The Class 2 Type V-A CRISPR effector protein Cas12a/Cpf1 has gained widespread attention in part because of the ease in achieving multiplexed genome editing, gene regulation, and DNA detection. Multiplexing derives from the ability of Cas12a alone to generate multiple guide RNAs from a transcribed CRISPR array encoding alternating conserved repeats and targeting spacers. While array design has focused on how to optimize guide-RNA sequences, little attention has been paid to sequences outside of the CRISPR array. Here, we show that a structured hairpin located immediately downstream of the 3ʹ repeat interferes with utilization of the adjacent encoded guide RNA by Francisella novicida (Fn)Cas12a. We first observed that a synthetic Rho-independent terminator immediately downstream of an array impaired DNA cleavage based on plasmid clearance in E. coli and DNA cleavage in a cell-free transcription-translation (TXTL) system. TXTL-based cleavage assays further revealed that inhibition was associated with incomplete processing of the transcribed CRISPR array and could be attributed to the stable hairpin formed by the terminator. We also found that the inhibitory effect partially extended to upstream spacers in a multi-spacer array. Finally, we found that removing the terminal repeat from the array increased the inhibitory effect, while replacing this repeat with an unprocessable terminal repeat from a native FnCas12a array restored cleavage activity directed by the adjacent encoded guide RNA. Our study thus revealed that sequences surrounding a CRISPR array can interfere with the function of a CRISPR nuclease, with implications for the design and evolution of CRISPR arrays.

Cas12a (Cpf1) is a CRISPR-associated nuclease with broad utility for synthetic genome engineering, agricultural genomics, and biomedical applications. While bacteria harboring CRISPR-Cas9 or CRISPR-Cas3 adaptive immune systems sometimes acquire mobile genetic elements encoding anti-CRISPR proteins that inhibit Cas9, Cas3, or the DNA-binding Cascade complex, no such inhibitors have been found for CRISPR-Cas12a. Here we employ a comprehensive bioinformatic and experimental screening approach to identify three different inhibitors that block or diminish CRISPR-Cas12a-mediated genome editing in human cells. We also find a widespread connection between CRISPR self-targeting and inhibitor prevalence in prokaryotic genomes, suggesting a straightforward path to the discovery of many more anti-CRISPRs from the microbial world.

Abstract. Levels of diversity vary strikingly among different phylogenetic lineages of ants. Rapid radiations in early ant evolution have often proven difficul

Significance Archaeogenomic analysis of scarlet macaw bones demonstrates that the genetic diversity of these birds acquired by people in the southwestern United States (SW) between 900 and 1200 CE was exceedingly low. Only one mitochondrial DNA haplogroup (Haplo6) is present of the five historically known haplogroups in the lowland forests of Mexico and Central America. Phylogenetic analyses indicate the ancient macaw lineage in the SW shared genetic affinities with this wild lineage. These data support the hypothesis that a translocated breeding colony of scarlet macaws belonging to only one haplogroup existed some distance north of their endemic range, and SW peoples continuously acquired these birds from this unknown location for nearly 3 centuries, as no evidence currently exists for macaw breeding in SW. , Hundreds of scarlet macaw ( Ara macao cyanoptera ) skeletons have been recovered from archaeological contexts in the southwestern United States and northwestern Mexico (SW/NW). The location of these skeletons, >1,000 km outside their Neotropical endemic range, has suggested a far-reaching pre-Hispanic acquisition network. Clear evidence for scarlet macaw breeding within this network is only known from the settlement of Paquimé in NW dating between 1250 and 1450 CE. Although some scholars have speculated on the probable existence of earlier breeding centers in the SW/NW region, there has been no supporting evidence. In this study, we performed an ancient DNA analysis of scarlet macaws recovered from archaeological sites in Chaco Canyon and the contemporaneous Mimbres area of New Mexico. All samples were directly radiocarbon dated between 900 and 1200 CE. We reconstructed complete or near-complete mitochondrial genome sequences of 14 scarlet macaws from five different sites. We observed remarkably low genetic diversity in this sample, consistent with breeding of a small founder population translocated outside their natural range. Phylogeographic comparisons of our ancient DNA mitogenomes with mitochondrial sequences from macaws collected during the last 200 years from their endemic Neotropical range identified genetic affinity between the ancient macaws and a single rare haplogroup (Haplo6) observed only among wild macaws in Mexico and northern Guatemala. Our results suggest that people at an undiscovered pre-Hispanic settlement dating between 900 and 1200 CE managed a macaw breeding colony outside their endemic range and distributed these symbolically important birds through the SW.

Empirical population genetic studies generally rely on sampling subsets of the population(s) of interest and of the nuclear or organellar genome targeted, assuming each are representative of the whole. Violations of these assumptions may impact population-level parameter estimation and lead to spurious inferences. Here we used targeted capture to sequence the full mitochondrial genome from 123 individuals of the Galapagos giant tortoise endemic to Pinzón Island (Chelonoidis duncanensis) sampled at two time points pre- and post-bottleneck (circa 1906 and 2014) to explicitly assess differences in diversity estimates and demographic reconstructions based on subsets of the mitochondrial genome versus the full sequences, and to evaluate potential biases associated with diversity estimates and demographic reconstructions from post-bottlenecked samples alone. Haplotypic diversities were equal between the temporal samples based on the full mitochondrial genome, but single gene estimates suggested either decreases or increases in diversity depending upon the region. Demographic reconstructions based on the full sequence were more similar between the temporal samples than those based on the control region alone, or a subset of three regions, where the trends in population size changes shifted in magnitude and direction between the temporal samples. In all cases, the estimated coalescent point was more distant for the historical than contemporary sample. In summary, our results empirically demonstrate the influence of sampling bias when interpreting population genetic patterns and punctuate the need for careful consideration of potentially conflicting evolutionary signal across the mitochondrial genome.

The musk ox (Ovibos moschatus) is the only surviving member of a group of Pleistocene North American musk ox genera (Praeovibos, Ovibos, Bootherium, Euceratherium, and Soergelia) whose taxonomy is uncertain. The helmeted musk ox (Bootherium bombifrons) and the woodland musk ox (Symbos cavifrons) have been synonymised as male and female forms of a single Nearctic species found from Alaska, in the north, to Texas, in the south. However, this reclassification has not been tested using molecular data, despite the potential to use ancient DNA to examine these late Pleistocene taxa. In the present study, we sequenced mitochondrial genomes from seven subfossil musk ox specimens (originally identified as Bootherium and/or Symbos), allowing us to evaluate the identity of these muskoxen, explore their phylogeography, and estimate the timeline for their evolution. We also used nuclear genomic data to determine the sex of six of our seven samples. Ultimately, our molecular data support the synonymisation of the North American muskoxen Bootherium and Symbos.

We investigated a pantropical sub-family and genus of damselfishes, the sergeant-majors (Pomacentridae: Abudefdufinae: Abudefduf), to identify the tempo and mechanisms of speciation in the lineage. We examined sequence capture data from 500 loci and 20 species, with multiple individuals sampled from across the geographic ranges of widespread species. Utilizing a maximum likelihood framework, as well as a time-calibrated Bayesian phylogeny, the following key questions are addressed: What is the historical tempo of speciation? What are the relative contributions of vicariant, peripatric and parapatric speciation to sergeant-major diversity? How is speciation related to major variation in trophic ecology? The approximately 20 species of sergeant-majors fall into three main lineages. The ancestral condition appears to be benthivory, which is predominant in two lineages comprising six species. The remaining species of sergeant-majors, of which there are at least 15, fall within a clade composed entirely of planktivores. This clade is sister to a benthivore clade that included one species, Abudefduf notatus, in transition to planktivory. Most speciation of sergeant-majors, which appeared ∼24 million years ago, occurred in the last 10 million years. Present distributional patterns indicate vicariant speciation precipitated by the closure of land barriers between both sides of the Atlantic and the Pacific, and the emergence of land between the Indian and Pacific Oceans. Within this backdrop, frequent oscillations in sea level over the last 10 million years also appear to have generated conditions suitable for both peripatric and vicariant speciation, and most speciation within the genus appears linked to these changes in sea level. Diversification within the genus has been concentrated in planktivorous seargeant-majors rather than benthivores. The root cause is unclear, but does not appear to be related to differences in dispersal potential, which is greater in the planktivorous species, due to the ability of their post-larval juveniles to raft with floating debris. This elevated speciation rate in planktivores and their propensity to form local endemics may reflect relaxation of selective pressures (e.g., on crypticity) that limit speciation in benthivorous sergeant-majors. Finally, our data allow us to clarify relationships of geminate sergeant-major species, indicating that there are subdivisions within the Atlantic for both benthivore and planktivore geminate pairs that may have misled previous studies.

Rome initiated several campaigns to expand, conquer, and enslave local Italic populations following the establishment of the republic in 504 BCE. However, the cultural and biological changes resulting from Roman subjugation across Italy remain a topic of intense historical debate. Although important, historic and archaeological lines of evidence fail to track the impact of forced enslavement and enculturation at individual and broader genetic scales and, more generally, offer fewer clues regarding the potential affinities of Roman period Italians to European, Near Eastern, western Asian and North African populations at this time.