Modeling human point mutation diseases inXenopus tropicaliswith a modified CRISPR/Cas9 system

Abstract

Precise single‐base editing mXenopus tropicalis would greatly expand the utility of this true diploid frog for modeling human genetic diseases caused by point mutations. Here, we report the efficient conversion of C‐to‐T or G‐to‐A in X. tropicalis using the rat apolipoprotein B mRNA editing enzyme catalytic subunit 1–XTEN‐clustered regularly interspaced short palindromic repeat‐associated protein 9 (Cas9) nickase‐uracil DNA glycosylase inhibitor‐nuclear localization sequence base editor [base editor 3 (BE3)]. Coinjection of guide RNA and the Cas9 mutant complex mRNA into 1‐cell stage X. tropicalis embryos caused precise C‐to‐T or G‐to‐A substitution in 14 out of 19 tested sites with efficiencies of 5–75%, which allowed for easy establishment of stable lines. Targeting the conserved T‐box 5 R237 and Tyr C28 residues in X. tropicalis with the BE3 system mimicked human Holt‐Oram syndrome and oculocutaneous albinism type 1A, respectively. Our data indicate that BE3 is an easy and efficient tool for precise base editing in X. tropicalis.—Shi, Z., Xin, H., Tian, D., Lian, J., Wang, J., Liu, G., Ran, R., Shi, S., Zhang, Z., Shi, Y., Deng, Y., Hou, C., Chen, Y. Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system. FASEB J. 33,6962–6968 (2019). http://www.fasebj.org

CRISPR-SID: identifying EZH2 as a druggable target for desmoid tumors viain vivodependency mapping

“…We modulated the Wnt signaling pathway using both classical CRISPR/Cas9 for introducing truncating INDEL mutations in the apc gene, thereby mimicking familial adenomatous polyposis, and CRISPR base editing, for establishing an activating missense mutation (S45F) in beta-catenin, associated with sporadic desmoid tumors (Komor et al, 2016;Shi et al, 2019).…”

“…Finally, desmoid tumors could also be induced by CRISPRbase editing (BE3) mediating a targeted C-to-T conversion in the ctnnb1 gene (Fig. 1C) (Komor et al, 2016;Shi et al, 2019). As such, we modulate the conserved S45 phosphorylation site of β-catenin in X. tropicalis, recapitulating a clinically relevant human S45F missense mutation (Fig.…”

Advancing genetic and genomic technologies deepen the pool for discovery in Xenopus tropicalis

“…X. tropicalis tadpoles have proven to be an outstanding high‐throughput model for this purpose, with considerable advantages in time and cost over mice . CRISPR‐mediated mutagenesis of candidate genes is rapid and efficient in F 0 animals, and gene products can be independently knocked down by morpholino oligonucleotides, allowing rapid orthogonal approaches to define the requirement for a gene in developmental processes such as autism, heart development, albinism, and craniofacial development . A uniquely powerful feature of Xenopus is that these reagents can be delivered to just one blastomere of the two‐cell embryo to inhibit gene function only on one half of the embryo, providing a contralateral control and allowing investigation of cell autonomy.…”

“…34,35 CRISPR-mediated mutagenesis of candidate genes is rapid and efficient in F 0 animals, and gene products can be independently knocked down by morpholino oligonucleotides, allowing rapid orthogonal approaches to define the requirement for a gene in developmental processes such as autism, heart development, albinism, and craniofacial development. [36][37][38][39] A uniquely powerful feature of Xenopus is that these reagents can be delivered to just one blastomere of the two-cell embryo to inhibit gene function only on one half of the embryo, providing a contralateral control and allowing investigation of cell autonomy. Another powerful genetic approach that can be carried out within days in the F 0 animal is to use CRISPR and/or morpholino knockdown to create a genetically depleted background for the factor of interest, followed by a rescue injections with either the wildtype or patient allele of the factor, allowing one to specifically investigate the functionality of patient gene versions without requiring generations of breeding or homologous recombination.…”

Simple embryo injection of long single‐stranded donor templates with the CRISPR/Cas9 system leads to homology‐directed repair in Xenopus tropicalis and Xenopus laevis

“…Since then, more advanced technologies have also been applied to the Xenopus system. For example, one can now introduce some point mutations via a Cas9‐linked cytidine deaminase in Xenopus (Park et al, 2017; Shi et al, 2019), but the possible variation of mutations one can introduce by this approach is constrained.…”