Reverse Genetics in Fisheries Research Credit Seminar on Reverse Genetics in Fisheries Research Major Advisor Dr. K Pani Prasad Shyam K U AAH-MA4-01

Introduction Mendel started with his mutant phenotype to come with his rules. Today, we know DNA sequences of many genes but not their phenotype. Reverse Genetics is a term coined to desirable processes where information flows in opposite direction, i.e., the gene is determined or altered directly and the resultant phenotype observed. It discovers the normal role of cryptic DNA or protein sequences by mutating the sequence in vitro and looking for changes at the phenotypic level.

Forward Genetics Vs Reverse Genetics Mutant Phenotype Mutant Phenotype Mutant Allele Mutant Allele FORWARD GENETICS REVERSE GENETICS DNA Sequence DNA Sequence Protein Sequence Protein Sequence

Forward Genetics Vs Reverse Genetics FG seeks to find the genetic basis of a phenotype or trait, RG seeks to find what phenotypes arise as a result of particular genetic sequences. FG screens are intrinsically limited in their effectiveness to isolate mutations of every single gene due to functional redundancy between different genes and the need to have measurable phenotypes. It is also quite time-consuming but without guarantees to identify the responsible mutated genes.

RG first identifies the gene of interest and subsequently obtain their mutations followed by the evaluation of the phenotypic effects of these mutations. Can investigate the impact of induced variation in a specific gene on the organism. RG is one of the modified application of recombinant DNA technology. Important tools for RG are in vitro mutagenesis and the gene disruption (gene knock out)

Reverse Genetics Techniques Large Scale Systematic Mutagenesis – Retrovirus mediated insertional mutagenesis,Tol2 Transposase system, Gene knock-out via TILLING Gene Targeting by Site-Specific Genome Modification – Gene targeting via ZFNs, TALENs Gene Knock-down via Morpholino nucleotides 4. Random Mutagenesis

Retrovirus Mediated Insertional Mutagenesis Easiest way of expressing the desired gene by engineering the retrovirus via cloning. Infecting embryonic stem cells with pseudo-typed retrovirus – integrates randomly into genome as a part of retroviral life cycle, causes the mutation. A pseudo-typed MLV-retrovirus with its envelope protein replaced by G-protein of VSV shown infectivity and germline transmission in zebra fish embryos. (Lin et al., 1994; Wu et al., 2003; Wang et al., 2007). A unique advantage of retroviral mutagenesis over chemical mutagens is that it allows rapid identification of mutated genes.

By using the retroviral sequence flanking the insertion site can be easily identified through inverse PCR or LM-PCR. 36748 essential genes for zebra fish have been successfully identified until 2016 (ZFIN Database., 2016; Amsterdam et al., 1999, 2004; Golling et al., 2002). Limitations :- genes that share functional redundancy or do not create visible phenotypes during early development are usually ignored. Its not practical for saturation mutagenesis screen due to its intensive labour and large fish maintenance requirement.

Injection of zebra fish embryo with retrovirus preparation Raising of founder embryo generation F1 generation by inbreeding/outbreeding of founder Identification of F1 fish with multiple insertions Raising F2 families by inbreeding selected F1 fishes Sperms were collected and archived from 2nd and 3rd generation DNA extracted from fin clips Insertion sites were identified by LM-PCR and sequencing Source : Huang et al., 2012

Tol2 transposon system It’s a most common transgenic approach in zebra fish. Tol2 transposable elements were originally discovered in the Japanese medaka, Oryzias latipes Co-injection of expression constructs containing transgenes flanked by the Tol2 elements with the transposase mRNAs. So, this will enhances the efficiency of trans-genesis and germ line transmission.

+ Transposon construct in plasmid Transposase mRNA Minimal Tol2 vector Transposase mRNA + Transposon construct in plasmid mRNA translate to Transposase Excision of construct from plasmid Integration of construct into genomic DNA Production of F1 generation & Detection by Green Fluorescence Source: Kawakami K., 2007

TILLING Targeting Induced Local Lesions IN Genome (TILLING) – 1st RG approach in Zebrafish Combined method of forward and RG based on chemical mutagenesis to isolate mutants harboring point mutations in genes of interest. TILLING - developed by the Henikoff laboratory to screen libraries of EMS- treated Arabidopsis for desired mutant alleles (Cobert et al., 2001; McCallum et al., 2000). TILLING in zebrafish was applied by Wienholds in 2002 (Wienholds et al., 2002; Weinholds et al., 2003b)

The fishes are mutagenized by ENU (N-ethyl N-Nitrosourea), a chemical mutagen. (Zebrafish male) Mutations in target genes (1 in 105 bp) are sought by sequencing the target regions. So it requires large scale sequencing, difficulty to conduct as a routine technique. Therefore community based mutation projects by TILLING were established by several consortia (eg: Sanger Institute – http://www.sanger.ac.uk/projects/D_rerio/zmp/: www.zfishtilling.org/zfish/ ). Eg: TILLING for rag1 gene in zebrafish.

Zinc Finger Nucleases (ZFN) ZFN are artificial endonuclease enzymes originally introduced as a hybrid restriction enzyme. It consists of two domains - Zinc Finger domain – recognizes a specific sequence of genomic DNA & FokI restriction enzyme domain – cuts dsDNA. ZFD will specifically bind a region where the FRED acts and FokI enzymes cuts the dsDNA region. A dsDNA break is repaired by homologous recombination or a non- homologous end joining (NHEJ).

Protein Zif268 (Blue) containing three ZF in complex with DNA (Orange) Zinc iron and coordinating amino acid (Green)

NHEJ is an error-prone process - It creates small deletion/insertion (indel) mutations in a site of lesion. By engineering the zinc finger domains to bind specific loci of genomes, possible to introduce mutations into genes of interest. Advantage : based on simple theory, viable tool for targeted mutagenesis. Disadvantage : technically challenging and costly, potential risk of off-target effects, complexity regarding the designing of DNA. Injected into fertilized eggs at the one or two cell stages Individual harbouring mutations in a target gene are subsequently identified These founder fish are outcrossed to obtain an F1 generation Confirmed F1 incrossed and resulting F2 generation analyzed

TALEN Transcription Activator-Like Effector Nucleases (TALENs). Same as ZFNs, two domains – restriction enzyme domain and DNA binding domain. But in TALENs, the DNA binding domain is derived from plant pathogenic bacteria with TALE modules recognizing specific bases. Advantages : more flexible and much simpler than ZFN. Disadvantage : Off-target effects.

Source : Ed Davis, Technical Note, Genecopoeia

Site-Specific Genome Modification via ZFN and TALEN A pair of mRNA encoding ZFN and TALEN are injected into embryo restriction enzyme digestion, CEL I or T7 endonuclease digestion, sequencing analysis, melting curve analysis Raising Mosaic embryos to founders F1 generation (outcross between Founder and wild) Fin clipping genotyping Incross between F1 carriers F2 generation and Phenotypic analysis Source: Huang et al., 2012

Morpholino knockdown Knockdown of gene function by morpholino antisense nucleotides -Most widely used RG technique in fish (zebrafish). Morpholinos are chemically synthesized nucleotides with morpholine rings. This will resist the breakdown by nucleases.

Mostly they are designed to bind in the vicinity of the start codon to block initiation of translation or to splice acceptor sequences to cause aberrantly spliced mRNAs. They are injected into freshly fertilized eggs and effectively block mRNA translation or splicing of gene up to 5dpf. It can also be used to block the maturation of miRNA as well as to inhibit their binding to the target mRNA. Advantages – easy to use and quick read out, high efficient. Disadvantage – require dose optimization, off –target effects.

RG in context of Virology Rescue of virus entirely from transfecting cloned cDNA plasmids encoding the viral components – Positional Cloning. Ability to manipulate a cDNA intermediate, exact copy of the viral RNA genome, opens the possibility of deleting genes for studying their functions. The use of recombinant DNA technology to convert viral genomes into complementary DNA and generate viruses from the cloned DNA. Introducing targeted mutations to determine potential genetic factors of virulence or inserting heterologous genes of interest and using these recombinant viruses as gene vectors.

RG in History of Virology Year Event Nature of Virus Reference 1981 Recovery of poliovirus from cDNA + ve sense ss RNA Racaniello and Baltimore, 1981 1994 Rescue of rhabdovirus rabies virus - ve sense ss RNA Schnell et al., 1994 1996 Rescue of trisegmented bunyavirus Bunyamwera virus ve sense ss RNA 3 segments Bridgen and Elliott., 1996 1998 Rescue of infectious pancreatic necrosis virus segmented ds RNA RNA virus infecting aquatic species Yao and Vakharia., 1998 2000 Rescued synthetic salmonid rhabdovirus minigenomes - ve sense ss RNA non-segmented Biacchesi S et al., 2000 2001 Developed RG system for SJNNV + ve segmented ss RNA Betanodavirus infecting fish Iwamoto T et al., 2001 2006 Recovered a rec SAV from rainbow trout Moriette C et al., 2006

Reverse Genetics System for Fish RNA viruses First success in RG for a positive RNA virus is the Poliovirus (Picornaviridae family) was in 1981. But after 13 years gap only RG system is used and revolutionized. First description of segmented dsRNA virus recovery entirely from cDNA by Mundt and Vakharia for IBDV. First RG system for a RNA virus infecting aquatic species is IPNV by Yao and Vakharia .

Fish RNA viruses RG system. Novirhabdovirus Infectious Haematopoietic Necrosis Virus (IHNV) Viral Haemorrhagic Septicemia Virus (VHSV) Alaphavirus Sleeping Disease Virus (SDV) Aquabirnavirus Infectious Pancreatic Necrosis Virus (IPNV) Nodavirus Striped Jack Nervous Necrosis Virus (SJNNV) Genome manipulations and rescue of + ve sense RNA viruses is easier. But – ve sense RNA viruses require RNP complex for infectivity.

Source : Biacchesi S., 2011

Potential Applications A powerful tool to study all the aspects of the virus biology and virus-host interaction. Gives the opportunity to use the viruses as a live vaccines or as gene vectors. Structure-Function study of individual viral genes….not by isolation in different systems (reductionist approach). To study molecular basis of pathogenicity of virus. To develop early, rapid and sensitive molecular diagnostics for emerging and existing viral pathogens. To develop a animal model system for studying gene function and human disease research.

Boundaries for RG Experiments Lethal mutations or mutations that are so detrimental that they are genetically unstable and cannot be easily examined. A major constrain is how viruses are packaged ? A more contentious issue is which viruses should be rescued Potential thread of misuse and weapon for bioterrorism. So, ethics based beneficial Reverse Genetics Research will always catalyze the modern research and its services are endless.

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