Drosophila melanogaster (Lee et al., ) and mouse Mus musculus (Sung et al., ). In this study, we chose Cas9 RNPs, the third method as described above, to edit the D. pulex genome.…”

Establishment of knockout adult sea urchins by using a CRISPR‐Cas9 system

“…Therefore, the timing of CRISPR‐Cas9 mediated genome editing is comparable to those of ZFN and TALEN. Ribonucleoprotein (RNP) consisting of the SpCas9 protein in complex with sgRNA has been used in cultured cells and animals (Kim, Kim, Cho, Kim, & Kim, ; Kotani, Taimatsu, Ohga, Ota, & Kawahara, ; Lee et al., ; Sakane et al., ; Shigeta et al., ; Sung et al., ); it shortens the timing of genome editing and overcomes the mosaic property (Kim et al., ; Kotani et al., ). We attempted to accelerate mutagenesis in H. pulcherrimus by using SpCas9 RNP.…”

TALENs and CRISPR/Cas9 fuel genetically engineered clinically relevant Xenopus tropicalis tumor models

“…Whereas antisense technologies previously only allowed for transient gene knockdown, TALEN and CRISPR/Cas9 now permit rapid gene knockout with germline transmission. This unlocks genetically engineered Xenopus tropicalis models (GEXM) as a platform providing unique possibilities for human disease modeling (Bhattacharya et al ., ; Lienkamp, ; Liu et al ., ; Shigeta et al ., ; Tandon et al ., ).…”

“…engineered Xenopus tropicalis models (GEXM) as a platform providing unique possibilities for human disease modeling (Bhattacharya et al, 2015;Lienkamp, 2016;Liu et al, 2016;Shigeta et al, 2016;Tandon et al, 2016).…”