Targeted Chromosomal Translocations and Essential Gene Knockout Using CRISPR/Cas9 Technology in Caenorhabditis elegans

Abstract

Many genes play essential roles in development and fertility; their disruption leads to growth arrest or sterility. Genetic balancers have been widely used to study essential genes in many organisms. However, it is technically challenging and laborious to generate and maintain the loss-of-function mutations of essential genes. The CRISPR/Cas9 technology has been successfully applied for gene editing and chromosome engineering. Here, we have developed a method to induce chromosomal translocations and produce genetic balancers using the CRISPR/Cas9 technology and have applied this approach to edit essential genes in Caenorhabditis elegans. The co-injection of dual small guide RNA targeting genes on different chromosomes resulted in reciprocal translocation between nonhomologous chromosomes. These animals with chromosomal translocations were subsequently crossed with animals that contain normal sets of chromosomes. The F1 progeny were subjected to a second round of Cas9-mediated gene editing. Through this method, we successfully produced nematode strains with specified chromosomal translocations and generated a number of loss-of-function alleles of two essential genes (csr-1 and mes-6). Therefore, our method provides an easy and efficient approach to generate and maintain loss-offunction alleles of essential genes with detailed genetic background information.KEYWORDS CRISPR/Cas9; chromosomal translocation; essential gene knockout; balancer E SSENTIAL genes are required for the development and fertility of organisms. Loss-of-function mutations of essential genes usually result in growth arrest or sterility. The production and maintenance of homozygous mutants of the essential genes are demanding and time-consuming. A series of strains with particular chromosomal rearrangements, such as duplications, translocations, and inversions, have been generated and applied to screen and grow lethal or sterile mutants. Conventional methods to elicit chromosomal rearrangements involved treating the animals with ion irradiation or chemical mutagens (Jones et al. 2011). However, the majority of existing balancer strains lack detailed sequence information. Additionally, many unintended mutations are introduced during mutagenesis and are difficult to eliminate by backcrossing with wild-type strains. Therefore, it is critical to develop a more efficient method to produce balancer strains with detailed sequence information and nominal background mutations. Furthermore, the development of more effective approaches to generate and maintain loss-offunction mutations of particular essential genes is required.Recent research in targeted genome editing has made inspiring progress in genome engineering, among which is the clustered regularly interspaced short palindromic repeats (CRISPR) technology (Cong et al. 2013;Jiang et al. 2013;Mali et al. 2013;Ran et al. 2013;Wang et al. 2013;Hsu et al. 2014;Shalem et al. 2014;Sternberg et al. 2014;Wang et al. 2014). In the CRISPR/Cas9 system, small guide RNA (sgRNA) targets its complementary...

Induction of Chromosomal Translocations with CRISPR-Cas9 and Other Nucleases: Understanding the Repair Mechanisms That Give Rise to Translocations

“…This new nuclease was quickly used to induce chromosomal translocations, including the previously described models of NPM1-ALK (Ghezraoui et al, 2014) and Ewing sarcoma (Torres et al, 2014) (Renouf et al, 2014) and new models of lung cancer translocations (Choi and Meyerson, 2014) and acute myelogenous leukemia (Renouf et al, 2014; Torres et al, 2014). CRISPR-Cas9 was also used by other teams to create chromosomal translocations in mouse embryonic stem cells (Jiang et al, 2016) and in mouse myoblasts, the latter modeling the human alveolar rhabdyomyosarcoma Pax3-Foxo1 (Lagutina et al, 2015), but also in other organisms, namely C. elegans (Chen et al, 2015) and Leishmania (Zhang et al, 2017). …”

Targeted genome engineering in Caenorhabditis elegans

“…Thereafter, large genomic fragments can be reversed, deleted, or translocated to other chromosomal loci. For example, our lab has reported the use of dual sgRNA strategy to direct reciprocal chromosomal translocations in C. elegans [58]. The nematode strains with specific chromosomal rearrangements can serve as genetic balancers for the screening and maintenance of essential genes [59].…”

“…It is pivotal to develop strategies to design sgRNAs with higher efficiency. The combination of multiple sgRNAs targeting the same gene was shown to improve cleavage efficiency [49, 58, 62]. Farboud and Meyer reported that guide RNAs with a GG motif at the 3′ end of their target sequences can dramatically improve the editing efficiency [63].…”

“…The PCR amplicons surrounding the sgRNA sites can be analyzed using T7 endonuclease I (T7E1) or restrictive endonuclease digestion. Moreover, simultaneous introduction of multiple sgRNAs lead to the removal of large DNA chunks between the sgRNAs, which simplifies the identification of deletion mutants by PCR amplification followed by agarose gel electrophoresis [58, 62]. The integrated transgenes can be identified by PCR amplified with appropriate primers as well.…”

“…Additionally, the benomyl resistance provides an alternative counter selection strategy for targeted knock-in of specific DNA fragments at the ben - 1 locus [44]. While wild-type animals exhibit a visible paralysis phenotype when exposed to benomyl at 25 °C, the loss of function of ben - 1 by targeted transgene insertion confers benomyl resistance [58]. …”