CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus

Abstract

Congenital malformations are the major cause of infant mortality in the US and Europe. Due to rapid advances in human genomics, we can now efficiently identify sequence variants that may cause disease in these patients. However, establishing disease causality remains a challenge. Additionally, in the case of congenital heart disease, many of the identified candidate genes are either novel to embryonic development or have no known function. Therefore, there is a pressing need to develop inexpensive and efficient technologies to screen these candidate genes for disease phenocopy in model systems and to perform functional studies to uncover their role in development. For this purpose, we sought to test F0 CRISPR based gene editing as a loss of function strategy for disease phenocopy in the frog model organism, Xenopus tropicalis. We demonstrate that the CRISPR/ Cas9 system can efficiently modify both alleles in the F0 generation within a few hours post fertilization, recapitulating even early disease phenotypes that are highly similar to knockdowns from morpholino oligos (MOs) in nearly all cases tested. We find that injecting Cas9 protein is dramatically more efficacious and less toxic than cas9 mRNA. We conclude that CRISPR based F0 gene modification in X. tropicalis is efficient and cost effective and readily recapitulates disease and MO phenotypes.

Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes

“…However, we speculate that the use of highly efficient doses of Cas9-sgRNA complexes or efficient doses of Cas9 mRNA plus sgRNA (see Nakayama et al, 2014) might have an additional advantage over titrated low doses of Cas9-3′UTR fusion mRNAs. The former approach is expected to yield mutations at earlier stages of development (Bhattacharya et al, 2015), resulting in a smaller diversity of alleles being transmitted in the F0 leapfrogged germline. In our gsc leapfrogging experiment, we recovered 10 different mutant alleles from the 37 F1 embryos analyzed (Fig.…”

High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus

“…CRISPR/Cas9 genome modification is a powerful approach for generating both targeted indel mutations by non-homologous endjoining (NHEJ) repair and more precise gene editing by the introduction of specific sequences through homology-directed repair (HDR) (Auer et al, 2014;Bassett et al, 2014;Bhattacharya et al, 2015;Blitz et al, 2013;Chen et al, 2013;Friedland et al, 2013;Guo et al, 2014;Hisano et al, 2015;Kimura et al, 2014;Kotani et al, 2015;Li et al, 2015;Nakayama et al, 2013Nakayama et al, , 2014Ran et al, 2013;Shi et al, 2015;Wang et al, 2015;Yang et al, 2014). While NHEJ-mediated indel mutations have proven to be effective in animal models including mouse, zebrafish and Xenopus (Blitz et al, 2013;Kotani et al, 2015;Nakayama et al, 2013;Wang et al, 2015;Xue et al, 2014), HDR-mediated targeted DNA insertion has proven more challenging.…”

Foxn4 promotes gene expression required for the formation of multiple motile cilia

“…To address these concerns, we asked whether similar Foxn4 phenotypes also occurred in F0 Xenopus embryos when targeted mutations were generated in the foxn4 genes using Cas9/CRISPR (Bhattacharya et al, 2015;Guo et al, 2014). Two independent gRNAs were designed that target conserved sequences encoding the forkhead domain within the third exon of all four foxn4 alleles in the pseudotetraploid X. laevis genome (Session et al, 2016).…”

“…CRISPR gRNAs for targeting foxn4 and foxj1 were generated in vitro from templates generated by PCR, as described previously (Bhattacharya et al, 2015). Briefly, cDNA sequences for foxj1 and foxn4 obtained from Xenbase were searched using Crisprdirect (http://crispr.dbcls.jp) for a 5′ GG-N(20) target sequence (Table S1, Fig.…”

“…This sequence was incorporated into a PCR primer that starts with a 5 bp stabilization sequence, the T7 promoter and a 20 bp overlap with a universal Cas9 PCR primer as described (Bhattacharya et al, 2015). PCR conditions for template generation followed those of Bhattacharya et al (2015) except that exTAQ (Takara Clontech) was used as the polymerase and 30 s was used for annealing. PCR products were column purified and used to generate gRNA using T7 polymerase (Promega) following the manufacturer's protocol and buffer conditions.…”