Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Summary

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F₁ generation. Here, the transplantation step is demonstrated.

Abstract

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to "cut and paste" embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F₀ animals carrying "leapfrog transplants" (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F₁ embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F₀ phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Introduction

How genotype encodes phenotype has been a major question in biology since the rediscovery of Mendel's laws. An understanding of the roles of genes, their regulation and interactions within gene networks, and the functions of encoded products promises to provide tools for uncovering new biology and ameliorating disease states. For over half a century¹, the African clawed frog, Xenopus, has been a leading model for studies on a wide variety of topics in basic biology and biomedicine, including the genetic control of development. Historically, most research on Xenopus has used the allotetraploid frog, X. laevis, but ....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee of the University of California, Irvine.

1. Preparations for PGC Transplantation

1. Prepare dissection tools in advance, as previously described²³.
   NOTE: Eyebrow hair knives are used to make incisions with the assistance of a hair loop to stabilize the embryo while performing the surgeries. Eyebrow hairs and hair loops are glued into borosilicate glass Pasteur pipettes that have first been drawn in a flame and broken on the thinned portion (see Figure 1A, arrowhead) to create ....

Results

Following transplantation, the qualitative determination of the efficacy of CRISPR/Cas9 mutagenesis should be performed before expending the effort in animal husbandry to grow and maintain the animals to sexual maturity. Because animals carrying leapfrog transplants are somatically wildtype and the germline is difficult to access for direct measurements, saving the carcasses of donor embryos becomes important. DNA analysis from the carcasses serves as a proxy for the extent of mutagenesis.......

Discussion

This report provides a detailed protocol for the transplantation of vegetal tissue containing PGCs. Transplantation of PGCs is used in conjunction with genome-editing technologies (e.g., CRISPR/Cas9) to modify the germline of an animal while maintaining nearly all of its somatic tissues as genetically wild type. For leapfrogging to be successful, there are a number of critical factors to consider prior to performing performing transplantations.

To ensure the complete replacement of th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dumont #5 forceps	Fine Science Tools	11252-20 or 11252-30
Eyebrow hair knife			Homemade
Hair loop			Homemade
Pasteur pipettes, borosilicate glass	Fisher Scientific	13-678-20A
Krazy Glue (Cyanoacrylate-based)	Elmer's Products, Inc.	KG581	To affix eyebrow hair and hair loops into Pasteur pipettes, other similar glues can be used.
Oligodeoxynucleotides, custom ordered	Integrated DNA Technologies	Custom ordered	Template oligos for sgRNA synthesis, see Nakayama et al., 2014 for design details.
Megascript T7 kit	Ambion/ThermoFisher	AM1334	Or use Megashortscript T7 (AM1354) kit
Phenol, Tris buffered			High quality distilled phenol from any commercial supplier
Chloroform			High quality chloroform from any commercial supplier
Ethanol			High quality ethanol from any commercial supplier
Diethylpyrocarbonate (DEPC)	Sigma Chemical Co.	D5758-50ML
Cas9 protein, with nuclear localization signal	PNA Bio, Inc.	CP01	Reconstituted using DEPC-treated water according to manufacturer's recommendations
10X Marc's Modified Ringers solution			Homemade. Recipe (ref 26) for 1X MMR (lacking EDTA) is 100mM NaCl, 2 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 5 mM HEPES. Solution is pH adjusted to 7.4.
Agarose			Any Molecular Biology grade agarose is sufficient
60 X15 mm Petri plates	Falcon	351007
24-well plates	Falcon	3047
L-Cysteine	Sigma Chemical Co.	C7352-100G	Free base, not HCl salt
Proteinase K	Roche	03 115 828 001	Typically ~20mg/ml from Roche
sera Micron	sera		Tadpole food. Resuspend in growth medium. Can be purchased from a variety of online retailers