Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains

Abstract

Transcription activator-like effectors (TALE) are sequence-specific DNA binding proteins that harbor modular, repetitive DNA binding domains. TALEs have enabled the creation of customizable designer transcriptional factors and sequence-specific nucleases for genome engineering. Here we report two improvements of the TALE toolbox for achieving efficient activation and repression of endogenous gene expression in mammalian cells. We show that the naturally occurring repeat variable diresidue (RVD) Asn-His (NH) has high biological activity and specificity for guanine, a highly prevalent base in mammalian genomes. We also report an effective TALE transcriptional repressor architecture for targeted inhibition of transcription in mammalian cells. These findings will improve the precision and effectiveness of genome engineering that can be achieved using TALEs.

Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells

“…The KRAB repressor domain [residues 1-75 from ZFN10 (Homo sapiens) (Cong et al, 2012), Addgene 42945] was subcloned to the 3′-end of the dCas9-NLS-3xHA to generate the dCas9-KRAB lentiviral expression construct. The VP64 activation domain [from Addgene 32188 (Zhang et al, 2011)] was subcloned to the 3′-end of the dCas9-NLS-3xHA to generate the dCas9-VP64 lentiviral expression construct.…”

A bistable genetic switch based on designable DNA-binding domains

“…The DNA-binding domains of zinc finger proteins, transcription-activator-like effectors (TALEs), or CRISPR/Cas nuclease can be genetically fused to transcriptional effector domains to function as designed site-specific transcriptional activators or repressors targeted against almost any DNA sequence [13][14][15][16][17][18][19][20] . The ability to design these synthetic transcription factors to target a large sequential diversity of DNA operators should support the construction of complex designed genetic circuits.…”

L’ingénierie des génomes par les TALEN

“…Les TALE s'associent alors à l'ADN en formant une hélice qui s'enroule autour de l'ADN où chaque répétition reconnaît un nucléotide de la séquence cible ( Figure 1B) [7,8]. Au sein des répétitions, ce sont les acides aminés 12 et 13 qui déterminent la spécificité de liaison aux nucléotides de chacun de ces domaines répétés [9][10][11] (Figure 1C Figure 3B). Ainsi, la technologie d'édition des génomes à l'aide des TALEN, en induisant un grand nombre de modifications génome par réparation de la cassure double-brin [13,18].…”