Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editing and mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator-like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.

Engineered nucleases target specific DNA sequences for gene disruption in nonmodel organisms.

Brassica rapa comprises several important cultivated vegetables and oil crops. Current reference genome assemblies of Brassica rapa are quite fragmented and not highly contiguous, thereby limiting extensive genetic and genomic analyses. Here, we report an improved assembly of the B. rapa genome (v3.0) using single-molecule sequencing, optical mapping, and chromosome conformation capture technologies (Hi-C). Relative to the previous reference genomes, our assembly features a contig N50 size of 1.45 Mb, representing a ~30-fold improvement. We also identified a new event that occurred in the B. rapa genome ~1.2 million years ago, when a long terminal repeat retrotransposon (LTR-RT) expanded. Further analysis refined the relationship of genome blocks and accurately located the centromeres in the B. rapa genome. The B. rapa genome v3.0 will serve as an important community resource for future genetic and genomic studies in B. rapa. This resource will facilitate breeding efforts in B. rapa, as well as comparative genomic analysis with other Brassica species.

The full-length sequences of Botrytiscinereamitovirus 1 (BcMV1) and an associated RNA (BcMV1-S) in strain CanBc-1c-78 of Botrytis cinerea were determined. Sequence analysis showed that BcMV1 is 2804 nt long and AU-rich (66.8%). BcMV1 shares 95% nucleotide sequence identity with Ophiostomanovo-ulmimitovirus 3b (OnuMV3b). However, it is 472 nt longer than OnuMV3b. Mitochondrial codon usage revealed that BcMV1 contains one open reading frame encoding RdRp, which is 96% identical to the RdRp of OnuMV3b. These findings confirm that BcMV1 belongs to the genus Mitovirus and is a strain of OnuMV3b. BcMV1-S is 2171 nt long and derived from BcMV1 through a single internal in-frame deletion of 633 nt, suggesting that it is a defective RNA of BcMV1. BcMV1-S was found to suppress the replication of BcMV1 and to be co-transmissible with BcMV1 through hyphal anastomosis. Its presence, however, did not alleviate the BcMV1-associated debilitation phenotypes of B. cinerea.