TY - JOUR

T1 - Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors

AU - Morbitzer, Robert

AU - Römer, Patrick

AU - Boch, Jens

AU - Lahaye, Thomas

N1 - Copyright: Copyright 2011 Elsevier B.V., All rights reserved.

PY - 2010/12/14

Y1 - 2010/12/14

N2 - Proteins that can be tailored to bind desired DNA sequences are key tools for molecular biology. Previous studies suggested that DNAbinding specificityof transcriptionactivator-like effectors (TALEs) from the bacterial genus Xanthomonas is defined by repeat-variable diresidues (RVDs) of tandem-arranged 34/35-amino acid repeat units. We have studied chimeras of two TALEs differing in RVDs and non-RVDs and found that, in contrast to the critical contributions by RVDs, non-RVDs had no major effect on the DNA-binding specificity of the chimeras. This finding suggests that one needs only to modify the RVDs to generate designer TALEs (dTALEs) to activate transcription of userdefined target genes. We used the scaffold of the TALE AvrBs3 and changed its RVDs to match either the tomato Bs4, the Arabidopsis EGL3, or the Arabidopsis KNAT1 promoter. All three dTALEs transcriptionally activated thedesired promoters ina sequence-specificmanner asmutations within the targeted DNA sequences abolished promoter activation. This study is unique in showing that chromosomal loci can be targeted specifically by dTALEs. We also engineered two AvrBs3 derivatives with four additional repeat units activating specifically either the pepper Bs3 or UPA20 promoter. Because AvrBs3 activates both promoters, our data show that addition of repeat units facilitates TALE-specificity fine-tuning. Finally, we demonstrate that the RVD NKmediates specific interaction withGnucleotides that thus far could not be targeted specifically by any known RVD type. In summary, our data demonstrate that the TALE scaffold can be tailored to target user-defined DNA sequences in whole genomes.

AB - Proteins that can be tailored to bind desired DNA sequences are key tools for molecular biology. Previous studies suggested that DNAbinding specificityof transcriptionactivator-like effectors (TALEs) from the bacterial genus Xanthomonas is defined by repeat-variable diresidues (RVDs) of tandem-arranged 34/35-amino acid repeat units. We have studied chimeras of two TALEs differing in RVDs and non-RVDs and found that, in contrast to the critical contributions by RVDs, non-RVDs had no major effect on the DNA-binding specificity of the chimeras. This finding suggests that one needs only to modify the RVDs to generate designer TALEs (dTALEs) to activate transcription of userdefined target genes. We used the scaffold of the TALE AvrBs3 and changed its RVDs to match either the tomato Bs4, the Arabidopsis EGL3, or the Arabidopsis KNAT1 promoter. All three dTALEs transcriptionally activated thedesired promoters ina sequence-specificmanner asmutations within the targeted DNA sequences abolished promoter activation. This study is unique in showing that chromosomal loci can be targeted specifically by dTALEs. We also engineered two AvrBs3 derivatives with four additional repeat units activating specifically either the pepper Bs3 or UPA20 promoter. Because AvrBs3 activates both promoters, our data show that addition of repeat units facilitates TALE-specificity fine-tuning. Finally, we demonstrate that the RVD NKmediates specific interaction withGnucleotides that thus far could not be targeted specifically by any known RVD type. In summary, our data demonstrate that the TALE scaffold can be tailored to target user-defined DNA sequences in whole genomes.

KW - AvrBs4

KW - AvrXa27

KW - Zinc-finger proteins

UR - http://www.scopus.com/inward/record.url?scp=78650739853&partnerID=8YFLogxK

U2 - 10.1073/pnas.1013133107

DO - 10.1073/pnas.1013133107

M3 - Article

C2 - 21106758

AN - SCOPUS:78650739853

VL - 107

SP - 21617

EP - 21622

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 50

ER -