Heterologous survey of 130 DNA transposons in human cells highlights their functional divergence and expands the genome engineering toolbox.

Experimental studies on DNA transposable elements (TEs) have been limited in scale, leading to a lack of understanding of the factors influencing transposition activity, evolutionary dynamics, and application potential as genome engineering tools. We predicted 130 active DNA TEs from 102 metazoan genomes and evaluated their activity in human cells. We identified 40 active (integration-competent) TEs, surpassing the cumulative number (20) of TEs found previously. With this unified comparative data, we found that the Tc1/mariner superfamily exhibits elevated activity, potentially explaining their pervasive horizontal transfers. Further functional characterization of TEs revealed additional divergence in features such as insertion bias. Remarkably, in CAR-T therapy for hematological and solid tumors, Mariner2_AG (MAG), the most active DNA TE identified, largely outperformed two widely used vectors, the lentiviral vector and the TE-based vector SB100X. Overall, this study highlights the varied transposition features and evolutionary dynamics of DNA TEs and increases the TE toolbox diversity.

Chasing Sequencing Perfection: Marching Toward Higher Accuracy and Lower Costs.

Next-generation sequencing (NGS), represented by Illumina platforms, has been an essential cornerstone of basic and applied research. However, the sequencing error rate of 1 per 1000 bp (10-3) represents a serious hurdle for research areas focusing on rare mutations, such as somatic mosaicism or microbe heterogeneity. By examining the high-fidelity sequencing methods developed in the past decade, we summarized three major factors underlying errors and the corresponding 12 strategies mitigating these errors. We then proposed a novel framework to classify 11 preexisting representative methods according to the corresponding combinatory strategies and identified three trends that emerged during methodological developments. We further extended this analysis to eight long-read sequencing methods, emphasizing error reduction strategies. Finally, we suggest two promising future directions that could achieve comparable or even higher accuracy with lower costs in both NGS and long-read sequencing.

Genome editing in plants using the TnpB transposase system.

The widely used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system is thought to have evolved from IS200/IS605 transposons. TnpB proteins, encoded by one type of IS200/IS605 transposon, are considered to be the evolutionary ancestors of Cas12 nucleases, which have been engineered to function as RNA-guided DNA endonucleases for genome editing in bacteria and human cells. TnpB nucleases, which are smaller than Cas nucleases, have been engineered for use in genome editing in animal systems, but the feasibility of this approach in plants remained unknown. Here, we obtained stably transformed genome-edited mutants in rice (Oryza sativa) by adapting three recently identified TnpB genome editing vectors, encoding distinct TnpB nucleases (ISAam1, ISDra2, and ISYmu1), for use in plants, demonstrating that the hypercompact TnpB proteins can effectively edit plant genomes. ISDra2 and ISYmu1 precisely edited their target sequences, with no off-target mutations detected, showing that TnpB transposon nucleases are suitable for development into a new genome editing tool for plants. Future modifications improving the genome-editing efficiency of the TnpB system will facilitate plant functional studies and breeding programs. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00172-6.

Low-input PacBio sequencing generates high-quality individual fly genomes and characterizes mutational processes.

Long-read sequencing, exemplified by PacBio, revolutionizes genomics, overcoming challenges like repetitive sequences. However, the high DNA requirement ( > 1 µg) is prohibitive for small organisms. We develop a low-input (100 ng), low-cost, and amplification-free library-generation method for PacBio sequencing (LILAP) using Tn5-based tagmentation and DNA circularization within one tube. We test LILAP with two Drosophila melanogaster individuals, and generate near-complete genomes, surpassing preexisting single-fly genomes. By analyzing variations in these two genomes, we characterize mutational processes: complex transpositions (transposon insertions together with extra duplications and/or deletions) prefer regions characterized by non-B DNA structures, and gene conversion of transposons occurs on both DNA and RNA levels. Concurrently, we generate two complete assemblies for the endosymbiotic bacterium Wolbachia in these flies and similarly detect transposon conversion. Thus, LILAP promises a broad PacBio sequencing adoption for not only mutational studies of flies and their symbionts but also explorations of other small organisms or precious samples.

Hagfish genome elucidates vertebrate whole-genome duplication events and their evolutionary consequences.

Polyploidy or whole-genome duplication (WGD) is a major event that drastically reshapes genome architecture and is often assumed to be causally associated with organismal innovations and radiations. The 2R hypothesis suggests that two WGD events (1R and 2R) occurred during early vertebrate evolution. However, the timing of the 2R event relative to the divergence of gnathostomes (jawed vertebrates) and cyclostomes (jawless hagfishes and lampreys) is unresolved and whether these WGD events underlie vertebrate phenotypic diversification remains elusive. Here we present the genome of the inshore hagfish, Eptatretus burgeri. Through comparative analysis with lamprey and gnathostome genomes, we reconstruct the early events in cyclostome genome evolution, leveraging insights into the ancestral vertebrate genome. Genome-wide synteny and phylogenetic analyses support a scenario in which 1R occurred in the vertebrate stem-lineage during the early Cambrian, and 2R occurred in the gnathostome stem-lineage, maximally in the late Cambrian-earliest Ordovician, after its divergence from cyclostomes. We find that the genome of stem-cyclostomes experienced an additional independent genome triplication. Functional genomic and morphospace analyses demonstrate that WGD events generally contribute to developmental evolution with similar changes in the regulatory genome of both vertebrate groups. However, appreciable morphological diversification occurred only in the gnathostome but not in the cyclostome lineage, calling into question the general expectation that WGDs lead to leaps of bodyplan complexity.