Cas9

Accepted Manuscript Establishing targeted carp TLR22 gene disruption via homologous recombination using CRISPR/Cas9 Vemulawada Chakrapani, Swagat Kumar Patra, Rudra Prasanna Panda, Kiran Dashrath Rasal, Pallipuram Jayasankar, Hirak Kumar Barman PII:

S0145-305X(16)30126-4

DOI:

10.1016/j.dci.2016.04.009

Reference:

DCI 2613

To appear in:

Developmental and Comparative Immunology

Received Date: 29 February 2016 Revised Date:

8 April 2016

Accepted Date: 8 April 2016

Please cite this article as: Chakrapani, V., Patra, S.K., Panda, R.P., Rasal, K.D., Jayasankar, P., Barman, H.K., Establishing targeted carp TLR22 gene disruption via homologous recombination using CRISPR/Cas9, Developmental and Comparative Immunology (2016), doi: 10.1016/j.dci.2016.04.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Short Communication

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

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Dashrath Rasal, Pallipuram Jayasankar, Hirak Kumar Barman*

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Vemulawada Chakrapani, Swagat Kumar Patra, Rudra Prasanna Panda, Kiran

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Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater

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Aquaculture, Bhubaneswar 751 002, Odisha, India

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*Corresponding Author: Hirak Kumar Barman, PhD

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E-mail: [email protected]; [email protected] Tel: +916742465407; +916742465414

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Fax:+916742465407.

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ACCEPTED MANUSCRIPT Abstract

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Recent advances in gene editing techniques have not been exploited in farmed fishes. We

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established a gene targeting technique, using the CRISPR/Cas9 system in Labeo rohita, a

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farmed carp (known as rohu). We demonstrated that donor DNA was integrated via

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homologous recombination (HR) at the site of targeted double-stranded nicks created by

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CRISPR/Cas9 nuclease. This resulted in the successful disruption of rohu Toll-like receptor

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22 (TLR22) gene, involved in innate immunity and exclusively present in teleost fishes and

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amphibians. The null mutant, thus, generated lacked TLR22 mRNA expression. Altogether,

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this is the first evidence that the CRISPR/Cas9 system is a highly efficient tool for targeted

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gene disruption via HR in teleosts for generating model large-bodied farmed fishes.

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Keywords:

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CarpTLR22

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Gene editing

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CRISPR/Cas9

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Microinjection

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Homologous recombination

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1. Introduction Toll-like receptors (TLRs) are major players for innate immunity (Baoprasertkul et

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al., 2007; Oshiumi et al., 2008; Panda et al., 2014). The TLRs are type 1 integral membrane

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glycoproteins, having leucine-rich repeat (LRR) domains in their extracellular regions for

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binding pathogen associated molecular patterns (PAMPs) and a Toll-interleukin-1 receptor

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domain (TIR), which transmits downstream signals into the cytosol by recruiting and

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activating a cascade of adaptor molecules (Akira, 2009; Panda et al., 2014). Together, the

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extracellular LRR domains constitute a horse-shoe shape and, mainly, act as pathogen

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recognition receptors (PRRs). In teleost fishes, most TLRs were identified as orthologs of

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mammalian TLRs (Byadgi et al., 2014; Reyes-Becerril et al., 2015). However, TLR5,

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TLR14, TLR19, TLR20, TLR21 and TLR22 are exclusively present in fish species (Aoki et

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al., 2008; Rebl et al., 2007; Reyes-Becerril et al., 2015) and, hence, are likely to play

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distinctive roles. Among these, TLR22 has been studied extensively in teleosts and

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amphibians (Ishii et al., 2007; Panda et al., 2014; Rebl et al., 2007; Reyes-Becerril et al.,

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2015; Roach et al., 2005; Samanta et al., 2014). It is present on the cell surface membrane

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and can recognize viral nucleic acids on the cell surface to transmit signals to induce

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cytokines (Aoki et al., 2008; Panda et al., 2014; Rebl et al., 2007). Recently, we cloned and

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characterized the TLR22 gene (Database ID: KC953874) in Labeo rohita (popularly known

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as rohu), a commercially important farmed carp species (Barman et al., 2003; Barman et al.,

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2011; Panda et al., 2011).

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profiling, provided the clue that it confers resistance against wide range of viral, bacterial

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and lice infections (Panda et al., 2014; Reyes-Becerril et al., 2015; Samanta et al., 2014).

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Documented evidences, based on mRNA/protein expression

Elucidation of exact immune-related mechanistic pathways has been made possible by

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establishing model animals to target disruption/integration of the selected gene. For example,

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the exact functions of TLRs, including other down regulated genes in mammals, were

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determined mainly by knock-out mice analysis (Alexopoulou et al., 2002; Zhang et al., 2007).

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Such knock-out models are lacking in teleost species (Schartl, 2014). Since the TLR22 gene

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is teleost-specific and targeting this gene in carp would be useful to enrich knowledge about

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defense mechanisms. This should have a larger impact on the progress of exploring molecular

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functions and its involvements in disease and host defense.

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Gene targeting by homologous recombination (HR) was made possible with

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mammalian embryonic stem (ES) cells and selective somatic chicken DT40 cell line (Barman

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et al., 2006; Barman et al., 2008; Sanematsu et al., 2006). In fish, such targeting efficient ES 3

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immunological pathways. The discovery of zinc-finger nucleases (ZFNs) based technology

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brought revolutionary change to gene editing/targeting in the predetermined position in the

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genome of wide ranging species, including cell lines (Klug, 2010). More recently, sequence-

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specific nuclease tools, such as ‘Transcription Activator-Like Effector Nucleases (TALENs)’

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and ‘Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated

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nucleases (Cas9)’ have impressively extended the genome editing possibilities in model

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organisms, including zebrafish and several cell lines (Auer et al., 2014; Bachu et al., 2015;

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Gaj et al., 2013; Hockemeyer et al., 2011; Sanjana et al., 2012; Tatsumi et al., 2014; Wang et

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al., 2015; Wang et al., 2013; Yang et al., 2014a). Both systems have recently been used to

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create knock-out allele efficienctly. Also,both the tools have been successfully employed in

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knock-in of DNA cassettes at defined loci via HR repair mechanism (Auer et al., 2014;

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Choulika et al., 1995; Sadelain et al., 2012).

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These nucleases are efficient in generating double-strand breaks within the precise

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locus in the genome that can be repaired by error-prone nonhomologous end joining leading

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to a functional knock-out of the targeted gene and can be used to integrate a foreign DNA

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sequence at a specific locus through homologous recombination. The Cas9 system has been

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shown to be most efficient in the generation genetically modified mice, rats, rabbits,

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zebrafish, medaka, atlantic salmon frog and tilapia (Auer et al., 2014; Edvardsen et al., 2014;

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Li et al., 2013; Li et al., 2014; Qiu et al., 2014; Wang et al., 2015; Wang H et al., 2013; Yang

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et al., 2014a).

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Fish generally have some inherent advantages for genetic engineering research,

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including enormous brood and in vitro fertilization, and ease of operation and observation.

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Even in such a scenario, recent advancements of genome editing technologies have not been

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fully implemented, which has limited research on in vivo gene function. HR mediated gene

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disruption is only limited to the model zebrafish (Zu et al., 2013). In this study, we

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successfully disrupted the LrTLR22 gene by enforcing HR mediated repair of double

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stranded nicks created by the Cas9 nuclease.

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2. Materials and methods

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2.1. Fish and breeding Labeo rohita (rohu) were collected from the farm of the ICAR-Central Institute of

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Freshwater Aquaculture, Bhubaneswar, Odisha, India. In vitro fertilization was performed in

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the hatchery, following the induced breeding protocol, using the ovaprim hormone as

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described by Mahapatra et al., (2006).

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2.2. Total RNA/genomic DNA extraction and cDNA preparation

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Total RNA was extracted using the TRIzol reagent (Invitrogen, USA) following an

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established protocol (Mohanta et al., 2014b; Panda et al., 2014; Patra et al., 2015). The

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extracted RNAs were purified and quantified as per standard protocol. The reverse

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transcriptase cDNA synthesis kit (Clontech, USA) was used to reverse transcribed RNA into

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cDNA (Mohanta et al., 2014b; Panda et al., 2011; Patra et al., 2015). Genomic DNA was

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isolated by standard phenol–chloroform extraction and ethanol precipitation methods

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(Mohanta et al., 2014a; Panda et al., 2014; Patra et al., 2015).

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2.3. Construction of Cas9 Smart Nuclease targeting vector with gene specific guide RNA

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The extracellular pathogen recognition site i.e. LRR of LrTLR22 (Panda et al., 2014)

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was chosen as an unique target site, and accordingly the guide RNA (gRNA) oligonucleotides

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were cloned into CRISPR-Cas9 vector named as EF1-hspCas9-H1-gRNA linearized

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SmartNuclease vector (Cat. No. CAS900A-1, SBI, USA) as per manufacturer’s guidelines.

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Briefly, the PAM sequence (5’-CGG-3’) immediately followed the 20 bp target sequence (5’-

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GTCTGAAGACAACTCGATCC-3’), spanning 1836 to 1858 bases of LRR domain of

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LrTLR22 (Database ID: KC953874). The possible off-target sites were scanned with the help

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of online tools, such as CHOPCHOP (https://chopchop.rc.fas.harvard.edu/), ZiFiT Targeter

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(http://zifi t.partners.org/ZiFiT/) and CRISPRdirect (http://crispr.dbcls.jp/). The structure of

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double stranded gRNA oligonucleotides contained desirable overhangs at 5’-termini (as

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shown in Fig. 1). The oligonucleotides were annealed by incubating at 95oC for 5 min to

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form a duplex. Subsequently, the duplex was ligated with the linearized CRISPR-Cas9 vector

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containing H1 polymerase III promoter as depicted in Fig. S1. The ligated products were

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transformed into the One Shot TOP10 chemically competent E. coli (Invitrogen, USA). The

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positive clones were screened and plasmid DNA (hereafter, known as LrTLR22Cas9) was

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extracted using QIAprep Spin Miniprep Kit (Qiagen, Germany).

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2.4. Construction of donor HR vector with homology arms of targeted site Two homology arms of LrTLR22 surrounding the gRNA target site were cloned into

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donor HR targeting vector (Cat. No. HR100PA-1, SBI, USA), containing a red fluorescence

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protein (RFP) reporter gene expression cassette. Briefly, respective modified primer sets were

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designed/synthesized with required restriction enzyme sites (Table S1) compatible with the

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HR vector. Homology arms were PCR-amplified (using Advantage polymerase, Clontech,

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USA) from LrTLR22 cDNA, restriction digested and purified by gel extraction, using

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NucleoSpin Extract II, MN, Germany. The purified PCR products were ligated using Ligate-

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IT Rapid Ligation kit, USB, USA, into restriction digested HR vector within respective

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multiple cloning sites I and II, one-by-one following established protocols described by

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(Barman et al., 2010; Mohanta et al., 2014a; Mohapatra and Barman, 2014; Mohapatra et al.,

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2014). The ligated DNA products were transformed into One Shot TOP10 chemically

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competent E. coli (Invitrogen, USA). The plasmids of screened clones were extracted by

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using Qiagen plasmid kit (Qiagen, USA).

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To examine the targeted mutagenesis as well as integration, genomic DNA, from

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experimental and control fishes, were PCR-amplified to obtain either surrounding the target

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site or the full length TLR22 gene, using primers as depicted in Table S1. The resulting PCR

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products were bi-directionally sequenced as described by (Mohapatra et al., 2010; Panda et

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al., 2014; Patra et al., 2015). The targeted integration should yield new restriction enzyme

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sites, not as that of wild-type sequences. Hence, the DNA sequence information, including

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orientations of donor HR vector (hereafter, known as LrTLR22HR) were confirmed/judged

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by the restriction mapping followed by repeated both-directional sequencing.

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2.5 Microinjection of rohu embryos LrTLR22Cas9 (33.3 pg) and linearized (ScaI digested) LrTLR22HR (66.3 pg) DNA

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constructs t were co-injected into rohu embryos at 1- to 4-cell stage as described (Gong et al.,

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2003; Ju et al., 1999) at a total concentration of 100 pg/µl/embryo using CellTram micro-

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injector (Eppendorf, Germany). Injected embryos along with controls were reared in 70%

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natural pond water containing 1% penicillin/streptomycin. After 6 days of post-fertilization,

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embryos were inspected under a fluorescence microscope (Leica, Germany) for RFP

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expression.

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3. Results and Discussion Because immune responsive TLR22 is specific to teleosts/amphibians and its intron-

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less gene structure in L. rohita (rohu carp) was available (Panda et al., 2014; Samanta et al.,

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2014); we intended to explore developing/generating model rohu fish lacking TLR22. We

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selected CRISPER/Cas9 system to disrupt the TLR22 locus, because of its ease of design

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with efficiency relative to ZFNs and TALE nucleases (Liang et al., 2015; Nakade et al., 2014;

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Ota et al., 2014; Tatsumi et al., 2014; Xiao et al., 2013; Zu et al., 2013). The unique LRR

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ecto-domains (act as PRRs) of TLR22 were targeted to enhance the specificity among the

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TLR family. Our previous findings revealed the LrTLR22 gene comprised nineteen LRR

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domains along with an intracellular conserved TIR domain (Panda et al., 2014). The gRNA

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sequence (upper panel of Fig. 1), containing PAM site (5’-CGG-3’), preceded by unique 20

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nucleotide bases (target site: 1836-1856 bases) as per (N)20NGG rule, was designed from the

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eighteenth LRR domain to construct a Cas9 target vector termed LrTLR22Cas9 (lower panel

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of Fig. 1). Above gRNA sequence was unique to LrTLR22, because no homology was

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detected with zebrafish (Danio rerio), human (Homo sapiens), mouse (Mus musculus) and

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chicken (Gallus gallus). This ruled out the possible off-target sites for CRISPR/Cas9

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nuclease mediated mutagenesis. To examine whether LrTLR22Cas9 can efficiently enforce

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precise gene targeting via HR, we designed experiments to co-inject the exogenous HR donor

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vector (LrTLR22HR), containing EF1α-RFP-T2A-Puro expression/selection cassette within

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ligated two homology arms (924 and 890 bp), respectively (Fig. 2A), immediately preceding

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the 924 bp spanning 912/1835 bases of LrTLR22 and following 890 bp positioned at 1860 -

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2750 bases of LrTLR22, gRNA target sequence of TRL22.

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LrTLR22Cas9 nuclease construct was co-injected with LrTLR22HR, linearized with

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ScaI, into 2-4 cell stages of rohu fertilized eggs (50 embryos). Out of which, seven embryos

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survived and grew to the fry stage (6 days). Among them, one individual (T2) was found to

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be positive, as detected by the expression of RFP in organs such as brain, spleen, liver,

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intestine and kidney (Fig. 2B). In line with this, LrTLR22 mRNA expression could not be

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detected only in RFP expressing embryo (T2), whereas others including non-injected control

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embryos expressed transcripts (Fig. 2C). Thus, it is most likely that there was an integration

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of exogenous donor DNA cassette of LrTLR22HR within LrTLR22 locus of T2 embryo.

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Next, the HR event was examined by PCR amplification for genomic DNA using

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primer sets for full-length cDNA (Table S1). The PCR amplification produced a PCR band

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(5900 bp) of expected size in RFP positive individuals (T2) for a successful HR event,

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whereas the rest of RFP negative embryos, including controls did not amplify this size rather 7

ACCEPTED MANUSCRIPT than wild-type (WT) 2700 bp band (Fig. 3A). Subsequent restriction mappings (BglII

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digested) of these PCR products also generated fragments of expected sizes of 300 bp, 1000

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bp and 4600 bp in RFP positive, in the case of HR event; whereas 300 bp and 2400 bp (WT)

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in the negative as well as control individuals (Fig. 3B). To further confirm integration by

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HR, junction sequences between target locus and knocked-in donors were analysed by bi-

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directional sequencing (Fig. 3C). Sequencing results of PCR amplified genomic DNA

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suggested the exogenous donor HR plasmid sequence was integrated efficiently within the

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expected cleavage site of Cas9. Together, above findings clearly demonstrated that Cas9-

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induced DSBs prompted the targeted integration of exogenous HR cassette in T2 embryos

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(Fig. 3B). In the absence of a TLR22 mRNA expression, the mutant individuals thus

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generated, should be considered as model fishes (∆LrTLR22 with knock-out allele) for

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undertaking basic research, particularly uncovering it’s in vivo functions.

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One concern with new genome editing tools is the potential for off-target sites as

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recently emphasised for CRISPR/Cas9 system (Fu et al., 2013). The possible off-target

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activities, as also reported in zebrafish, mice, rabbits and human cells (Ota et al., 2014; Yang

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et al., 2014b; Yasue et al., 2014; Zou et al., 2009), could not be ruled out. Evidence also was

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found of double nickase, which potentially could reduce the likelihood of off-target

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modifications (Cho et al., 2014; Ran et al., 2013). Based on our sequencing data, off-target

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alteration was not detected in the LrTLR22 gene. Off-target mutations could not be detected

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in other carp TLRs, such as TLR21 and TLR23 (data not presented), specific to teleosts. This

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is in line with the fact that off-targets sites could not be predicted with genomes of zebrafish,

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human, mouse and chicken. However, it is essential to determine other off-target sites, if any,

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while generating model rohu fishes lacking TLR22 gene expression. Nevertheless, this is the

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first report of developing the successful gene (TLR22) disruption technique mediated by HR

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in large-bodied teleost (rohu carp, L. rohita) by using CRISPR/Cas9 system. Development of

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such type of model carps would enrich basic understanding of participatory distinctive roles

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being played by TLR22 in relation to its effects on pathogenic dsRNA viruses and bacteria,

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including lice infections. Our study shows the utility of the CRISPR/Cas9 system as a genetic

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tool in creating model fish species, potentially in the area of genome editing as well as

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transgenic research.

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Acknowledgements

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This work was supported by a grant from the National Agricultural Science Fund (NASF),

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Indian Council of Agricultural Research, Union Ministry of Agriculture, Government of

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India. Thanks to the Director of this Institute for providing required facilities to carry out this

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research.

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Figure legends

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Fig. 1. Step-wise depiction of Construction of CRISPR/Cas9 Nuclease target vector. Each

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complementary guide RNA strands were designed, synthesized and annealed. The double-

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stranded guide RNA was cloned into Cas9 nuclease vector to prepare LrTLR22Cas9

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construct.

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Fig. 2. Gene targeting of TLR22 locus, RFP expression and assessment of TLR22 gene

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expression. (A) Schematic representation of targeting HR donor construct (top), intact

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(middle) and targeted (bottom) TLR22 alleles. Only relevant restriction sites (BglII site) and

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possible relevant fragments with their lengths in kb are shown. (B) The RFP was expressed in

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one (T2) of the targeted embryos, but not in other embryos. (C) A representative figure of

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mRNA expression profiling that documented lack of TLR22 expression in T2 embryo.

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T1/T2/T3, injected; control, non-injected.

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Fig. 3. HR mediated gene integration at a pre-determined LrTLR22 locus. (A) A

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representative gel of PCR amplifications (for LrTLR22 gene) from genomic DNA generating

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bands of the expected sizes in targeted vs non-targeted individuals. (B) Restriction (BglII)

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mapping of respective above PCR-amplified fragments showing expected digested banding

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patterns of targeted (T2) and non-targeted individuals. (C) Genotyping by nucleotide

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sequencing (of above PCR-amplified fragment of the targeted T2 embryo) validated the

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successful integration of HR donor vector at targeted sites of LrTLR22 gene. Sequences in

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italic and non-italic represent HR vector and LrTLR22, respectively. T1/T2/T3/T4/T5,

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injected; control, non-injected.

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Fig. S1. LrTLR22Cas9 vector construct bearing TLR22 targeted guide RNA.

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Highlights 1. Establishment of a TLR22 gene editing technique using CRISPR/Cas9 system in Labeo

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rohita carp.

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

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3. Gene edited mutant carp lacked TLR22 mRNA expression evidencing targeted TLR22

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gene disruption.