High Frequency Targeted Mutagenesis Using Engineered Endonucleases and DNA-End Processing Enzymes

Abstract

Targeting DNA double-strand breaks is a powerful strategy for gene inactivation applications. Without the use of a repair plasmid, targeted mutagenesis can be achieved through Non-Homologous End joining (NHEJ) pathways. However, many of the DNA breaks produced by engineered nucleases may be subject to precise re-ligation without loss of genetic information and thus are likely to be unproductive. In this study, we combined engineered endonucleases and DNA-end processing enzymes to increase the efficiency of targeted mutagenesis, providing a robust and efficient method to (i) greatly improve targeted mutagenesis frequency up to 30-fold, and; (ii) control the nature of mutagenic events using meganucleases in conjunction with DNA-end processing enzymes in human primary cells.

Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology

“…We observed bi-allelic mutations in 29% (7/24) of the subclones derived from a colony with 15% overall TM. In agreement with our previous study on the processivity of the scTrex2 exonuclease 27 , small deletions of 1 to 4 nucleotides were predominantly found at the double-strand break site (Fig. 1c).…”

“…The ability of MNs to induce TM was assessed by cotransforming Pt with a MN-encoding plasmid, an autonomous selection cassette plasmid (NAT gene to select for nourseothricin (NAT) resistance), and a plasmid encoding the DNA processing enzyme scTrex2, which has previously been shown to increase about 10-fold the TM frequency induced by MNs in mammalian cells 27,28 . Twelve NAT-resistant colonies transformed with Mn17181 were analysed for the presence of mutations using locus-specific PCR followed by deep sequencing.…”

Compact designer TALENs for efficient genome engineering

“…Remarkably, although FUT8::TevI was ~6-fold less effective than FUT8::FokI, activity could essentially be rescued (FUT8::TevI(ε), 16%) by an enhancer molecule such as three-prime repair exonuclease 2 (TREX2). As DNA double-strand cleavage by the TevI CD liberates 2-nt 3′ overhangs, end processing by TREX2 can be used to drive mutagenic NHEJ before the precise rejoining of the induced breaks4647. For all TALEN monomers, however, NHEJ activity could not be observed (only cFUT8L::TevI shown) even with added TREX2, presumably because at least one DNA strand remains intact.…”

Transgenic farm animals: status of the current researches and the future

“…Среди сайт-специфических нуклеаз первоначально для трансгенеза животных, главным образом, с целью нокаутагенов получили распространение так называ-емые нуклеазы «цинковых пальцев» (ZFN) , эффекторные нуклеазы, подобные акти-ваторам транскрипции (TALEN) и хо-мингмегануклеазы (Delacote et al, 2013), индуцирующие в целевых последовательностях генома мутации в форме инсерций или делеций небольшого размера посредством репарации разрывов ДНК негомологичным соедине-нием концов (NHIJ) или гомологичной рекомбинаци-ей. Используя технологии ZNF и TALEN, был успешно «выключен» ген α1,3-галактозилтрансферазы (GGTA1) у свиней, что является первым шагом в создании транс-генных свиней -доноров (Hauschild et al, 2011;Xin et al, 2013).…”