Illuminating DNA replication during Drosophila development using TALE-lights

phase is a key determinant of timing and is required for cycle 14 MBT events [7]. In the early, fast S phases, all of the DNA sequences replicate at the same time without exhibiting the typical eukaryotic subdivision in which euchromatin replicates early and heterochromatin replicates late. As the S phase incrementally increases in duration during cycles 12 and 13, there is a slight delay in the timing of replication of satellite sequences, the so-called constitutively heterochromatic sequences [8]. The abrupt prolongation of S phase in cycle 14 is triggered by down-regulation of G2 cyclin–Cdk1 activity, a kinase capable of driving early replication of otherwise late replicating satellite sequences [9]. Thereafter the replication of satellite sequences is substantially delayed and different satellites replicate with different delays [8]. To understand this program, we need to identify the developmental signal triggering these events, and we need to understand the control of replication timing. To begin to investigate the latter with temporal precision, we wanted methods to follow replication timing live. To do this we have made TALE-lights that mark specific blocks of satellite sequence. Two TALE-lights recognize sequences within a 359 base-pair sequence that is repeated about 30,000 times on the X-chromosome, and another recognizes two repeats of a 10 base-pair sequence present in pericentric arrays called 1.686 on each of two large autosomes (Figure 1A,B and see Experimental Procedures in Supplemental Information, published with this article online). We injected in vitro produced RNAs into the embryos to express the TALE-lights (not shown), or have directly injected bacterially expressed TALE-light proteins into the embryos. Figure 1C shows three nuclei of a blastoderm embryo as they progress through cell cycle 13. The embryo was injected with TALE-lights, 359–RFP and 1.686–GFP, during the previous cycle. A single spot for 359–RFP is seen, indicating that this is a male embryo (one X chromosome) and four spots can generally be seen for 1.686 (two homologs for each of the two chromosomes carrying this repeat). We confirmed the specificity by comparison of the TALE-light signal to the in situ signal obtained with New discoveries allow systematic engineering of DNA sequence recognition using the modular recognition units of the transcription activator-like effectors (TALEs) or the guide RNA of the CRISPRs. The engineered specificity offers the potential to guide a wide range of activities to particular sequences [1–3], and targeted nucleases cause directed mutagenesis [4–6]. Here we have tagged …