Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Summary

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Abstract

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Introduction

Forward genetic screens have been widely used to generate genetic mutants disrupting various biological processes in model organisms such as Caenorhabditis elegans (C. elegans)¹. Analyses of such mutants lead to the discovery of functionally important genes and their signaling pathways^1,2. Traditionally, mutagenesis in C. elegans is achieved using mutagenic chemicals, radiation, or transposons². Chemicals such as ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) are toxic to humans; gamma-ray or ultraviolet (UV) radiation mutagenesis requires special equipment; and transposon-active strain....

Protocol

1. Construction of the LED Illuminator

NOTE: The required LED equipment is summarized in the List of Materials. The entire LED setup is small and can be placed anywhere in the lab, although we recommend it be placed in a dark room to control light exposure of worms⁴.

1. Connect the LED to a controller with cables (Figure 1A, D).
2. Connect the controller to a digital function generator/amplifier with a BNC cable (Figure 1A, C, D).
3. Fix the LED light 10 cm above the stage using a custom holder (Figure 1A).
   NOTE: We made the holder with metal parts.
4. C....

Results

We performed a forward genetic screen with CZ20310 juSi164 unc-119(ed3) using 30 min light exposure following the standard protocol described in Sections 2 and 3. Among 60 F₁ plates corresponding to 120 mutagenized haploid genomes, we found 8 F₁ plates with F₂ worms displaying visible phenotypes such as body size defect (Figure 3B), uncoordinated movement (Figure 3C), and adult lethality (Figure 3D

Discussion

Here, we describe a detailed procedure of optogenetic mutagenesis using His-mSOG expressed in the germline and provide an example of a small scale forward genetic screen. Compared to standard chemical mutagenesis, this optogenetic mutagenesis method has several advantages. Firstly, it is nontoxic, thereby keeping lab personnel away from toxic chemical mutagens such as EMS, ENU and trimethylpsoralen with ultraviolet light (TMP/UV). Secondly, the experimental procedure of mutagenesis can be used for a small number of the P.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ultra High Power LED light source	Prizmatix	UHP-mic-LED-460	460 ± 5 nm
LED controller	Prizmatix	UHPLCC-01
Digital function generator/amplifier	PASCO	PI-9587C	PI-9587C is no longer available. The replacement is PI-8127.
BNC cable male/male	THORLABS	CA3136
USB-TTL interface	Prizmatix		Optional
Photometer	THORLABS	PM50 and Model D10MM
Filter paper	Whatman	1001-110
Stereomicroscope	Leica	MZ95
NGM plate			Dissolve 5 g NaCl, 2.5 g Peptone, 20 g Agar, 10 µg/mL cholesterol in 1 L H2O. After autoclaving, add 1 mM CaCl2, 1 mM MgSO4, 25 mM KH2PO4(pH 6.0).
Lysis solution			10 mM Tris(pH 8.8), 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl2, 100 µg/mL Proteinase K