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Platelet and fibrin clots occlude blood vessels in hemostasis and thrombosis. Here we report a noncanonical mechanism for vascular occlusion based on neutrophil extracellular traps (NETs), DNA fibers released by neutrophils during inflammation. We investigated which host factors control NETs in vivo and found that two deoxyribonucleases (DNases), DNase1 and DNase1-like 3, degraded NETs in circulation during sterile neutrophilia and septicemia. In the absence of both DNases, intravascular NETs formed clots that obstructed blood vessels and caused organ damage. Vascular occlusions in patients with severe bacterial infections were associated with a defect to degrade NETs ex vivo and the formation of intravascular NET clots. DNase1 and DNase1-like 3 are independently expressed and thus provide dual host protection against deleterious effects of intravascular NETs.

The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins 1 . Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases 2 , 3 . TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR–Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA–target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR–Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR–Cas12 effectors. Cryo-electron microscopy analysis of the Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA provides insights into the mechanism of TnpB function and the evolution of CRISPR–Cas12 effectors.

Conventional CRISPR–Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a notable inversion of this paradigm, in which bacterial Tn 7 -like transposons have co-opted nuclease-deficient CRISPR–Cas systems to catalyse RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn 6677 in Escherichia coli requires CRISPR- and transposon-associated molecular machineries, including a co-complex between the DNA-targeting complex Cascade and the transposition protein TniQ. Integration of donor DNA occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep-sequencing experiments reveal highly specific, genome-wide DNA insertion across dozens of unique target sites. This discovery of a fully programmable, RNA-guided integrase lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair. A programmable transposase integrates donor DNA at user-defined genomic target sites with high fidelity, revealing a new approach for genetic engineering that obviates the need for DNA double-strand breaks and homologous recombination. 

Engineered Sp Cas9s and As Cas12a cleave fewer off-target genomic sites than wild-type (wt) Cas9. However, understanding their fidelity, mechanisms and cleavage outcomes requires systematic profiling across mispaired target DNAs. Here we describe NucleaSeq—nuclease digestion and deep sequencing—a massively parallel platform that measures the cleavage kinetics and time-resolved cleavage products for over 10,000 targets containing mismatches, insertions and deletions relative to the guide RNA. Combining cleavage rates and binding specificities on the same target libraries, we benchmarked five Sp Cas9 variants and As Cas12a. A biophysical model built from these data sets revealed mechanistic insights into off-target cleavage. Engineered Cas9s, especially Cas9-HF1, dramatically increased cleavage specificity but not binding specificity compared to wtCas9. Surprisingly, As Cas12a cleavage specificity differed little from that of wtCas9. Initial DNA cleavage sites and end trimming varied by nuclease, guide RNA and the positions of mispaired nucleotides. More broadly, NucleaSeq enables rapid, quantitative and systematic comparisons of specificity and cleavage outcomes across engineered and natural nucleases. The enzymatic properties of RNA-guided nucleases are revealed through massively parallel analysis.

DNA double-strand break (DSB) repair by homologous recombination is initiated by DNA end resection, a process involving the controlled degradation of the 5′-terminated strands at DSB sites 1 , 2 . The breast cancer suppressor BRCA1–BARD1 not only promotes resection and homologous recombination, but it also protects DNA upon replication stress 1 , 3 , 4 , 5 , 6 , 7 , 8 – 9 . BRCA1–BARD1 counteracts the anti-resection and pro-non-homologous end-joining factor 53BP1, but whether it functions in resection directly has been unclear 10 , 11 , 12 , 13 , 14 , 15 – 16 . Using purified recombinant proteins, we show here that BRCA1–BARD1 directly promotes long-range DNA end resection pathways catalysed by the EXO1 or DNA2 nucleases. In the DNA2-dependent pathway, BRCA1–BARD1 stimulates DNA unwinding by the Werner or Bloom helicase. Together with MRE11–RAD50–NBS1 and phosphorylated CtIP, BRCA1–BARD1 forms the BRCA1–C complex 17 , 18 , which stimulates resection synergistically to an even greater extent. A mutation in phosphorylated CtIP (S327A), which disrupts its binding to the BRCT repeats of BRCA1 and hence the integrity of the BRCA1–C complex 19 , 20 – 21 , inhibits resection, showing that BRCA1–C is a functionally integrated ensemble. Whereas BRCA1–BARD1 stimulates resection in DSB repair, it paradoxically also protects replication forks from unscheduled degradation upon stress, which involves a homologous recombination-independent function of the recombinase RAD51 (refs. 4 , 5 – 6 , 8 ). We show that in the presence of RAD51, BRCA1–BARD1 instead inhibits DNA degradation. On the basis of our data, the presence and local concentration of RAD51 might determine the balance between the pronuclease and the DNA protection functions of BRCA1–BARD1 in various physiological contexts. BRCA1–BARD1 directly promotes double-strand break repair by stimulating long-range DNA end resection pathways.

Mitochondria are inherited maternally in most animals, but the mechanisms of selective paternal mitochondrial elimination (PME) are unknown. While examining fertilization in Caenorhabditis elegans, we observed that paternal mitochondria rapidly lose their inner membrane integrity. CPS-6, a mitochondrial endonuclease G, serves as a paternal mitochondrial factor that is critical for PME. We found that CPS-6 relocates from the intermembrane space of paternal mitochondria to the matrix after fertilization to degrade mitochondrial DNA. It acts with maternal autophagy and proteasome machineries to promote PME. Loss of cps-6 delays breakdown of mitochondrial inner membranes, autophagosome enclosure of paternal mitochondria, and PME. Delayed removal of paternal mitochondria causes increased embryonic lethality, demonstrating that PME is important for normal animal development. Thus, CPS-6 functions as a paternal mitochondrial degradation factor during animal development.

CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis . This study greatly expands the targeting scope of Cas12a for crop genome engineering. CRISPR-Cas12a is a promising system for targeting AT-rich regions of the genome. Here the authors identify Cas12a orthologs with expanded targeting scope and develop a highly multiplexable editing system in rice.

The CRISPR-Cas12a system has revolutionized nucleic acid testing (NAT) with its rapid and precise capabilities, yet it traditionally required RNA pre-amplification. Here we develop rapid fluorescence and lateral flow NAT assays utilizing a split Cas12a system (SCas12a), consisting of a Cas12a enzyme and a split crRNA. The SCas12a assay enables highly sensitive, amplification-free, and multiplexed detection of miRNAs and long RNAs without complex secondary structures. It can differentiate between mature miRNA and its precursor (pre-miRNA), a critical distinction for precise biomarker identification and cancer progression monitoring. The system’s specificity is further highlighted by its ability to detect DNA and miRNA point mutations. Notably, the SCas12a system can quantify the miR-21 biomarker in plasma from cervical cancer patients and, when combined with RPA, detect HPV at attomole levels in clinical samples. Together, our work presents a simple and cost-effective SCas12a-based NAT platform for various diagnostic settings. CRISPR-Cas12a nucleic acid detection largely involves pre-amplification and is limited in its ability to directly detect RNA. Here, authors present a split Cas12a system that enables direct, highly sensitive, and cost-effective detection of both DNA and RNA without amplification.

The ability to write a stable record of identified molecular events into a specific genomic locus would enable the examination of long cellular histories and have many applications, ranging from developmental biology to synthetic devices. We show that the type I-E CRISPR (clustered regularly interspaced short palindromic repeats)–Cas system of Escherichia coli can mediate acquisition of defined pieces of synthetic DNA. We harnessed this feature to generate records of specific DNA sequences into a population of bacterial genomes. We then applied directed evolution so as to alter the recognition of a protospacer adjacent motif by the Cas1-Cas2 complex, which enabled recording in two modes simultaneously. We used this system to reveal aspects of spacer acquisition, fundamental to the CRISPR-Cas adaptation process. These results lay the foundations of a multimodal intracellular recording device.

We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.