Research Progress of CRISPR-Cas3

Werner syndrome is an autosomal recessive disorder associated with premature aging and cancer predisposition caused by mutations of the WRN gene. WRN is a member of the RecQ DNA helicase family with functions in maintaining genome stability. Sir2, an NAD-dependent histone deacetylase, has been proven to extend life span in yeast and Caenorhabditis elegans. Mammalian Sir2 (SIRT1) has also been found to regulate premature cellular senescence induced by the tumor suppressors PML and p53. SIRT1 plays an important role in cell survival promoted by calorie restriction. Here we show that SIRT1 interacts with WRN both in vitro and in vivo; this interaction is enhanced after DNA damage. WRN can be acetylated by acetyltransferase CBP/p300, and SIRT1 can deacetylate WRN both in vitro and in vivo. WRN acetylation decreases its helicase and exonuclease activities, and SIRT1 can reverse this effect. WRN acetylation alters its nuclear distribution. Down-regulation of SIRT1 reduces WRN translocation from nucleoplasm to nucleoli after DNA damage. These results suggest that SIRT1 regulates WRN-mediated cellular responses to DNA damage through deacetylation of WRN.

For more than a decade, Luciano Marraffini has played a major role in the CRISPR revolution. As a postdoc with

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems, fundamental to prokaryotic adaptive immunity, are divided into two major classes based on their effector protein composition.Class 1 systems, including types I, III, and IV, utilize multi-subunit complexes to carry out interference (Wang et al., 2022), while Class 2 systems, encompassing types II, V, and VI, rely on single protein effectors to accomplish the same defence task (Dong et al., 2016(Dong et al., , 2017;;Makarova et al., 2020).The candidate type VII CRISPR-Cas system, a recent addition to Class 1, features a ribonucleoprotein complex composed of Cas5 and Cas7 proteins, guided by CRISPR RNA (crRNA) (Altae-Tran et al., 2023) (Fig. 1A).However, what truly sets this system apart is the recruitment of Cas14, a distinct nuclease with a -CASP (CPSF-Artemis-SNM1-Pso2) domain homologous to RNase J, which executes RNA cleavage-a function typically performed by other effectors in different CRISPR systems.In their study, Yang et al. ( 2024) systematically dissected the structural and functional components of the type VII CRISPR-Cas system.They began by utilizing cryo-electron microscopy to resolve seven distinct structures of the Cas14-bound interference complex, capturing it in various functional states.These structures revealed that the type VII system utilizes a ribonucleoprotein complex formed by Cas5 and Cas7 proteins, guided by CRISPR RNA (crRNA), to target RNA (Fig. 1B).Unlike in other type III CRISPR systems, where Cas7 or Cas11 are responsible for RNA cleavage (Guo et al., 2019;

Background: High microsatellite instability (MSI-H) is highly prevalent across many cancer types including endometrium, gastric, ovary and colorectal cancer. It is well known that there is a clear synthetic lethality (SL) interaction between MSI-H and Werner syndrome protein (WRN) inhibition. WRN is a member of RecQ helicase family and involved in the maintenance of genome integrity. Different from other RecQ family members, WRN has two activity centers responsible for helicase and exonuclease activity, respectively. Further studies confirmed that an inhibition on helicase activity, but not exonuclease activity, is responsible for SL interaction with MSI-H. Also, Recent clinical observations supported that though loss-of-function of WRN could cause an early-aged disease Werner syndrome, selective loss of helicase but not exonuclease leads to only mild to moderate symptoms, suggesting a selective inhibitor of helicase activity has the potential to induce the SL interaction with MSI-H and keep patients safe. Methods: Biochemical assays and cell-based assays were performed to evaluate in vitro activity and selectivity of PH027-1. The in vivo anti-tumor efficacy was tested in two cell-derived xenograft (CDX) mouse models with tumors harboring MSI-H. The in vivo pharmacokinetics (PK) properties were evaluated in mice, rats and dogs, and other in vitro and in vivo PK and safety properties were assessed with corresponding assay methods. Results: Biochemical assays showed that PH027-1 potently inhibited helicase activity of WRN (IC50, 0.8 nM), but had no effect on other RecQ family members even at the highest tested concentration of 10 μM (IC50, all≥10 μM for BLM, RecQ4 and RecQ5). At the same time, PH027-1 did not affect the exonuclease activity of WRN at all tested concentrations. In cell-based assays, PH027-1 potently inhibited cell viability of MSI-H cancer cells SW48 and HCT116 at tens of nM, but had no effect on MSS cells (HT29 cancer cells and CD34+ hematopoietic stem cells) even at 10 μM. The tests were further expanded to 18 cancer cell lines covering uterus, gastric, ovary and colorectal cancer. Nine of twelve MSI-H cell lines were very sensitive to WRN inhibition, and all six MSS cell lines were totally resistant to the treatment. PH027-1 possessed excellent PK profiles as shown by in vivo PK studies, with absolute bioavailability of 58%~102% in mice, rats and dogs. In two CDX models (SW48 and HCT116), oral administration of PH027-1 dose-dependently inhibited tumor growth, and a nearly complete inhibition was observed at a dose of 20 mg/kg QD. Tumor regression was clearly shown at higher doses. All other in vitro and in vivo PK and safety tests showed favorable results and supported for preclinical development of PH027-1. Conclusion: As a highly selective and potent inhibitor on helicase activity of WRN, PH027-1 has the potential to become a novel and safe approach for treatment of MSI-H tumors. Citation Format: Feng Gao, Bin Liu, Liandong Jing, Jing Wang, Yongyong Wu, Jun Wan, Pengzhi Zhang, Yu Gao, Zhizhong Li, Yongqi Guo. PH027-1, a potent and selective small-molecule WRN inhibitor that targets MSI-H tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5921.

RecJ/cell division cycle 45 (Cdc45) proteins are widely conserved in the three domains of life, i.e. in bacteria, Eukarya, and Archaea. Bacterial RecJ is a 5'-3' exonuclease and functions in DNA repair pathways by using its 5'-3' exonuclease activity. Eukaryotic Cdc45 has no identified enzymatic activity but participates in the CMG complex, so named because it is composed of Cdc45, minichromosome maintenance protein complex (MCM) proteins 2-7, and GINS complex proteins (Sld5, Psf11-3). Eukaryotic Cdc45 and bacterial/archaeal RecJ share similar amino acid sequences and are considered functional counterparts. In Archaea, a RecJ homolog in Thermococcus kodakarensis was shown to associate with GINS and accelerate its nuclease activity and was, therefore, designated GAN (GINS-associated nuclease); however, to date, no archaeal RecJ·MCM·GINS complex has been isolated. The thermophilic archaeon Thermoplasma acidophilum has two RecJ-like proteins, designated TaRecJ1 and TaRecJ2. TaRecJ1 exhibited DNA-specific 5'-3' exonuclease activity, whereas TaRecJ2 had 3'-5' exonuclease activity and preferred RNA over DNA. TaRecJ2, but not TaRecJ1, formed a stable complex with TaGINS in a 2:1 molar ratio. Furthermore, the TaRecJ2·TaGINS complex stimulated activity of TaMCM (T. acidophilum MCM) helicase in vitro, and the TaRecJ2·TaMCM·TaGINS complex was also observed in vivo However, TaRecJ2 did not interact with TaMCM directly and was not required for the helicase activation in vitro These findings suggest that the function of archaeal RecJ in DNA replication evolved divergently from Cdc45 despite conservation of the CMG-like complex formation between Archaea and Eukarya.

Natural CRISPR Systems and Targets in the Human Microbiome

The clustered regularly interspaced short palindromic repeat (CRISPR) system represents a fundamental tool for biotechnological applications. However, the ability to engineer intrinsic components of the CRISPR system to regulate its activity remains limited. Here, we present a strategy for the regulation of CRISPR-Cas12a by engineering an enhanced split CRISPR RNA (split en-crRNA) variant. Reassembly of this variant autonomously restores the full activity of Cas12a nuclease without the need for chemical modifications or exogenous activation stimuli. Leveraging this strategy, we developed CREST (CRISPR Reactions Elicited by Split en-crRNA Transcription) for molecular diagnostics. CREST integrates biochemical circuits that ingeniously harness isothermal amplification to trigger the on-demand synthesis and reassembly of a universal split en-crRNA, addressing longstanding challenges in CRISPR diagnostics, including the low efficiency of one-pot assays, protospacer adjacent motif restriction, the need for crRNA reoptimization, and limited reagent stability. The CREST assay enables robust detection of attomolar DNAs or RNAs within 15 to 20 min and demonstrates capability for identifying bacterial infections in patients. This study provides new insights into Cas12a regulation, which is expected to advance the development of controllable CRISPR technologies for diagnostics as well as broader applications such as genome editing and programmable gene expression.

PurposeCRISPR gene-editing technology has the potential to transform the diagnosis and treatment of infectious diseases, but most clinicians are unaware of its broad applicability. Derived from an ancient microbial defence system, these so-called “molecular scissors” enable precise gene editing with a low error rate. However, CRISPR systems can also be targeted against pathogenic DNA or RNA sequences. This potential is being combined with innovative delivery systems to develop new therapeutic approaches to infectious diseases.MethodsWe searched Pubmed and Google Scholar for CRISPR-based strategies in the diagnosis and treatment of infectious diseases. Reference lists were reviewed and synthesized for narrative review.ResultsCRISPR-based strategies represent a novel approach to many challenging infectious diseases. CRISPR technologies can be harnessed to create rapid, low-cost diagnostic systems, as well as to identify drug-resistance genes. Therapeutic strategies, such as CRISPR systems that cleave integrated viral genomes or that target resistant bacteria, are in development. CRISPR-based therapies for emerging viruses, such as SARS-CoV-2, have also been proposed. Finally, CRISPR systems can be used to reprogram human B cells to produce neutralizing antibodies. The risks of CRISPR-based therapies include off-target and on-target modifications. Strategies to control these risks are being developed and a phase 1 clinical trials of CRISPR-based therapies for cancer and monogenic diseases are already underway.ConclusionsCRISPR systems have broad applicability in the field of infectious diseases and may offer solutions to many of the most challenging human infections.

With the arrival of CRISPR‒Cas systems, transformative pathways for gene therapy have been created to bring genetic changes of precise gene alterations to treat a range of genetic, oncological, and infectious diseases. However, despite its potential, CRISPR technology’s clinical application is currently limited by delivery inefficiencies, off-target effects, and immunogenicity. Newly formed nanocarriers are providing improved CRISPR delivery, targeting, and precision editing. This review describes how nanomaterials such as lipid-based nanoparticles, polymeric carriers, and metallic nanoparticles, provide more efficient delivery and improve therapeutic efficacy, thus overcoming these obstacles. These nanoparticles improve cellular uptake, diminish immune responses, and facilitate targeted delivery overcoming common barriers to CRISPR delivery. We explore several types of nanomaterial to target different CRISPR delivery methods, such as lipid nanoparticles for RNA-based CRISPR systems, polymeric particles to deliver DNA, or protein, for CRISPR delivery and discuss their advantages for improving CRISPR precision and reducing the off-target effect. The controlled release and maximum therapeutic outcome is also explored with hybrid nanomaterials and responsive nanocarriers. We also review current preclinical applications for cancer, cystic fibrosis, and genetic disorders and illustrate their therapeutic potential in disease-specific contexts using nanotechnology-enabled CRISPR systems. In addition to providing safety and regulatory considerations pertinent to clinical translation and ethical questions arising from the convergence of nanotechnology and gene editing, the review takes a critical look at the progress in the field. In the future, we expect that continued advancement in nanomaterial design and CRISPR engineering will help bring this combined approach to clinical viability and enable the development of personalized, targeted therapies not only with increased precision but increased safety as well. This novel approach, pairing CRISPR gene editing with nanotechnology delivery opens the door to this transformative way of tuning gene therapy throughout medicine.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and Cas proteins represent an adaptive microbial immunity system against viruses and plasmids. Cas3 proteins have been proposed to play a key role in the CRISPR mechanism through the direct cleavage of invasive DNA. Here, we show that the Cas3 HD domain protein MJ0384 from Methanocaldococcus jannaschii cleaves endonucleolytically and exonucleolytically (3'-5') single-stranded DNAs and RNAs, as well as 3'-flaps, splayed arms, and R-loops. The degradation of branched DNA substrates by MJ0384 is stimulated by the Cas3 helicase MJ0383 and ATP. The crystal structure of MJ0384 revealed the active site with two bound metal cations and together with site-directed mutagenesis suggested a catalytic mechanism. Our studies suggest that the Cas3 HD nucleases working together with the Cas3 helicases can completely degrade invasive DNAs through the combination of endo- and exonuclease activities.

Genome engineering has made remarkable strides, evolving from DNA-binding proteins such as zinc fingers and transcription activator-like effectors to CRISPR-Cas systems. CRISPR technology has revolutionized the field through its simplicity and ability to target specific genome regions via guide RNA and Cas proteins. Progress in CRISPR tools-CRISPR nucleases, base editors and prime editors-has expanded the toolkit to induce targeted insertions or deletions, nucleotide conversions and a wider array of genetic alterations. Nevertheless, variations in editing outcomes across cell types and unintended off-target effects still present substantial hurdles. Artificial intelligence (AI), which has seen rapid advances, provides high-level solutions to these problems. By leveraging large datasets from diverse experiments, AI enhances guide RNA design, predicts off-target activities and improves editing efficiency. In addition, AI aids in discovering and designing novel CRISPR systems beyond natural limitations. These developments provide new modalities essential for the innovation of personalized therapies and help to ensure efficiency, precision and safety. Here we discuss the transformative role of AI in advancing CRISPR technology. We highlight how AI contributes to refining nuclease-based editing, base editing and prime editing. Integrating AI with CRISPR technology enhances existing tools and opens doors to next-generation medicine for gene therapy.

Werner syndrome (WS) is a genetic premature aging disorder in which patients appear much older than their chronological age. The gene mutated in WS encodes a nuclear protein (WRN) which possesses 3'-5' exonuclease and ATPase-dependent 3'-5' helicase activities. The genomic instability associated with WS cells and the biochemical characteristics of WRN suggest that WRN plays a role in DNA metabolic pathways such as transcription, replication, recombination and repair. Recently we have identified poly(ADP-ribose) polymerase-1 (PARP-1) as a new WRN interacting protein. In this paper, we further mapped the interacting domains. We found that PARP-1 bound to the N-terminus of WRN and to the C-terminus containing the RecQ-conserved (RQC) domain. WRN bound to the N-terminus of PARP-1 containing DNA binding and BRCA1 C-terminal (BRCT) domains. We show that unmodified PARP-1 inhibited both WRN exonuclease and helicase activities, and to our knowledge is the only known WRN protein partner that inactivates both of the WRN's catalytic activities suggesting a biologically significant regulation. Moreover, this dual inhibition seems to be specific for PARP-1, as PARP-2 did not affect WRN helicase activity and only slightly inhibited WRN exonuclease activity. The differential effect of PARP-1 and PARP-2 on WRN catalytic activity was not due to differences in affinity for WRN or the DNA substrate. Finally, we demonstrate that the inhibition of WRN by PARP-1 was influenced by the poly(ADP-ribosyl)ation state of PARP-1. The biological relevance of the specific modulation of WRN catalytic activities by PARP-1 are discussed in the context of pathways in which these proteins may function together, namely in the repair of DNA strand breaks.

The Werner syndrome protein has separable recombination and survival functions

Pharmaceutical Patent AnalystVol. 7, No. 4 CommentaryDisruption and development: the evolving CRISPR patent and technology landscapeDaniel LimDaniel Lim*Author for correspondence: Tel.: +44 20 3088 3769; E-mail Address: Daniel.lim@allenovery.com Allen & Overy LLP, One Bishops Square, London E1 6AD, UKSearch for more papers by this authorPublished Online:8 Jun 2018https://doi.org/10.4155/ppa-2018-0010AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleKeywords: Broad InstituteCas9CRISPREPOEuropegene editinggene therapylicensingpatentspriorityrevocationReferences1 The Broad Institute, Inc., Massachusetts Institute of Technology, President and Fellows of Harvard College: EP2771468 (2015).Google Scholar2 Gaudelli NM, Komor AC, Rees HA et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. 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The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download

CRISPR-Cas systems (Clustered regularly interspaced short palindromic repeats and associated proteins) are adaptive immune systems that protect bacteria and archaea against invading phages. Spacer sequences derived from invader genome are arranged between repeat sequences in the CRISPR array. Upgrading the CRISPR array with new spacers is a crucial step to ensure bacterial systems are at pace with rapidly evolving phages. This is carried out by Cas1 and Cas2 proteins, universally present in most CRISPR systems. Many systems require proteins in addition to Cas1 and Cas2 to maintain the efficiency and fidelity of adaptation. Cas4 is one such protein and is part of the core family of Cas proteins in over half of known CRISPR systems. Cas4 carries a RecB like nuclease domain and initial studies on Cas4 characterized its function as a 5’ to 3’ exonuclease. Recent in-vivo and in-vitro studies have shown that Cas4 plays a role in selection, processing, and integration of functional spacers. Our lab has shown that Cas4 performs endonucleolytic processing of 3’ overhangs of prespacers in the presence of Cas1 and Cas2. Using negative stain EM, we have shown that Cas4 forms a complex with Cas1 and Cas2 in the presence of a potential spacer substrate. However, the exact mechanistic steps in Cas4-Cas1-Cas2 mediated integration remain unknown. Moreover, some systems encode Cas4-1 fusion proteins that provide an increased efficiency of spacer acquisition observed in-vivo. These fusions remain to be characterized biochemically. With this work, we aim to determine the sequence of events in integration in systems that have Cas4 and understand the structure function relationship of Cas4 in fusion with Cas1 and complex with Cas1-Cas2.