Homologous Recombination

• the ideal transformation would be gene replacement by homologous recombination (HR), also « gene targeting »

• integration of a DNA fragment involves repairing DS DNA breaks >>

• 2 mechanisms, Homologous recombination (HR) and Non – homologous end-joining (NHEJ)

Non-Homologous

End-Joining (NHEJ, IR)

Homologous Recombination

DS break repair

•However, HR is much less efficient than IR (NHEJ) in plants (HR is ~10-5 as opposed to 10-2 for mouse)

Methods of Gene Targeting

Homologous Recombination

• very low frequencies of gene targeting observed >lower than NHEJ background> currently impracticable in higher plants

Options?• promotion of DNA repair by mutations or

transgenes ?• Exploit the moss model?• Alternatives for excision of unwanted

transgenes – HR or otherwise?

DS DNA break repair by HR involves many gene products in plants

High frequency gene targeting by over-expressionof Rad54 in Arabidopsis

PNAS 102, 34, 12265 (2005)

Targeting assay: integration of GFP downstream from a seed promoter

Proof of targeting: PCR

to show GFP at the correct integration site

targeting frequency increased by 27-fold

→ 30% of transformants were targeted

Current Opinion in Genetics & Development : Volume 17, Issue 2, April 2007, Pages 126-131

Chromatin remodeling is involved in homologous recombination

Copyright ©2006 American Society of Plant Biologists

Kirik, A., et al. Plant Cell 2006;18:2431-2442

The fas1-4 MutantThe Chromatin Assembly Factor (CAF-1)

Subunit FASCIATA1 Is Involved in Homologous Recombination in Plants

A model for targeting nucleosome assembly to DNA

transactions.

Trends in Biochemical Sciences

Volume 31, Issue 7, July 2006, Pages 395-401

Copyright ©2006 American Society of Plant Biologists

Kirik, A., et al. Plant Cell 2006;18:2431-2442

HR Is Stimulated in fas1-4

Copyright ©2006 American Society of Plant Biologists

Kirik, A., et al. Plant Cell 2006;18:2431-2442

fas1-4 Causes Loss of Heterochromatin

Copyright ©2006 American Society of Plant Biologists

Kirik, A., et al. Plant Cell 2006;18:2431-2442

fas1-4 Affects Chromatin Conformation

In moss (Physcomitrella patens) gene targeting is efficient

Towards creation of new varieties using Reverse genetics

• non-transgenic eg., TILLING, Fast neutron

• transgenic - technology challenges!

Towards creation of new varieties using Reverse genetics

Problems associated with T-DNA transformants:

• Certain selectable markers (herbicide resistance, drug resistance)

• Site of transgene integration and stability, copy number

gene silencingrisk of secondary effects on phenotype

Towards creation of new varieties using Reverse genetics

• Problems:

• transgene transcription and translationlow level transcriptionlittle product accumulation

Towards creation of new varieties using Reverse genetics

• Possible solutions:

• Novel plant vectors (environmentally friendly selection systems)

Co-transform with marker and segregate outuse of excisable insertsZinc finger endonucleasesuse of novel non-drug selectable markers

Co-transformation procedure to generate marker-free transgenic plants

use of excisable inserts:

The Cre-Lox system for excising T-DNA fragments in planta

Cre-lox recombination: Creative tools for plant biotechnology  Gilbertson L ,  TRENDS IN BIOTECH 21 (12): 550-555 DEC 2003

Could also be used to integrate DNA at a LOX site

Lox repeat substrate

Substrate after excision

Mode of action of a Zn-finger nuclease

Uses:

- Promote homologous recombination at a specific site when cleaved (~100 x )

- site-specific excision

Trends in Plant Science 11, 159

Principle: construction and employment of a nuclease recognizing a defined (and rare) site

use of novel non-drug selectable markers

The dsdA gene from Escherichia coli provides a novel selectable marker for plant transformationOskar Erikson, Magnus Hertzberg and Torgny Näsholm

Plants are sensitive to D-serine, but expression of the dsdA gene, encoding D-serine ammonia lyase from Escherichia coli, can alleviate this toxicity.

Plant Molecular Biology :  Volume 57, Number 3 Date:  February 2005 Pages: 425 - 433

Selection on D-Ser

Selection on Kanamycin

Towards creation of new varieties using Reverse genetics

• Ideally, homologous recombination in plants ?• advantages:• gene replacement/gene targeting • precise positioning of insert• Problems remaining:• How to increase efficiency without deleterious

consequences for the plant• High background of NHEJ• No ‘universal’ or ‘one-step’ method

Objectif du TP TILLING: identifier mutations dans pools d’ADN de plantes

mutées

PCR produits fournis

Digestion avec CelI

Filtration sur Sephadex G-50

Concentrer par évaporation

Déposer sur la Gel

Séparation, analyse

démonstrations:

préparation d’un gel

analyse informatique des séquences

production des plantes

• References:

• Krysan PJ, Young JC, Sussman MR. T-DNA as an insertional • mutagen in Arabidopsis. Plant Cell. 1999. Dec;11(12):2283-90.

• 2. Li X, Song Y, Century K, Straight S, Ronald P, Dong X, Lassner M, • Zhang Y. A fast neutron deletion mutagenesis-based reverse genetics • system for plants. Plant J. 2001 Aug;27(3):235-42.

• 3. Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, • Burtner C, Odden AR, Young K, Taylor NE, Henikoff JG, Comai L, • Henikoff S. Large-scale discovery of induced point mutations with • high-throughput TILLING. Genome Res. 2003 Mar;13(3):524-30.

•

• • Helpful links for getting the full text:

• PubMed: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi• UBC ejournal: http://toby.library.ubc.ca/ejournals/ejournals.cfm

• useful sites

• http://www.licor.com/bio/Tilling/Tilling4.jsp

• Anand, A; Krichevsky, A; Schomack, S; Lahaye, T; Tzfira, T; Tang, YH; Citovsky, V; Mysore, KS Arabidopsis VIRE2 INTERACTING PROTEIN2 is required for Agrobacterium T-DNA integration in plants

PLANT CELL 1040-4651 2007 191695 1708

• Terada, R; Johzuka-Hisatomi, Y; Saitoh, M; Asao, H; Iida, S Gene targeting by homologous recombination as a biotechnological tool for rice functional genomics PLANT PHYSIOLOGY 2007 144

846 856• Li, J; Hsia, AP; Schnable, PS Recent advances in plant recombination CURRENT OPINION IN

PLANT BIOLOGY 2007 2 131 135

• Smith, J; Grizot, S; Arnould, S; Duclert, A; Epinat, JC; Chames, P; Prieto, J; Redondo, P; Blanco, FJ; Bravo, J; Montoya, G; Paques, F; Duchateau, P A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences NUCLEIC ACIDS RESEARCH 2006 22

e149

• Endo, M; Ishikawa, Y; Osakabe, K; Nakayama, S; Kaya, H; Araki, T; Shibahara, KI; Abe, K; Ichikawa, H; Valentine, L; Hohn, B; Toki, S Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants EMBO JOURNAL 2006 23 55795590

• Kamisugi, Y; Schlink, K; Rensing, SA; Schween, G; von Stackelberg, M; Cuming, AC; Reski, R; Cove, DJThe mechanism of gene targeting in Physcomitrella patens: homologous recombination,

concatenation and multiple integration NUCLEIC ACIDS RESEARCH 2006 216205 6214

• Kirik, A; Pecinka, A; Wendeler, E; Reiss, B The chromatin assembly factor subunit FASCIATA1 is involved in homologous recombination in plants PLANT CELL 2006 10 24312442