Hannon Laboratory

Since their discovery about 20 years ago, small RNAs have been shown to play a critical role in a myriad of biological processes. The greater availability of high-throughput sequencing has been invaluable to furthering our understanding of small RNAs as regulatory molecules. In particular, these sequencing technologies have been crucial in understanding the role of small RNAs in reproductive tissues, where millions of individual sequences are generated. In this context, high-throughput sequencing provides the requisite level of resolution that other procedures, like northern blotting, would not be able to achieve. Here, we describe a protocol for the preparation of small RNA libraries for sequencing using the Solexa/Illumina technology.

The Drosophila piRNA pathway provides an RNA-based immune system that defends the germline genome against selfish genetic elements. Two interrelated branches of the piRNA system exist: somatic cells that support oogenesis only employ Piwi, whereas germ cells utilize a more elaborate pathway centered on the three gonad-specific Argonaute proteins (Piwi, Aubergine, and Argonaute 3). While several key factors of each branch have been identified, our current knowledge is insufficient to explain the complex workings of the piRNA machinery. Here, we report a reverse genetic screen spanning the ovarian transcriptome in an attempt to uncover the full repertoire of genes required for piRNA-mediated transposon silencing in the female germline. Our screen reveals key factors of piRNA-mediated transposon silencing, including the piRNA biogenesis factors CG2183 (GASZ) and Deadlock. Our data uncover a previously unanticipated set of factors preferentially required for repression of different transposon types.

CRISPR-Cas systems defend prokaryotes against viruses and plasmids. Short DNA segments of the invader, known as spacers, are stored in the CRISPR array as immunological memories. New spacers are added invariably to the 5′ end of the array; therefore, the first spacer matches the latest foreign threat. Whether this highly polarized order of spacer insertion influences CRISPR-Cas immunity has not been explored. Here we show that a conserved sequence located immediately upstream of the CRISPR array specifies the site of new spacer integration. Mutation of this sequence results in erroneous incorporation of new spacers into the middle of the array. We show that spacers added through polarized acquisition give rise to more robust CRISPR-Cas immunity than spacers added to the middle of the array. This study demonstrates that the CRISPR-Cas system specifies the site of spacer integration to optimize the immune response against the most immediate threat to the host.

Many bacteria and archaea have the unique ability to heritably alter their genomes by incorporating small fragments of foreign DNA, called spacers, into CRISPR loci. Once transcribed and processed into individual CRISPR RNAs, spacer sequences guide Cas effector nucleases to destroy complementary, invading nucleic acids. Collectively, these two processes are known as the CRISPR-Cas immune response. In this Progress article, we review recent studies that have advanced our understanding of the molecular mechanisms underlying spacer acquisition and that have revealed a fundamental link between the two phases of CRISPR immunity that ensures optimal immunity from newly acquired spacers. Finally, we highlight important open questions and discuss the potential basic and applied impact of spacer acquisition research.

Type II CRISPR-Cas systems defend prokaryotes from bacteriophage infection through the acquisition of short viral DNA sequences known as spacers, which are transcribed into short RNA guides to specify the targets of the Cas9 nuclease. To counter the potentially devastating propagation of escaper phages with mutations in the target sequences, the host population acquires many different spacers. Whether and how pre-existing spacers in type II systems affect the acquisition of new ones is unknown. Here, we demonstrate that previously acquired spacers promote additional spacer acquisition from the vicinity of the target DNA site cleaved by Cas9. Therefore, CRISPR immune cells acquire additional spacers at the same time as they destroy the infecting virus. This anticipates the rise of escapers or related viruses that could escape targeting by the first spacer acquired. Our results thus reveal Cas9's role in the generation of immunological memories.

Rickettsiae are obligate intracellular bacteria that can cause life-threatening illnesses and are among the oldest known vector-borne pathogens. Members of this genus are extraordinarily diverse and exhibit a broad host range. To establish intracellular infection, Rickettsia species undergo complex, multistep life cycles that are encoded by heavily streamlined genomes. As a result of reductive genome evolution, rickettsiae are exquisitely tailored to their host cell environment but cannot survive extracellularly. This host-cell dependence makes for a compelling system to uncover novel host-pathogen biology, but it has also hindered experimental progress. Consequently, the molecular details of rickettsial biology and pathogenesis remain poorly understood. With recent advances in molecular biology and genetics, the field is poised to start unraveling the molecular mechanisms of these host-pathogen interactions. Here, we review recent discoveries that have shed light on key aspects of rickettsial biology. These studies have revealed that rickettsiae subvert host cells using mechanisms that are distinct from other better-studied pathogens, underscoring the great potential of the Rickettsia genus for revealing novel biology. We also highlight several open questions as promising areas for future study and discuss the path toward solving the fundamental mysteries of this neglected and emerging human pathogen.