Short communication
Establishing targeted carp TLR22 gene disruption via homologous recombination using CRISPR/Cas9

https://doi.org/10.1016/j.dci.2016.04.009Get rights and content

Highlights

  • Establishment of a TLR22 gene editing technique using CRISPR/Cas9 system in Labeo rohita carp.

  • Successful integration of donor DNA via HR at a targeted Cas9 nuclease site of TLR22 gene.

  • Gene edited mutant carp lacked TLR22 mRNA expression evidencing targeted TLR22 gene disruption.

Abstract

Recent advances in gene editing techniques have not been exploited in farmed fishes. We established a gene targeting technique, using the CRISPR/Cas9 system in Labeo rohita, a farmed carp (known as rohu). We demonstrated that donor DNA was integrated via homologous recombination (HR) at the site of targeted double-stranded nicks created by CRISPR/Cas9 nuclease. This resulted in the successful disruption of rohu Toll-like receptor 22 (TLR22) gene, involved in innate immunity and exclusively present in teleost fishes and amphibians. The null mutant, thus, generated lacked TLR22 mRNA expression. Altogether, this is the first evidence that the CRISPR/Cas9 system is a highly efficient tool for targeted gene disruption via HR in teleosts for generating model large-bodied farmed fishes.

Introduction

Toll-like receptors (TLRs) are major players for innate immunity (Baoprasertkul et al., 2007, Oshiumi et al., 2008, Panda et al., 2014). The TLRs are type 1 integral membrane glycoproteins, having leucine-rich repeat (LRR) domains in their extracellular regions for binding pathogen associated molecular patterns (PAMPs) and a Toll-interleukin-1 receptor domain (TIR), which transmits downstream signals into the cytosol by recruiting and activating a cascade of adaptor molecules (Akira, 2009, Panda et al., 2014). Together, the extracellular LRR domains constitute a horse-shoe shape and, mainly, act as pathogen recognition receptors (PRRs). In teleost fishes, most TLRs were identified as orthologs of mammalian TLRs (Byadgi et al., 2014, Reyes-Becerril et al., 2015). However, TLR5, TLR14, TLR19, TLR20, TLR21 and TLR22 are exclusively present in fish species (Aoki et al., 2008, Rebl et al., 2007, Reyes-Becerril et al., 2015) and, hence, are likely to play distinctive roles. Among these, TLR22 has been studied extensively in teleosts and amphibians (Ishii et al., 2007, Panda et al., 2014, Rebl et al., 2007, Reyes-Becerril et al., 2015, Roach et al., 2005, Samanta et al., 2014). It is present on the cell surface membrane and can recognize viral nucleic acids on the cell surface to transmit signals to induce cytokines (Aoki et al., 2008, Panda et al., 2014, Rebl et al., 2007). Recently, we cloned and characterized the TLR22 gene (Database ID: KC953874) in Labeo rohita (popularly known as rohu), a commercially important farmed carp species (Barman et al., 2003, Barman et al., 2011, Panda et al., 2011). Documented evidences, based on mRNA/protein expression profiling, provided the clue that it confers resistance against wide range of viral, bacterial and lice infections (Panda et al., 2014, Reyes-Becerril et al., 2015, Samanta et al., 2014).

Elucidation of exact immune-related mechanistic pathways has been made possible by establishing model animals to target disruption/integration of the selected gene. For example, the exact functions of TLRs, including other down regulated genes in mammals, were determined mainly by knock-out mice analysis (Alexopoulou et al., 2002, Zhang et al., 2007). Such knock-out models are lacking in teleost species (Schartl, 2014). Since the TLR22 gene is teleost-specific and targeting this gene in carp would be useful to enrich knowledge about defense mechanisms. This should have a larger impact on the progress of exploring molecular functions and its involvements in disease and host defense.

Gene targeting by homologous recombination (HR) was made possible with mammalian embryonic stem (ES) cells and selective somatic chicken DT40 cell line (Barman et al., 2006, Barman et al., 2008, Sanematsu et al., 2006). In fish, such targeting efficient ES cell or somatic cells are not available, impeding research progress in physiological or immunological pathways. The discovery of zinc-finger nucleases (ZFNs) based technology brought revolutionary change to gene editing/targeting in the predetermined position in the genome of wide ranging species, including cell lines (Klug, 2010). More recently, sequence-specific nuclease tools, such as ‘Transcription Activator-Like Effector Nucleases (TALENs)’ and ‘Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nucleases (Cas9)’ have impressively extended the genome editing possibilities in model organisms, including zebrafish and several cell lines (Auer et al., 2014, Bachu et al., 2015, Gaj et al., 2013, Hockemeyer et al., 2011, Sanjana et al., 2012, Tatsumi et al., 2014, Wang et al., 2013a, Wang et al., 2015, Yang et al., 2014a). Both systems have recently been used to create knock-out allele efficiently. Also, both the tools have been successfully employed in knock-in of DNA cassettes at defined loci via HR repair mechanism (Auer et al., 2014, Choulika et al., 1995, Sadelain et al., 2012).

These nucleases are efficient in generating double-strand breaks within the precise locus in the genome that can be repaired by error-prone nonhomologous end joining leading to a functional knock-out of the targeted gene and can be used to integrate a foreign DNA sequence at a specific locus through homologous recombination. The Cas9 system has been shown to be most efficient in the generation genetically modified mice, rats, rabbits, zebrafish, medaka, atlantic salmon frog and tilapia (Auer et al., 2014, Edvardsen et al., 2014, Li et al., 2013, Li et al., 2014, Qiu et al., 2014, Wang et al., 2015, Wang et al., 2013a, Yang et al., 2014a).

Fish generally have some inherent advantages for genetic engineering research, including enormous brood and in vitro fertilization, and ease of operation and observation. Even in such a scenario, recent advancements of genome editing technologies have not been fully implemented, which has limited research on in vivo gene function. HR mediated gene disruption is only limited to the model zebrafish (Zu et al., 2013). In this study, we successfully disrupted the LrTLR22 gene by enforcing HR mediated repair of double stranded nicks created by the Cas9 nuclease.

Section snippets

Fish and breeding

Labeo rohita (rohu) were collected from the farm of the ICAR-Central Institute of Freshwater Aquaculture, Bhubaneswar, Odisha, India. In vitro fertilization was performed in the hatchery, following the induced breeding protocol, using the ovaprim hormone as described by Mahapatra et al. (2006).

Total RNA/genomic DNA extraction and cDNA preparation

Total RNA was extracted using the TRIzol reagent (Invitrogen, USA) following an established protocol (Panda et al., 2014, Patra et al., 2015). The extracted RNAs were purified and quantified as per

Results and discussion

Because immune responsive TLR22 is specific to teleosts/amphibians and its intron-less gene structure in Labeo rohita (rohu carp) was available (Panda et al., 2014, Samanta et al., 2014); we intended to explore developing/generating model rohu fish lacking TLR22. We selected CRISPR/Cas9 system to disrupt the TLR22 locus, because of its ease of design with efficiency relative to ZFNs and TALE nucleases (Liang et al., 2015, Nakade et al., 2014, Ota et al., 2014, Tatsumi et al., 2014, Xiao et al.,

Acknowledgements

This work was supported by a grant from the National Agricultural Science Fund (NASF), Indian Council of Agricultural Research, Union Ministry of Agriculture, Government of India (NASF/BS-3017). Thanks to the Director of this Institute for providing required facilities to carry out this research.

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