Gene Site-Specific Insertion in Plants

Abstract

Gene site-speci fi c insertion is an important tool to precisely integrate DNA molecules at a de fi ned genomic location. Gene Targeting has many important implications in plant biotechnology such as creation of novel plant traits or the study of gene function. It requires the insertion of an extrachromosomal DNA into the plant cell that will be further integrated at a de fi ned genomic locus through the use of the plant endogenous recombination machinery. Extensive efforts have been made to understand the mechanisms governing gene targeting and for establishing ef fi cient system to achieve precise and ef fi cient targeting. In particular the recent development of engineered nucleases represents an important step forward towards allowing the insertion of a transgene at any given locus in the genome.

Advances in New Technology for Targeted Modification of Plant Genomes

“…Similar to AtRAD51 itself, its paralogs RAD51C and XXRC3 are essential for SDSA, but not for SSA. It is tempting to speculate that the same holds true for the other paralogs, RAD51B, RAD51D and XRCC2, as general defi ciencies of HR have been reported (Da Ines et al 2013 ;Durrant et al 2007 ). Apart from RAD54, other ATPases appear to play a role in SDSA, including AtRAD5A, which is involved in "post-replicative" strand switching (Mannuss et al 2010 ), and AtFANCM , which is involved in the control of the recombination reaction (Knoll et al 2012 ).…”

“…The integration of an introduced transgene by somatic HR to yield true gene targeting (TGT) with the expected sequence alterations occurs in the order of 10 −3 to 10 −6 compared with random integration, and the overwhelming occurrence of random integration of transgenes by nonhomologous end joining (NHEJ) relative to targeted HR is the main obstacle in the development of an effi cient GT system for fl owering plants (Vergunst and Hooykaas 1999 ;Iida and Terada 2005 ;Da Ines and White 2013 ). In addition to random integration, aberrant recombination events associated with GT, called one-sided invasion (OSI) and ectopic gene targeting (EGT), have occasionally been detected (Fig.…”

“…2 , 3 , 4 , and 9 ); (2) engineering of the host recombination and/or repair machinery (Lieberman-Lazarovich and Levy 2011 ;Waterworth et al 2011 ;Da Ines and White 2013 ), or expressing of foreign genes involving HR (e.g., Shaked et al 2005 ), for enhancing HR processes or for suppressing NHEJ processes; and (3) effi cient enrichment by effective selection systems, namely, genespecifi c selection and positive-negative selection (Table 6.1 ; Endo et al 2007 ;Terada et al 2002 ).…”

“…6.1a ), GT by gene-specifi c selection has been applied to only a few endogenous genes (Iida and Terada 2005 ;Da Ines and White 2013 ). The fi rst reproducible GT of an endogenous gene by gene-specifi c selection was the PPO gene encoding protoporphyrinogen oxidase in Arabidopsis, and appropriate base substitutions in PPO conferred herbicide resistance (Hanin et al 2001 ).…”

“…GT refers to the alteration of a specifi c The anticipated TGT is thought to occur via double crossovers at fl anking homologous regions, and brackets under the maps indicate junction fragments generated by crossovers and used for screening HR events by PCR. The most effi cient mode of transgene integration in Agrobacterium -mediated transformation is BARI, but the resulting transformants are to be killed by the expressed DT-A proteins DNA sequence in an endogenous gene at its original locus in the genome by homologous recombination (HR) (Iida and Terada 2005 ;Da Ines and White 2013 ). GT provides a powerful tool for both the functional analysis of the gene of interest and the molecular breeding of crop plants, because it enables us in principle to convert an endogenous genomic sequence to desired sequences exactly as expected.…”