Exploiting activation and inactivation mechanisms in type I-C CRISPR-Cas3 for genome-editing applications.

Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets and the nuclease-helicase Cas3 to degrade them. Among the seven subtypes, type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here, we use four cryoelectron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9 that strongly inhibit Neisseria lactamica I-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through allosteric inhibition, whereas AcrIC9 achieves so through direct competition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for type I CRISPR eukaryotic genome engineering.

Engineering a transposon-associated TnpB-ωRNA system for efficient gene editing and phenotypic correction of a tyrosinaemia mouse model.

Transposon-associated ribonucleoprotein TnpB is known to be the ancestry endonuclease of diverse Cas12 effector proteins from type-V CRISPR system. Given its small size (408 aa), it is of interest to examine whether engineered TnpB could be used for efficient mammalian genome editing. Here, we showed that the gene editing activity of native TnpB from Deinococcus radiodurans (ISDra2 TnpB) in mouse embryos was already higher than previously identified small-sized Cas12f1. Further stepwise engineering of noncoding RNA (ωRNA or reRNA) component of TnpB significantly elevated the nuclease activity of TnpB. Notably, an optimized TnpB-ωRNA system could be efficiently delivered in vivo with single adeno-associated virus (AAV) and corrected the disease phenotype in a tyrosinaemia mouse model. Thus, the engineered miniature TnpB system represents a new addition to the current genome editing toolbox, with the unique feature of the smallest effector size that facilitate efficient AAV delivery for editing of cells and tissues.

Bioinspired Flexible Wearable Sensor with High Self-Cleaning and Antibacterial Performance for Human Motion Sensing.

Flexible wearable strain sensors have shown great potential in monitoring human motion, due to their ability to flexibly fit to multiple surfaces, which can realize the monitoring of human motions and external stimulation. However, the utilization of the sensor in extreme conditions such as low or high temperatures still poses a risk of signal output distortion. Moreover, the continuous usage of the sensor may result in extensive bacterial growth at the interface between the sensor and the skin, posing a threat to human health. Herein, a hydrophobic flexible antibacterial strain sensor (CGP) based on carbon black-PDMS was prepared, inspired by the superhydrophobic surface of a lotus leaf. The CGP sensor demonstrates exceptional sensitivity, with a gauge factor (GF) of 0.467 in the strain range of 0-15% and a fast response time (65.4 ms, 5% strain). Additionally, it exhibits a high conductivity of 1.2 mS cm-1 at -20 °C and 2.0 mS cm-1 at 100 °C, indicating its ability to function effectively even in extreme temperatures. The static water contact angle of CGP measures 121.7°, and self-cleaning experiments have confirmed its excellent self-cleaning performance. Furthermore, the CGP displays distinct response characteristics to movements of human fingers, wrists, and knees, making it an ideal choice for monitoring various joints in the human body. In terms of antibacterial properties, CGP has demonstrated an antibacterial rate of over 99% against E. coli and S. aureus. Possessing high sensitivity, superior electrical conductivity in harsh environments, and super antibacterial capabilities, CGP holds significant potential for applications in human motion monitoring and other fields.

Exploring the Anti-Cancer Effects of Fish Bone Fermented Using Monascus purpureus: Induction of Apoptosis and Autophagy in Human Colorectal Cancer Cells.

Fish bone fermented using Monascus purpureus (FBF) has total phenols and functional amino acids that contribute to its anti-oxidant and anti-inflammatory properties. Colorectal cancer, one of the most prevalent cancers and the third largest cause of death worldwide, has become a serious threat to global health. This study investigates the anti-cancer effects of FBF (1, 2.5 or 5 mg/mL) on the cell growth and molecular mechanism of HCT-116 cells. The HCT-116 cell treatment with 2.5 or 5 mg/mL of FBF for 24 h significantly decreased cell viability (p < 0.05). The S and G2/M phases significantly increased by 88-105% and 25-43%, respectively (p < 0.05). Additionally, FBF increased the mRNA expression of caspase 8 (38-77%), protein expression of caspase 3 (34-94%), poly (ADP-ribose) polymerase (PARP) (31-34%) and induced apoptosis (236-773%) of HCT-116 cells (p < 0.05). FBF also increased microtubule-associated protein 1B light chain 3 (LC3) (38-48%) and phosphoinositide 3 kinase class III (PI3K III) (32-53%) protein expression, thereby inducing autophagy (26-52%) of HCT-116 cells (p < 0.05). These results showed that FBF could inhibit HCT-116 cell growth by inducing S and G2/M phase arrest of the cell cycle, apoptosis and autophagy. Thus, FBF has the potential to treat colorectal cancer.

Exploiting Activation and Inactivation Mechanisms in Type I-C CRISPR-Cas3 for Genome Editing Applications.

Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets, and the nuclease-helicase Cas3 to degrade them. Among seven subtypes, Type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here we use four cryo-electron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling efficient NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9, that strongly inhibit N. lactamica I-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through direct competition, whereas AcrIC9 achieves so through allosteric inhibition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for controllable Type I CRISPR genome engineering.

Isolation of feline panleukopenia virus from Yanji of China and molecular epidemiology from 2021 to 2022.

BACKGROUND: Feline panleukopenia virus (FPV) is a widespread and highly infectious pathogen in cats with a high mortality rate. Although Yanji has a developed cat breeding industry, the variation of FPV locally is still unclear. OBJECTIVES: This study aimed to isolate and investigate the epidemiology of FPV in Yanji between 2021 and 2022. METHODS: A strain of FPV was isolated from F81 cells. Cats suspected of FPV infection (n = 80) between 2021 and 2022 from Yanji were enrolled in this study. The capsid protein 2 (VP2) of FPV was amplified. It was cloned into the pMD-19T vector and transformed into a competent Escherichia coli strain. The positive colonies were analyzed via VP2 Sanger sequencing. A phylogenetic analysis based on a VP2 coding sequence was performed to identify the genetic relationships between the strains. RESULTS: An FPV strain named YBYJ-1 was successfully isolated. The virus diameter was approximately 20-24 nm, 50% tissue culture infectious dose = 1 × 10-4.94/mL, which caused cytopathic effect in F81 cells. The epidemiological survey from 2021 to 2022 showed that 27 of the 80 samples were FPV-positive. Additionally, three strains positive for CPV-2c were unexpectedly found. Phylogenetic analysis showed that most of the 27 FPV strains belonged to the same group, and no mutations were found in the critical amino acids. CONCLUSIONS: A local FPV strain named YBYJ-1 was successfully isolated. There was no critical mutation in FPV in Yanji, but some cases with CPV-2c infected cats were identified.

Anti-inflammatory effects of fish bone fermented using Monascus purpureus in LPS-induced RAW264.7 cells by regulating NF-κB pathway.

Fish bones are the by-products of aquatic and fishery processing, which are often discarded. However, it has been considered having health-promoting by containing many essential nutrients. This study investigates the anti-inflammatory effect of fish bone fermented by Monascus purpureus (FBF) and the NF-κB pathway regulation mechanism in lipopolysaccharides (LPS)-induced RAW 264.7 cells. FBF has inhibited the production of PGE2 (prostaglandin E2), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in LPS-induced RAW264.7 cells. The FBF has significantly inhibited mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, FBF has suppressed activation of NF-κB (nuclear factor kappa-B) by increasing IκB mRNA expression and reduced of p65, p50 mRNA expression, as well as nuclear NF-κB DNA binding activity in LPS-induced RAW 246.7 cells. These findings demonstrate that FBF has inhibited LPS-induced inflammation by subsiding the activation of NF-κB in RAW 246.7 cells, implying that FBF could be employed as a promising natural product.

Reconstitution and biochemical characterization of the RNA-guided helicase-nuclease protein Cas3 from type I-A CRISPR-Cas system.

Type I is the most prevalent CRISPR system found in nature. It can be further defined into six subtypes, from I-A to I-G. Among them, the Type I-A CRISPR-Cas systems are almost exclusively found in hyperthermophilic archaeal organisms. The system achieves RNA-guided DNA degradation through the concerted action of a CRISPR RNA containing complex Cascade and a helicase-nuclease fusion enzyme Cas3. Here, we summarize assays to characterize the biochemical behavior of Cas3. A steep temperature-dependency was found for the helicase component of Cas3HEL, but not the nuclease component HD. This finding enabled us to establish the correct experimental condition to carry out I-A CRISPR-Cas based genome editing in human cells with extremely high efficiency.

Craspase is a CRISPR RNA-guided, RNA-activated protease.

The CRISPR-Cas type III-E RNA-targeting effector complex gRAMP/Cas7-11 is associated with a caspase-like protein (TPR-CHAT/Csx29) to form Craspase (CRISPR-guided caspase). Here, we use cryo-electron microscopy snapshots of Craspase to explain its target RNA cleavage and protease activation mechanisms. Target-guide pairing extending into the 5' region of the guide RNA displaces a gating loop in gRAMP, which triggers an extensive conformational relay that allosterically aligns the protease catalytic dyad and opens an amino acid side-chain-binding pocket. We further define Csx30 as the endogenous protein substrate that is site-specifically proteolyzed by RNA-activated Craspase. This protease activity is switched off by target RNA cleavage by gRAMP and is not activated by RNA targets containing a matching protospacer flanking sequence. We thus conclude that Craspase is a target RNA-activated protease with self-regulatory capacity.

Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools.

Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme Cas3 is then recruited in trans for processive DNA degradation. Contrary to this model, here, we show that type I-A Cascade and Cas3 function as an integral effector complex. We provide four cryoelectron microscopy (cryo-EM) snapshots of the Pyrococcus furiosus (Pfu) type I-A effector complex in different stages of DNA recognition and degradation. The HD nuclease of Cas3 is autoinhibited inside the effector complex. It is only allosterically activated upon full R-loop formation, when the entire targeted region has been validated by the RNA guide. The mechanistic insights inspired us to convert Pfu Cascade-Cas3 into a high-sensitivity, low-background, and temperature-activated nucleic acid detection tool. Moreover, Pfu CRISPR-Cas3 shows robust bi-directional deletion-editing activity in human cells, which could find usage in allele-specific inactivation of disease-causing mutations.

Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9.

Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated ωRNA plays the combined role of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) to guide double-stranded DNA (dsDNA) cleavage. Here we report a 2.78-angstrom cryo-electron microscopy structure of IscB-ωRNA bound to a dsDNA target, revealing the architectural and mechanistic similarities between IscB and Cas9 ribonucleoproteins. Target-adjacent motif recognition, R-loop formation, and DNA cleavage mechanisms are explained at high resolution. ωRNA plays the equivalent function of REC domains in Cas9 and contacts the RNA-DNA heteroduplex. The IscB-specific PLMP domain is dispensable for RNA-guided DNA cleavage. The transition from ancestral IscB to Cas9 involved dwarfing the ωRNA and introducing protein domain replacements.

The Antimicrobial Effects of Bacterial Cellulose Produced by Komagataeibacter intermedius in Promoting Wound Healing in Diabetic Mice.

As a conventional medical dressing, medical gauze does not adequately protect complex and hard-to-heal diabetic wounds and is likely to permit bacterial entry and infections. Therefore, it is necessary to develop novel dressings to promote wound healing in diabetic patients. Komagataeibacter intermedius was used to produce unmodified bacterial cellulose, which is rarely applied directly to diabetic wounds. The produced cellulose was evaluated for wound recovery rate, level of inflammation, epidermal histopathology, and antimicrobial activities in treated wounds. Diabetic mices' wounds treated with bacterial cellulose healed 1.63 times faster than those treated with gauze; the values for the skin indicators in bacterial cellulose treated wounds were more significant than those treated with gauze. Bacterial cellulose was more effective than gauze in promoting tissue proliferation with more complete epidermal layers and the formation of compact collagen in the histological examination. Moreover, wounds treated with bacterial cellulose alone had less water and glucose content than those treated with gauze; this led to an increase of 6.82 times in antimicrobial protection, lower levels of TNF-α and IL-6 (39.6% and 83.2%), and higher levels of IL-10 (2.07 times) than in mice wounds treated with gauze. The results show that bacterial cellulose produced using K. intermedius beneficially affects diabetic wound healing and creates a hygienic microenvironment by preventing inflammation. We suggest that bacterial cellulose can replace medical gauze as a wound dressing for diabetic patients.

Pterostilbene and Its Derivative 3'-Hydroxypterostilbene Ameliorated Nonalcoholic Fatty Liver Disease Through Synergistic Modulation of the Gut Microbiota and SIRT1/AMPK Signaling Pathway.

Nonalcoholic fatty liver disease (NAFLD) is a recent chronic liver disease common in many developed countries and is closely associated with metabolic syndrome, such as obesity and insulin resistance. The present study was performed to investigate the effects of pterostilbene (Pt) and its derivative 3'-hydroxypterostilbene (OHPt) on free fatty acids (FFA)-induced lipid accumulation in HepG2 cells and high-fat diet (HFD)-induced NAFLD in C57BL/6J mice. The results showed that Pt and OHPt significantly ameliorated FFA-induced steatosis in HepG2 cells and enhanced lipolysis through the upregulation of SIRT1/AMPK and insulin signaling pathways. In the in vivo study, Pt and OHPt treatment resulted in reduced hepatic lipid droplets accumulation. The data showed that Pt and OHPt upregulated the SIRT1/AMPK pathway and subsequently downregulated the protein expression of SREBP-1 to activate fatty acid (FA) ß-oxidation to inhibit FA synthesis. Pt and OHPt administration activated the insulin signaling pathway and further ameliorated the insulin resistance and liver function in the HFD-fed mice. Furthermore, Pt and OHPt markedly increased the numbers of Oscillospira and decreased the numbers of Allobaculum, Phascolarctobacterium, and Staphylococcus compared with those in the HFD group. These robust results indicate that Pt and OHPt are able to possess potential health benefits in improving insulin resistance and hepatic steatosis by promoting healthy populations or abundances of considered vital microbiota. Besides, OHPt is more effective than Pt, which might have promising chemotherapeutic effects for future clinical application.

Introducing Large Genomic Deletions in Human Pluripotent Stem Cells Using CRISPR-Cas3.

CRISPR-Cas systems provide researchers with eukaryotic genome editing tools and therapeutic platforms that make it possible to target disease mutations in somatic organs. Most of these tools employ Type II (e.g., Cas9) or Type V (e.g., Cas12a) CRISPR enzymes to create RNA-guided precise double-strand breaks in the genome. However, such technologies are limited in their capacity to make targeted large deletions. Recently, the Type I CRISPR system, which is prevalent in microbes and displays unique enzymatic features, has been harnessed to effectively create large chromosomal deletions in human cells. Type I CRISPR first uses a multisubunit ribonucleoprotein (RNP) complex called Cascade to find its guide-complementary target site, and then recruits a helicase-nuclease enzyme, Cas3, to travel along and shred the target DNA over a long distance with high processivity. When introduced into human cells as purified RNPs, the CRISPR-Cas3 complex can efficiently induce large genomic deletions of varying lengths (1-100 kb) from the CRISPR-targeted site. Because of this unique editing outcome, CRISPR-Cas3 holds great promise for tasks such as the removal of integrated viral genomes and the interrogation of structural variants affecting gene function and human disease. Here, we provide detailed protocols for introducing large deletions using CRISPR-Cas3. We describe step-by-step procedures for purifying the Type I-E CRISPR proteins Cascade and Cas3 from Thermobifida fusca, electroporating RNPs into human cells, and characterizing DNA deletions using PCR and sequencing. We focus here on human pluripotent stem cells due to their clinical potential, but these protocols will be broadly useful for other cell lines and model organisms for applications including large genomic deletion, full-gene or -chromosome removal, and CRISPR screening for noncoding elements, among others. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of Tfu Cascade RNP Support Protocol 1: Expression and purification of TfuCas3 protein Support Protocol 2: Culture of human pluripotent stem cells Basic Protocol 2: Introduction of Tfu Cascade RNP and Cas3 protein into hPSCs via electroporation Basic Protocol 3: Characterization of genomic DNA lesions using long-range PCR, TOPO cloning, and Sanger sequencing Alternate Protocol: Comprehensive analysis of genomic lesions by Tn5-based next-generation sequencing Support Protocol 3: Single-cell clonal isolation.

Effect of Tempeh on Gut Microbiota and Anti-Stress Activity in Zebrafish.

Rhizopus oryzae is a fungus used to ferment tempeh in Indonesia and is generally recognized as safe (GRAS) for human consumption by the USA FDA. We previously assessed the effect of a tempeh extract on cortisol levels in zebrafish but did not include behavioral studies. Here, we measured the GABA content in three strains of Rhizopus oryzae, two isolated by us (MHU 001 and MHU 002) and one purchased. We then investigated the effect of tempeh on cortisol and the gut microbiota in a zebrafish experimental model. GABA concentration was the highest in MHU 002 (9.712 ± 0.404 g kg-1) followed by our MHU 001 strain and the purchased one. The fish were divided into one control group fed a normal diet and three experimental groups fed soybean tempeh fermented with one of the three strains of Rhizopus oryzae. After two weeks, individual fish were subjected to unpredicted chronic stress using the novel tank diving test and the tank light-dark test. Next-generation sequencing was used to analyze gut microbial communities and RT-PCR to analyze the expression of BDNF (brain-derived neurotrophic factor) gene and of other genes involved in serotonin signaling/metabolism in gut and brain. Tempeh-fed zebrafish exhibited increased exploratory behavior (less stress) in both tank tests. They also had significantly reduced gut Proteobacteria (include E. coli) (51.90% vs. 84.97%) and significantly increased gut Actinobacteria (include Bifidobacterium spp.) (1.80% vs. 0.79%). The content of Bifidobacteriumadolescentis, a "psychobiotic", increased ten-fold from 0.04% to 0.45%. Tempeh also increases BDNF levels in zebrafish brain. Rhizopus oryzae MHU 001 greatly improved the anti-stress effect of tempeh and microbiota composition in zebrafish gut.

Mechanism for Cas4-assisted directional spacer acquisition in CRISPR-Cas.

Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array1. Spacer insertion is carried out by the Cas1-Cas2 integrase complex2-4. A substantial fraction of CRISPR-Cas systems use a Fe-S cluster containing Cas4 nuclease to ensure that spacers are acquired from DNA flanked by a protospacer adjacent motif (PAM)5,6 and inserted into the CRISPR array unidirectionally, so that the transcribed CRISPR RNA can guide target searching in a PAM-dependent manner. Here we provide a high-resolution mechanistic explanation for the Cas4-assisted PAM selection, spacer biogenesis and directional integration by type I-G CRISPR in Geobacter sulfurreducens, in which Cas4 is naturally fused with Cas1, forming Cas4/Cas1. During biogenesis, only DNA duplexes possessing a PAM-embedded 3'-overhang trigger Cas4/Cas1-Cas2 assembly. During this process, the PAM overhang is specifically recognized and sequestered, but is not cleaved by Cas4. This 'molecular constipation' prevents the PAM-side prespacer from participating in integration. Lacking such sequestration, the non-PAM overhang is trimmed by host nucleases and integrated to the leader-side CRISPR repeat. Half-integration subsequently triggers PAM cleavage and Cas4 dissociation, allowing spacer-side integration. Overall, the intricate molecular interaction between Cas4 and Cas1-Cas2 selects PAM-containing prespacers for integration and couples the timing of PAM processing with the stepwise integration to establish directionality.

Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly.

Telomeres protect chromosome ends from inappropriately activating the DNA damage and repair responses. Primary microcephaly is a key clinical feature of several human telomere disorder syndromes, but how microcephaly is linked to dysfunctional telomeres is not known. Here, we show that the microcephalin 1/BRCT-repeats inhibitor of hTERT (MCPH1/BRIT1) protein, mutated in primary microcephaly, specifically interacts with the TRFH domain of the telomere binding protein TRF2. The crystal structure of the MCPH1-TRF2 complex reveals that this interaction is mediated by the MCPH1 330YRLSP334 motif. TRF2-dependent recruitment of MCPH1 promotes localization of DNA damage factors and homology directed repair of dysfunctional telomeres lacking POT1-TPP1. Additionally, MCPH1 is involved in the replication stress response, promoting telomere replication fork progression and restart of stalled telomere replication forks. Our work uncovers a previously unrecognized role for MCPH1 in promoting telomere replication, providing evidence that telomere replication defects may contribute to the onset of microcephaly.

Effects of Red-Bean Tempeh with Various Strains of Rhizopus on GABA Content and Cortisol Level in Zebrafish.

Tempeh is traditionally produced by fermenting soybean with the fungus Rhizopus oligosporus found in banana leafs. We wanted to investigate if Taiwan's flavorful red bean could be used as a healthy substitute for soybeans in tempeh. One bioactive component of tempeh is γ-Aminobutyric acid (GABA). We measured GABA content and shelf-life-related antimicrobial activity in red-bean tempeh made with four strains of Rhizopus, one purchased strain of Rhizopus, and an experimental co-cultured group (Rhizopus and Lactobacillus rhamnosus BCRC16000) as well as cortisol in red-bean-tempeh-treated zebrafish. GABA was highest in the co-culture group (19.028 ± 1.831 g kg-1), followed by screened Strain 1, the purchased strain, and screened Strain 4. All strains had antibacterial activity on S. aureus and B. cereus. The extract significantly reduced cortisol in zebrafish. However, Strain 1, with less GABA than some of the other strains, had the best effect on cortisol level, suggesting that other components in red-bean tempeh may also affect stress-related cortisol. We found the benefits of red-bean tempeh to be similar to those reported for soybean-produced tempeh, suggesting that it could be produced as an alternative product. Considering the Taiwanese appreciation of the red-bean flavor, it might find a welcoming market.

Real-time observation of CRISPR spacer acquisition by Cas1-Cas2 integrase.

Cas1 integrase associates with Cas2 to insert short DNA fragments into a CRISPR array, establishing nucleic acid memory in prokaryotes. Here we applied single-molecule FRET methods to the Enterococcus faecalis (Efa) Cas1-Cas2 system to establish a kinetic framework describing target-searching, integration, and post-synapsis events. EfaCas1-Cas2 on its own is not able to find the CRISPR repeat in the CRISPR array; it only does so after prespacer loading. The leader sequence adjacent to the repeat further stabilizes EfaCas1-Cas2 contacts, enabling leader-side integration and subsequent spacer-side integration. The resulting post-synaptic complex (PSC) has a surprisingly short mean lifetime. Remarkably, transcription effectively resolves the PSC, and we predict that this is a conserved mechanism that ensures efficient and directional spacer integration in many CRISPR systems. Overall, our study provides a complete model of spacer acquisition, which can be harnessed for DNA-based information storage and cell lineage tracing technologies.