Genome expansion by a CRISPR trimmer-integrase.

CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity1. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1-Cas2 integrase is necessary but not sufficient2-5. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation6,7, but many CRISPR-Cas systems lack Cas48. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1-Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited9,10 exonucleases for faithful acquisition of new CRISPR immune sequences.

A Functional Mini-Integrase in a Two-Protein-type V-C CRISPR System.

CRISPR-Cas immunity requires integration of short, foreign DNA fragments into the host genome at the CRISPR locus, a site consisting of alternating repeat sequences and foreign-derived spacers. In most CRISPR systems, the proteins Cas1 and Cas2 form the integration complex and are both essential for DNA acquisition. Most type V-C and V-D systems lack the cas2 gene and have unusually short CRISPR repeats and spacers. Here, we show that a mini-integrase comprising the type V-C Cas1 protein alone catalyzes DNA integration with a preference for short (17- to 19-base-pair) DNA fragments. The mini-integrase has weak specificity for the CRISPR array. We present evidence that the Cas1 proteins form a tetramer for integration. Our findings support a model of a minimal integrase with an internal ruler mechanism that favors shorter repeats and spacers. This minimal integrase may represent the function of the ancestral Cas1 prior to Cas2 adoption.

CRISPR technology: A decade of genome editing is only the beginning.

The advent of clustered regularly interspaced short palindromic repeat (CRISPR) genome editing, coupled with advances in computing and imaging capabilities, has initiated a new era in which genetic diseases and individual disease susceptibilities are both predictable and actionable. Likewise, genes responsible for plant traits can be identified and altered quickly, transforming the pace of agricultural research and plant breeding. In this Review, we discuss the current state of CRISPR-mediated genetic manipulation in human cells, animals, and plants along with relevant successes and challenges and present a roadmap for the future of this technology.

Structural biology of CRISPR-Cas immunity and genome editing enzymes.

CRISPR-Cas systems provide resistance against foreign mobile genetic elements and have a wide range of genome editing and biotechnological applications. In this Review, we examine recent advances in understanding the molecular structures and mechanisms of enzymes comprising bacterial RNA-guided CRISPR-Cas immune systems and deployed for wide-ranging genome editing applications. We explore the adaptive and interference aspects of CRISPR-Cas function as well as open questions about the molecular mechanisms responsible for genome targeting. These structural insights reflect close evolutionary links between CRISPR-Cas systems and mobile genetic elements, including the origins and evolution of CRISPR-Cas systems from DNA transposons, retrotransposons and toxin-antitoxin modules. We discuss how the evolution and structural diversity of CRISPR-Cas systems explain their functional complexity and utility as genome editing tools.

Structural coordination between active sites of a CRISPR reverse transcriptase-integrase complex.

CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation of CRISPR RNAs (crRNAs). In some CRISPR systems, a reverse transcriptase (RT) fusion to the Cas1 integrase and Cas6 maturase creates a single protein that enables concerted sequence integration and crRNA production. To elucidate how the RT-integrase organizes distinct enzymatic activities, we present the cryo-EM structure of a Cas6-RT-Cas1-Cas2 CRISPR integrase complex. The structure reveals a heterohexamer in which the RT directly contacts the integrase and maturase domains, suggesting functional coordination between all three active sites. Together with biochemical experiments, our data support a model of sequential enzymatic activities that enable CRISPR sequence acquisition from RNA and DNA substrates. These findings highlight an expanded capacity of some CRISPR systems to acquire diverse sequences that direct CRISPR-mediated interference.

DABs are inorganic carbon pumps found throughout prokaryotic phyla.

Bacterial autotrophs often rely on CO2 concentrating mechanisms (CCMs) to assimilate carbon. Although many CCM proteins have been identified, a systematic screen of the components of CCMs is lacking. Here, we performed a genome-wide barcoded transposon screen to identify essential and CCM-related genes in the γ-proteobacterium Halothiobacillus neapolitanus. Screening revealed that the CCM comprises at least 17 and probably no more than 25 genes, most of which are encoded in 3 operons. Two of these operons (DAB1 and DAB2) contain a two-gene locus that encodes a domain of unknown function (Pfam: PF10070) and a putative cation transporter (Pfam: PF00361). Physiological and biochemical assays demonstrated that these proteins-which we name DabA and DabB, for DABs accumulate bicarbonate-assemble into a heterodimeric complex, which contains a putative ß-carbonic anhydrase-like active site and functions as an energy-coupled inorganic carbon (Ci) pump. Interestingly, DAB operons are found in a diverse range of bacteria and archaea. We demonstrate that functional DABs are present in the human pathogens Bacillus anthracis and Vibrio cholerae. On the basis of these results, we propose that DABs constitute a class of energized Ci pumps and play a critical role in the metabolism of Ci throughout prokaryotic phyla.

A phenotype for genetic studies of successful cognitive aging.

Survival from Alzheimer's disease (AD) and other dementias into late old age may be a useful phenotype for genetic studies of successful cognitive aging. To support molecular genetics studies for successful cognitive aging, we conducted a two-stage study to determine an optimal age phenotype for successful cognitive aging. First, risk of AD was evaluated, through informant interviews, in 4,794 parents and siblings of 976 elderly nondemented probands who were divided into three different proband age groups: those aged 60-74, 75-89, and 90+. Relatives of probands aged 90+ had a significantly lower risk than the relatives of the other two proband groups. Second, this sample was combined with an earlier sample (combined nondemented elderly probands: n = 2,025; relatives: n = 10,506), and a series of proband age groups (i.e., 75-79, 80-84, 85-89, 90+) were used to determine which optimally identifies a group of relatives with low AD risk. Using the relatives of the nondemented proband aged 60-74 as the reference group, there were reductions in cumulative risk among relatives of probands aged 85-89 and 90+, but only the latter group also showed significant reductions to the relatives of probands aged 75-79, 80-84, and 85-89. This pattern of results varied little by sex. Finally, cumulative AD risk curves were similar between relatives of probands aged 90-94 and 95+. These results suggest that age 90 is an optimal age threshold to use for both men and women in genetic studies seeking to identify genes associated with successful cognitive aging.

Increased longevity in offspring of mothers with Alzheimer's disease.

Life expectancy is a familial trait. However, the effectiveness of using the age at death of a deceased parent to estimate life expectancy in their offspring can vary depending on whether death in the parent was due to extrinsic versus intrinsic causes, as well as demographic characteristics such as sex. While Alzheimer's disease (AD) risk increases with increased age, mortality for individuals with AD is increased in contrast to comparably aged individuals without AD. Yet in most cases it is not the defining neuropathology of AD that directly terminates life but instead conditions and illnesses extrinsic to AD pathology that nevertheless have increased likelihood in its presence. For this reason, we hypothesized that offspring of AD mothers would have greater longevity than offspring of mothers without AD (insufficient numbers prevented a comparable analysis using fathers with AD). The longevity of 345 offspring of 100 deceased 60+ year old AD mothers was compared with 5,465 offspring in 1,312 deceased 60+ year old non-AD mothers. We used a proportional hazards model that accounted for clustered (nonindependent) observations due to the inclusion of several offspring from the same family. In both an unadjusted model and one that adjusted for the age at death in the mother, and the sex and birth year in the offspring we found evidence for increased longevity in the offspring of AD mothers. The results suggest that, in addition to genes that might directly affect pathways leading to AD, there may be familial/genetic factors not connected to specific pathophysiological processes in AD but instead associated with increased longevity that contribute to the familial aggregation observed in AD.