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Targeted DNA editing has great potential to cure some genetic diseases; however, the use of artificial nucleases such as CRISPR-Cas9 and TALEN in gene therapy can potentially cause severe side effects due to off-target DNA cleavages. Single-stranded (ss) DNAs and 5'-tailed duplexes (TDs) can achieve target base substitutions when introduced without artificial nucleases into cultured cells and mouse liver. In this study, ss DNA and TD were separately co-introduced into human U2OS cells, together with a target plasmid DNA bearing an inactivated lacZα gene, and the gene correction efficiencies were compared. Unlike the genes examined in previous studies, ss DNA and TD showed similar efficiencies. Therefore, ss DNAs might be as useful as TD for gene correction, depending on the target sequence. [ABSTRACT FROM AUTHOR]

Cpf1-linked base editors broaden the targeting scope of programmable cytidine deaminases by recognizing thymidine-rich protospacer-adjacent motifs (PAM) without inducing DNA double-strand breaks (DSBs). Here we present an unbiased in vitro method for identifying genome-wide off-target sites of Cpf1 base editors via whole genome sequencing. First, we treat human genomic DNA with dLbCpf1-BE ribonucleoprotein (RNP) complexes, which convert C-to-U at on-target and off-target sites and, then, with a mixture of E. coli uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease VIII, which removes uracil and produces single-strand breaks (SSBs) in vitro. Whole-genome sequencing of the resulting digested genome (Digenome-seq) reveals that, on average, dLbCpf1-BE induces 12 SSBs in vitro per crRNA in the human genome. Off-target sites with an editing frequency as low as 0.1% are successfully identified by this modified Digenome-seq method, demonstrating its high sensitivity. dLbCpf1-BEs and LbCpf1 nucleases often recognize different off-target sites, calling for independent analysis of each tool. Cas12a-linked base editors can broaden the targeting scope of programmable cytidine deaminases. Here the authors assess their target specificity in an in vitro genome-wide assay. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is commonly associated with biofilm-related infections and contributes to the large financial loss that accompany nosocomial infections. The micrococcal nuclease Nuc1 enzyme limits biofilm formation via cleavage of eDNA, a structural component of the biofilm matrix. Solid surface hydrophobicity influences bacterial adhesion forces and may as well influence eDNA production. Therefore, it is hypothesized that the impact of Nuc1 activity is dependent on surface characteristics of solid surfaces. For this reason, this study investigated the influence of solid surface hydrophobicity on S. aureus Newman biofilms where Nuc1 is constitutively produced. To this end, biofilms of both a wild-type and a nuc1 knockout mutant strain, grown on glass, salinized glass and Pluronic F-127-coated silanized glass were analysed. Results indicated that biofilms can grow in the presence of Nuc1 activity. Also, Nuc1 and solid surface hydrophobicity significantly affected the biofilm 3D-architecture. In particular, biofilm densities of the wild-type strain on hydrophilic surfaces appeared higher than of the mutant nuc1 knockout strain. Since virulence is related to bacterial cell densities, this suggests that the virulence of S. aureus Newman biofilms is increased by its nuclease production in particular on a hydrophilic surface. [ABSTRACT FROM AUTHOR]

The CRISPR RNA (crRNA)–guided nuclease Cas13 recognizes complementary viral transcripts to trigger the degradation of both host and viral RNA during the type VI CRISPR-Cas antiviral response. However, how viruses can counteract this immunity is not known. We describe a listeriaphage (fLS46) encoding an anti-CRISPR protein (AcrVIA1) that inactivates the type VI-A CRISPR system of Listeria seeligeri. Using genetics, biochemistry, and structural biology, we found that AcrVIA1 interacts with the guide-exposed face of Cas13a, preventing access to the target RNA and the conformational changes required for nuclease activation. Unlike inhibitors of DNA-cleaving Cas nucleases, which cause limited immunosuppression and require multiple infections to bypass CRISPR defenses, a single dose of AcrVIA1 delivered by an individual virion completely dismantles type VI-A CRISPR-mediated immunity. [ABSTRACT FROM AUTHOR]

The inclusion of a nuclease, a new‐to‐laundry enzyme class in detergent formulations, reduces the concentration of recalcitrant soils on dirty clothing. Nucleases target extracellular DNA that, albeit present in minor quantities in soiled laundry items, disproportionately contributes to the accumulation of sebaceous soils and other compounds that affect the appearance of clothes. These lingering soils accumulate over multiple wear cycles and provoke a dingy appearance in clothing, characterized by dull colors, yellow staining, and an unpleasant malodor. The single and continued use of a nuclease in detergent formulations offers increased removal of these recalcitrant soils to produce less malodorous and visually preferred textiles. Furthermore, the nuclease is effective across different detergent forms, as well as in cotton short washing cycles, enabling improved cleaning in more sustainable laundry washing conditions. [ABSTRACT FROM AUTHOR]

The nuclease/helicase DNA2 plays important roles in DNA replication, repair and processing of stalled replication forks. DNA2 contains an iron-sulphur (FeS) cluster, conserved in eukaryotes and in a related bacterial nuclease. FeS clusters in DNA maintenance proteins are required for structural integrity and/or act as redox-sensors. Here, we demonstrate that loss of the FeS cluster affects binding of human DNA2 to specific DNA substrates, likely through a conformational change that distorts the central DNA binding tunnel. Moreover, we show that the FeS cluster is required for DNA2's nuclease, helicase and ATPase activities. Our data also establish that oxidation of DNA2 impairs DNA binding in vitro, an effect that is reversible upon reduction. Unexpectedly, though, this redox-regulation is independent of the presence of the FeS cluster. Together, our study establishes an important structural role for the FeS cluster in human DNA2 and discovers a redox-regulatory mechanism to control DNA binding. Mariotti et al. show that the iron-sulphur cluster in human DNA2 is required for its nuclease, helicase and ATPase activities. This study highlights the structural importance of the iron-sulphur cluster in human DNA2 and presents a separate redox-regulatory mechanism that controls DNA binding. [ABSTRACT FROM AUTHOR]

CRISPR-Cas9 nucleases are powerful genome engineering tools, but unwanted cleavage at off-target and previously edited sites remains a major concern. Numerous strategies to reduce unwanted cleavage have been devised, but all are imperfect. Here, we report that off-target sites can be shielded from the active Cas9•single guide RNA (sgRNA) complex through the co-administration of dead-RNAs (dRNAs), truncated guide RNAs that direct Cas9 binding but not cleavage. dRNAs can effectively suppress a wide-range of off-targets with minimal optimization while preserving on-target editing, and they can be multiplexed to suppress several off-targets simultaneously. dRNAs can be combined with high-specificity Cas9 variants, which often do not eliminate all unwanted editing. Moreover, dRNAs can prevent cleavage of homology-directed repair (HDR)-corrected sites, facilitating scarless editing by eliminating the need for blocking mutations. Thus, we enable precise genome editing by establishing a flexible approach for suppressing unwanted editing of both off-targets and HDR-corrected sites. Numerous strategies exist to limit the off-target activity of CRISPR-Cas9 nucleases. Here the authors co-administer truncated gRNAs that block both Cas9 and high-fidelity Cas9 variants from cleaving at off-target sites. [ABSTRACT FROM AUTHOR]

The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9-gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9-gRNA, SaCas9-gRNA, and FnCas9-gRNA, respectively) and of three engineered SpCas9-gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a "beacon" assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9-gRNA binding to fluorescently labeled target DNA derivatives called "Cas9 beacons." We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9-gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency. [ABSTRACT FROM AUTHOR]

The ribosome biogenesis factor Las1 is an essential endoribonuclease that is well-conserved across eukaryotes and a newly established member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain-containing nuclease family. HEPN nucleases participate in diverse RNA cleavage pathways and share a short HEPN nuclease motif (RΦXXXH) important for RNA cleavage. Most HEPN nucleases participate in stress-activated RNA cleavage pathways; Las1 plays a fundamental role in processing pre-rRNA. Underscoring the significance of Las1 function in the cell, mutations in the human LAS1L (LAS1-like) gene have been associated with neurological dysfunction. Two juxtaposed HEPN nuclease motifs create Las1's composite nuclease active site, but the roles of the individual HEPN motif residues are poorly defined. Here using a combination of in vivo experiments in Saccharomyces cerevisiae and in vitro assays, we show that both HEPN nuclease motifs are required for Las1 nuclease activity and fidelity. Through in-depth sequence analysis and systematic mutagenesis, we determined the consensus HEPN motif in the Las1 subfamily and uncovered its canonical and specialized elements. Using reconstituted Las1 HEPN-HEPN' chimeras, we defined the molecular requirements for RNA cleavage. Intriguingly, both copies of the Las1 HEPN motif were important for nuclease function, revealing that both HEPN motifs participate in coordinating the RNA within the Las1 active site. We also established that conformational flexibility of the two HEPN domains is important for proper nuclease function. The results of our work reveal critical information about how dual HEPN domains come together to drive Las1-mediated RNA cleavage. [ABSTRACT FROM AUTHOR]

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Background: CRISPR/Cas has recently become a widely used genome editing tool in various organisms, including plants. Applying CRISPR/Cas often requires delivering multiple expression units into plant and hence there is a need for a quick and easy cloning procedure. The modular cloning (MoClo), based on the Golden Gate (GG) method, has enabled development of cloning systems with standardised genetic parts, e.g. promoters, coding sequences or terminators, that can be easily interchanged and assembled into expression units, which in their own turn can be further assembled into higher order multigene constructs. Results: Here we present an expanded cloning toolkit that contains 103 modules encoding a variety of CRISPR/Cas-based nucleases and their corresponding guide RNA backbones. Among other components, the toolkit includes a number of promoters that allow expression of CRISPR/Cas nucleases (or any other coding sequences) and their guide RNAs in monocots and dicots. As part of the toolkit, we present a set of modules that enable quick and facile assembly of tRNA-sgRNA polycistronic units without a PCR step involved. We also demonstrate that our tRNA-sgRNA system is functional in wheat protoplasts. Conclusions: We believe the presented CRISPR/Cas toolkit is a great resource that will contribute towards wider adoption of the CRISPR/Cas genome editing technology and modular cloning by researchers across the plant science community. [ABSTRACT FROM AUTHOR]

Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer-adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG that is capable of targeting an expanded set of NGN PAMs, and we further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN and to a lesser extent NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications. [ABSTRACT FROM AUTHOR]

5′‐nucleotidases (5′‐NTs) are enzymes that catalyze the hydrolysis of nucleoside monophosphates to produce nucleosides and phosphate. Since the identification of adenosine synthase A (AdsA) in Staphylococcus aureus in 2009, several other 5′‐NTs have been discovered in Gram‐positive cocci, mainly in streptococci. Despite some differences in substrate specificity, pH range and metal ion requirements, all characterized 5′‐NTs use AMP and ADP, and in some cases ATP, to produce the immunosuppressive adenosine, which dampens pro‐inflammatory immune responses. Several 5′‐NTs are also able to use dAMP as substrate to generate deoxy‐adenosine which is cytotoxic for macrophages. A synergy between 5′‐NTs and exonucleases which are commonly expressed in Gram‐positive cocci has been described, where the nucleases provide dAMP as a cleavage product from DNA. Some of these nucleases produce dAMP by degrading the DNA backbone of neutrophil extracellular traps (NETs) resulting in a "double hit" strategy of immune evasion. This Micro Review provides an overview of the biochemical properties of Gram‐positive cell wall‐anchored 5′‐NTs and their role as virulence factors. A potential use of 5′‐NTs for vaccine development is also briefly discussed. [ABSTRACT FROM AUTHOR]

Bacterial non-specific nucleases are ubiquitously distributed and involved in numerous intra- and extracellular processes. Although all nucleases share the basic chemistry for the hydrolysis of phosphodiester bonds in nucleic acid molecules, the catalysis comprises diverse modes of action, which offers great potential for versatile biotechnological applications. A major criterium for their differentiation is substrate specificity. Specific endonucleases are widely used as restriction enzymes in molecular biology approaches, whereas the main applications of non-specific nucleases (NSNs) are the removal of nucleic acids from crude extracts in industrial downstream processing and the prevention of cell clumping in microfabricated channels. In nature, the predominant role of NSNs is the acquisition of nutrient sources such as nucleotides and phosphates. The number of extensively characterized NSNs and available structures is limited. Moreover, their applicability is mostly challenged by the presence of metal chelators that impede the hydrolysis of nucleic acids in a metal ion–dependent manner. However, a few metal ion–independent NSNs that tolerate the presence of metal chelators have been characterized in recent years with none being commercially available to date. The classification and biotechnological potential of bacterial NSNs with a special focus on metal ion–independent nucleases are presented and discussed. Key Points • Bacterial phospholipases (PLD-family) exhibit nucleolytic activity. • Bacterial nucleases of the PLD-family are metal ion-independent. • NSNs can be used in downstream processing approaches. [ABSTRACT FROM AUTHOR]

Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application. [ABSTRACT FROM AUTHOR]

A range of parasites (cultured promastigotes of Leishmania tropica and L. donovani, adult Fasciola gigantica, protoscoleces of Echinococcus granulosus, adult Hymenolepis nana, Moniezia expansa, Joyeuxiella pasqualei and adult Toxocara cati) have been surveyed for the presence of four phosphatase (nitrophenyl phosphatase, naphtho -1- phosphatase, glycerol -2- phosphatase and glucose -6- phosphatase) one galactosidase, one proteinase, one lipase and two nucleases (ribonuclease and deoxyribonuclease) enzymes. Depending on subcellular fractionation and cytochemical localization the specific common feature was observed including the association of all these enzymes with the lysosomes. It is concluded that these acid hydrolases can be used as lysosomal markers in these parasites. [ABSTRACT FROM AUTHOR]

Cpf1, an endonuclease of the class 2 CRISPR family, fills the gaps that were previously faced in the world of genome engineering tools, which include the TALEN, ZFN, and CRISPR/Cas9. Other simultaneously discovered nucleases were not able to carry out re-engineering at the same region due to the loss of a target site after first-time engineering. Cpf1 acts as a dual nuclease, functioning as an endoribonuclease to process crRNA and endodeoxyribonuclease to cleave target sequences and generate double-stranded breaks. Additionally, Cpf1 allows for multiplexed genome editing, as a single crRNA array transcript can target multiple loci in the genome. The CRISPR/Cpf1 system enables gene deletion, insertion, base editing, and locus tagging in monocot as well as in dicot plants with fewer off-target effects. This tool has been efficiently demonstrated into tobacco, rice, soybean, wheat, etc. This review covers the development and applications of Cpf1 mediated genome editing technology in plants. [ABSTRACT FROM AUTHOR]

Bacterial and archaeal CRISPR-Cas systems provide RNA-guided immunity against genetic invaders such as bacteriophages and plasmids. Upon target RNA recognition, type III CRISPR-Cas systems produce cyclic-oligoadenylate second messengers that activate downstream effectors, including Csm6 ribonucleases, via their CARF domains. Here, we show that Enteroccocus italicus Csm6 (EiCsm6) degrades its cognate cyclic hexa-AMP (cA6) activator, and report the crystal structure of EiCsm6 bound to a cA6 mimic. Our structural, biochemical, and in vivo functional assays reveal how cA6 recognition by the CARF domain activates the Csm6 HEPN domains for collateral RNA degradation, and how CARF domain-mediated cA6 cleavage provides an intrinsic off-switch to limit Csm6 activity in the absence of ring nucleases. These mechanisms facilitate rapid invader clearance and ensure termination of CRISPR interference to limit self-toxicity. Upon target RNA recognition, type III CRISPR-Cas systems produce cyclic oligoadenylates that activate effectors such as Csm6 ribonucleases. Here, Garcia-Doval et al. show that Enteroccocus italicus Csm6 degrades its cyclic hexa-AMP activator, and report the crystal structure of the protein bound to an activator mimic. [ABSTRACT FROM AUTHOR]

Life sciences have been revolutionized by genome editing (GE) tools, including zinc finger nucleases, transcription activator‐Like effector nucleases, and CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas (CRISPR‐associated) systems, which make the targeted modification of genomic DNA of all organisms possible. CRISPR/Cas systems are being widely used because of their accuracy, efficiency, and cost‐effectiveness. Various classes of CRISPR/Cas systems have been developed, but their extensive use may be hindered by off‐target effects. Efforts are being made to reduce the off‐target effects of CRISPR/Cas9 by generating various CRISPR/Cas systems with high fidelity and accuracy. Several approaches have been applied to detect and evaluate the off‐target effects. Here, the current GE tools, the off‐target effects generated by GE technology, types of off‐target effects, mechanisms of off‐target effects, major concerns, and outcomes of off‐target effects in plants and animals are summarized. The methods to detect off‐target effects, tools for single‐guide RNA (sgRNA) design, evaluation and prediction of off‐target effects, and strategies to increase the on‐target efficiency and mitigate the off‐target impact on intended genome‐editing outcomes are summarized. [ABSTRACT FROM AUTHOR]

The CRISPR‐Cas technologies derived from bacterial and archaeal adaptive immune systems have emerged as a series of groundbreaking nucleic acid‐guided gene editing tools, ultimately standing out among several engineered nucleases because of their high efficiency, sequence‐specific targeting, ease of programming and versatility. Facilitated by the advancement across multiple disciplines such as bioinformatics, structural biology and high‐throughput sequencing, the discoveries and engineering of various innovative CRISPR‐Cas systems are rapidly expanding the CRISPR toolbox. This is revolutionizing not only genome editing but also various other types of nucleic acid‐guided manipulations such as transcriptional control and genomic imaging. Meanwhile, the adaptation of various CRISPR strategies in multiple settings has realized numerous previously non‐existing applications, ranging from the introduction of sophisticated approaches in basic research to impactful agricultural and therapeutic applications. Here, we summarize the recent advances of CRISPR technologies and strategies, as well as their impactful applications. [ABSTRACT FROM AUTHOR]

Methodologies of genome editing are rapidly developing with the improvement of gene science and technology, mechanism‐based understanding, and urgent needs. In addition to the specificity and efficiency of on‐target sites, one of the most important issues is to find and avoid off‐targets before clinical application of gene editing as a therapy. Various algorithms, modified nucleases, and delivery vectors are developed to localize and minimize off‐target sites. The present review aimed to clarify off‐targets of various genome editing and explore potentials of clinical application by understanding structures, mechanisms, clinical applications, and off‐target activities of genome editing systems, including CRISPR/Cas9, CRISPR/Cas12a, zinc finger nucleases, transcription activator‐like effector nucleases, meganucleases, and recent developments. Current genome editing in cancer therapy mainly targeted immune systems in tumor microenvironment by ex vivo modification of the immune cells in phases I/II of clinical trials. We believe that genome editing will be the critical part of clinical precision medicine strategy and multidisciplinary therapy strategy by integrating gene sequencing, clinical transomics, and single cell biomedicine. There is an urgent need to develop on/off‐target‐specific biomarkers to monitor the efficacy and side‐effects of gene therapy. Thus, the genome editing will be an alternative of clinical therapies for cancer with the rapid development of methodology and an important part of clinical precision medicine strategy. [ABSTRACT FROM AUTHOR]

Introduction: At present, infections induced by staphylococci, especially methicillin-resistant Staphylococcus aureus (MRSA) are one of key therapeutic and epidemiological problems. Aim: The assessment of Staphylococcus aureus carrier state occurrence among a healthy adult population as well as determination of phenotypic and genotypic properties of the isolated strains. Material and methods: The study included 100 healthy individuals. Material for bacteriological evaluation was collected from the posterior pharyngeal wall and tonsils, nasal vestibule and the skin of anterior nares using a sterile swab. The isolates identified as Staphylococcus aureus were analysed further, towards slime-forming capacity and the presence of genes mecA and nuc. Results: The analysis included 300 samples obtained from the posterior vault of the pharynx and tonsils, nasal vestibule and the skin of nares. Pharyngeal and vestibular S. aureus carriage was determined in 20% of the examined adults, whereas in 11 people with recognized positive throat colonization, the concurrent presence of golden staph was detected in the vestibule of the nose and on the skin, in the nose region. Identification process indicated the occurrence of strains defective in clumping factor synthesis (5% of isolates) and lack of the coa gene (11% of examined isolates). The PCR technique used to screen for the presence of the mec gene, did not confirm it in any of the strains under study. All the isolates had the gene encoding the thermostable nuclease nuc. Conclusions: S. aureus is a pervasive pathogen in community settings with constantly changing trends. [ABSTRACT FROM AUTHOR]

Cell-free DNA (cf. DNA) is a powerful noninvasive biomarker for cancer and prenatal testing, and it circulates in plasma as short fragments. To elucidate the biology of cf. DNA fragmentation, we explored the roles of deoxyribonuclease 1 (DNASE1), deoxyribonuclease 1 like 3 (DNASE1L3), and DNA fragmentation factor subunit beta (DFFB) with mice deficient in each of these nucleases. By analyzing the ends of cf. DNA fragments in each type of nuclease-deficient mice with those in wild-type mice, we show that each nuclease has a specific cutting preference that reveals the stepwise process of cf. DNA fragmentation. Essentially, we demonstrate that cf. DNA is generated first intracellularly with DFFB, intracellular DNASE1L3, and other nucleases. Then, cf. DNA fragmentation continues extracellularly with circulating DNASE1L3 and DNASE1. With the use of heparin to disrupt the nucleosomal structure, we also show that the 10 bp periodicity originates from the cutting of DNA within an intact nucleosomal structure. Altogether, this work establishes a model of cf. DNA fragmentation. [ABSTRACT FROM AUTHOR]

Discovered as an adaptive immune system of prokaryotes, CRISPR-Cas provides many promising applications. DNA-cleaving Cas enzymes like Cas9 and Cas12a, are of great interest for genome editing. The specificity of these DNA nucleases is determined by RNA guides, providing great targeting adaptability. Besides this general method of programmable DNA cleavage, these nucleases have different biochemical characteristics, that can be exploited for different applications. Although Cas nucleases are highly promising, some room for improvement remains. New developments and discoveries like base editing, prime editing, and CRISPR-associated transposons might address some of these challenges. [ABSTRACT FROM AUTHOR]

• Anti-CRISPR proteins (Acrs) allow phages to inhibit CRISPR-Cas immune systems. • Our method uses a cell-free TXTL system to rapidly characterize Acrs. • In the method, Acrs block targeting of a GFP reporter plasmid by a Cas nuclease. • Pre-expressed Acrs, Cas nucleases, and guide RNAs can be frozen and stored. • Diluting the pre-expressed Acr can eliminate non-specific inhibition of the reporter. The characterization of CRISPR-Cas immune systems in bacteria was quickly followed by the discovery of anti-CRISPR proteins (Acrs) in bacteriophages. These proteins block different steps of CRISPR-based immunity and, as some inhibit Cas nucleases, can offer tight control over CRISPR technologies. While Acrs have been identified against a few CRISPR-Cas systems, likely many more await discovery and application. Here, we report a rapid and scalable method for characterizing putative Acrs against Cas nucleases using an E. coli -derived cell-free transcription-translation system. Using known Acrs against type II Cas9 nucleases as models, we demonstrate how the method can be used to measure the inhibitory activity of individual Acrs in under two days. We also show how the method can overcome non-specific inhibition of gene expression observed for some Acrs. In total, the method should accelerate the interrogation and application of Acrs as CRISPR-Cas inhibitors. [ABSTRACT FROM AUTHOR]

• Medium-throughput method for DNA cleavage quantification. • Sensitive quantification of DNA cleavage by CRISPR-Cas. • No need for expensive equipment. Quantifying DNA cleavage by CRISPR-Cas nucleases is usually done by separating the cleaved products from the non-cleaved target by agarose gel electrophoresis. We devised a method that eliminates the quantification from band intensity on agarose gel, and uses a target with a fluorescent dye on the one end and a biotin on the other. Cleavage of the target will separate the dye from the biotin, and cause the dye to stay in solution when streptavidin beads are introduced. All non-cleaved target will be eliminated from solution and no longer contribute to detectable fluorescence. Cleavage will therefore increase the fluorescent signal. A control, which has no streptavidin treatment, is taken along to correct for any errors that might have been introduced by pipetting, inactivation of the fluorescent dye or release of the biotin during several steps of the procedure. With this method we were able to quantify the fraction of active Cas12a in a purification sample and assess the cleavage rate. [ABSTRACT FROM AUTHOR]

• crRNA biogenesis relies on diverse endoribonucleases depending on the system. • Most CRISPR-Cas systems employ dedicated, specialized Cas nucleases such as Cas6. • Some systems use cellular host-encoded endoribonucleases, e.g. RNase III or RNase E. • High throughput techniques and molecular methods allow the identification of crRNAs. • Biochemical and genetic methods are used to characterize crRNA processing nucleases. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins provide an inheritable and adaptive immune system against phages and foreign genetic elements in many bacteria and archaea. The three stages of CRISPR-Cas immunity comprise adaptation, CRISPR RNA (crRNA) biogenesis and interference. The maturation of the pre-crRNA into mature crRNAs, short guide RNAs that target invading nucleic acids, is crucial for the functionality of CRISPR-Cas defense systems. Mature crRNAs assemble with Cas proteins into the ribonucleoprotein (RNP) effector complex and guide the Cas nucleases to the cognate foreign DNA or RNA target. Experimental approaches to characterize these crRNAs, the specific steps toward their maturation and the involved factors, include RNA-seq analyses, enzyme assays, methods such as cryo-electron microscopy, the crystallization of proteins, or UV-induced protein-RNA crosslinking coupled to mass spectrometry analysis. Complex and multiple interactions exist between CRISPR- cas -encoded specific riboendonucleases such as Cas6, Cas5d and Csf5, endonucleases with dual functions in maturation and interference such as the enzymes of the Cas12 and Cas13 families, and nucleases belonging to the cell's degradosome such as RNase E, PNPase and RNase J, both in the maturation as well as in interference. The results of these studies have yielded a picture of unprecedented diversity of sequences, enzymes and biochemical mechanisms. [ABSTRACT FROM AUTHOR]

Genome editing tools have greatly facilitated the functional analysis of genes of interest by targeted mutagenesis. Many usable genome editing tools, including different site-specific nucleases and editor databases that allow single-nucleotide polymorphisms (SNPs) to be introduced at a given site, are now available. These tools can be used to generate high allelic diversity at a given locus to facilitate gene function studies, including examining the role of a specific protein domain or a single amino acid. We compared the effects, efficiencies and mutation types generated by our LbCPF1, SpCAS9 and base editor (BECAS9) constructs for the OsCAO1 gene. SpCAS9 and LbCPF1 have similar efficiencies in generating mutations but differ in the types of mutations induced, with the majority of changes being single-nucleotide insertions and short deletions for SpCAS9 and LbCPF1, respectively. The proportions of heterozygotes also differed, representing a majority in our LbCPF1, while with SpCAS9, we obtained a large number of biallelic mutants. Finally, we demonstrated that it is possible to specifically introduce stop codons using the BECAS9 with an acceptable efficiency of approximately 20%. Based on these results, a rational choice among these three alternatives may be made depending on the type of mutation that one wishes to introduce, the three systems being complementary. SpCAS9 remains the best choice to generate KO mutations in primary transformants, while if the desired gene mutation interferes with regeneration or viability, the use of our LbCPF1 construction will be preferred, because it produces mainly heterozygotes. LbCPF1 has been described in other studies as being as effective as SpCAS9 in generating homozygous and biallelic mutations. It will remain to be clarified in the future, whether the different LbCFP1 constructions have different efficiencies and determine the origin of these differences. Finally, if one wishes to specifically introduce stop codons, BECAS9 is a viable and efficient alternative, although it has a lower efficiency than SpCAS9 and LbCPF1 for creating KO mutations. [ABSTRACT FROM AUTHOR]

Background: Delivery of CRISPR reagents into cells as ribonucleoprotein (RNP) complexes enables transient editing, and avoids CRISPR reagent integration in the genomes. Another technical advantage is that RNP delivery can bypass the need of cloning and vector construction steps. In this work we compared efficacies and types of edits for three Cas9 (WT Cas9 nuclease, HiFi Cas9 nuclease, Cas9 D10A nickase) and two Cas12a nucleases (AsCas12a and LbCas12a), using the rice phytoene desaturase (PDS) gene as a target site. Findings: Delivery of two Cas9 nucleases (WT Cas9, and HiFi Cas9) and one Cas12a nuclease (LbCas12a) resulted in targeted mutagenesis of the PDS gene. LbCas12a had a higher editing efficiency than that of WT Cas9 and HiFi Cas9. Editing by Cas9 enzymes resulted in indels (1–2 bp) or larger deletions between 20-bp to 30-bp, which included the loss of the PAM site; whereas LbCas12a editing resulted in deletions ranging between 2 bp to 20 bp without the loss of the PAM site. Conclusions: In this work, when a single target site of the rice gene OsPDS was evaluated, the LbCas12a RNP complex achieved a higher targeted mutagenesis frequency than the AsCas12a or Cas9 RNPs. [ABSTRACT FROM AUTHOR]

In this study, we evaluated DNase activity of rainbow trout oocyte using an in vitro and in vivo study. First, synthetic single strand and natural double strand DNA from Eukaryotic and prokaryotic sources as well as naked DNA were i n vitro incubated with the oocyte cytoplasm. Results showed that the DNase activity of rainbow trout oocyte is strong enough to degrade any type of DNA at the onset of the incubation. Then, we evaluated if similar to the mammalian species, dead spermatozoa from rainbow trout can protect exogenous DNA from oocyte DNases. A series of dead spermatozoa was incubated with pDB2, carrying EGFP transgene, for 30 min followed by the ooplasm treatment for an additional 30 min. Not only did oocyte DNases completely degrade the exogenous DNA, but also it degraded the compact genome of spermatozoa. In conclusion, in vitro results clearly showed that strong DNase activity of ooplasm could degrade any types of foreign DNAs including oligonucleotides and intensively compact sperm genome. The strong DNase activity of rainbow trout ooplasm could be a stumbling block for genetic modification using plasmids in salmonids. • Strong nuclease activity of ooplasm could degrade any types of foreign DNAs. • Dead spermatozoa cannot protect exogenous DNA from the ooplasm nucleases. • Oplasm nuclease is a hurdle for genetic modification of salmonids. [ABSTRACT FROM AUTHOR]

The ability to create desirable gene variants through targeted changes offers tremendous opportunities for the advancement of basic and applied plant research. Gene editing technologies have opened new avenues to perform such precise gene modifications in diverse biological systems. These technologies use sequence-specific nucleases, such as homing endonucleases, zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (CRISPR/Cas) complexes to enable targeted genetic manipulations. Among these, the CRISPR/Cas system has emerged as a broadly applicable and valued gene editing system for its ease of use and versatility. The adaptability of the CRISPR/Cas system has facilitated rapid and continuous innovative developments to the precision and applications of this technology, since its introduction less than a decade ago. Although developed in animal systems, the simple and elegant CRISPR/Cas gene editing technology has quickly been embraced by plant researchers. From early demonstration in model plants, the CRISPR/Cas system has been successfully adapted for various crop species and enabled targeting of agronomically important traits. Although the approach faces several efficiency and delivery related challenges, especially in recalcitrant crop species, continuous advances in the CRISPR/Cas system to address these limitations are being made. In this review, we discuss the CRISPR/Cas technology, its myriad applications and their prospects for crop improvement. [ABSTRACT FROM AUTHOR]

The past decade has witnessed unprecedented development in genome engineering, a process that enables targeted modification of genomes. The identification of sequence-specific nucleases such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas system, in particular, has led to precise and efficient introduction of genetic variations into genomes of various organisms. Since the CRISPR/Cas system is highly versatile, cost-effective and much superior to ZFNs and TALENs, its widespread adoption by the research community has been inevitable. In plants, a number of studies have shown that CRISPR/Cas could be a potential tool in basic research where insertion, deletion and/or substitution in the genetic sequence could help answer fundamental questions about plant processes, and in applied research these technologies could help build or reverse-engineer plant systems to make them more useful. In this review article, we summarize technologies for precise editing of genomes with a special focus on the CRISPR/Cas system, highlight the latest developments in the CRISPR/Cas system and discuss the challenges and prospects in using the system for plant biology research. [ABSTRACT FROM AUTHOR]

Limited DNA end resection is the key to impaired homologous recombination in BRCA1-mutant cancer cells. Here, using a loss-of-function CRISPR screen, we identify DYNLL1 as an inhibitor of DNA end resection. The loss of DYNLL1 enables DNA end resection and restores homologous recombination in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and inhibitors of poly(ADP-ribose) polymerase. Low BRCA1 expression correlates with increased chromosomal aberrations in primary ovarian carcinomas, and the junction sequences of somatic structural variants indicate diminished homologous recombination. Concurrent decreases in DYNLL1 expression in carcinomas with low BRCA1 expression reduced genomic alterations and increased homology at lesions. In cells, DYNLL1 limits nucleolytic degradation of DNA ends by associating with the DNA end-resection machinery (MRN complex, BLM helicase and DNA2 endonuclease). In vitro, DYNLL1 binds directly to MRE11 to limit its end-resection activity. Therefore, we infer that DYNLL1 is an important anti-resection factor that influences genomic stability and responses to DNA-damaging chemotherapy. DYNLL1 antagonizes end resection of DNA double-strand breaks, thereby inhibiting homologous repair, and the loss of DYNLL1 correlates with poor progression-free survival of patients with BRCA1-mutant ovarian cancer., Author(s): Yizhou Joseph He [sup.1] , Khyati Meghani [sup.1] , Marie-Christine Caron [sup.2] [sup.3] , Chunyu Yang [sup.1] , Daryl A. Ronato [sup.2] [sup.3] , Jie Bian [sup.1] , Anchal [...]

Mutations of the mitochondrial genome (mtDNA) underlie a substantial portion of mitochondrial disease burden. These disorders are currently incurable and effectively untreatable, with heterogeneous penetrance, presentation and prognosis. To address the lack of effective treatment for these disorders, we exploited a recently developed mouse model that recapitulates common molecular features of heteroplasmic mtDNA disease in cardiac tissue: the m.5024C>T tRNA.sup.Ala mouse. Through application of a programmable nuclease therapy approach, using systemically administered, mitochondrially targeted zinc-finger nucleases (mtZFN) delivered by adeno-associated virus, we induced specific elimination of mutant mtDNA across the heart, coupled to a reversion of molecular and biochemical phenotypes. These findings constitute proof of principle that mtDNA heteroplasmy correction using programmable nucleases could provide a therapeutic route for heteroplasmic mitochondrial diseases of diverse genetic origin. Mitochondrially targeted zinc-finger nucleases reduce mutational burden and correct biochemical defects in a mouse model of mitochondrial disease., Author(s): Payam A. Gammage [sup.1] , Carlo Viscomi [sup.1] , Marie-Lune Simard [sup.2] , Ana S. H. Costa [sup.3] , Edoardo Gaude [sup.3] , Christopher A. Powell [sup.1] , Lindsey [...]

Nucleosome positioning is critical to chromatin accessibility and is associated with gene expression programs in cells.sup.1-3. Previous nucleosome mapping methods assemble profiles from cell populations and reveal a cell-averaged pattern: nucleosomes are positioned and form a phased array that surrounds the transcription start sites of active genes.sup.3-6 and DNase I hypersensitive sites.sup.7. However, even in a homogenous population of cells, cells exhibit heterogeneity in expression in response to active signalling.sup.8,9 that may be related to heterogeneity in chromatin accessibility.sup.10-12. Here we report a technique, termed single-cell micrococcal nuclease sequencing (scMNase-seq), that can be used to simultaneously measure genome-wide nucleosome positioning and chromatin accessibility in single cells. Application of scMNase-seq to NIH3T3 cells, mouse primary naive CD4 T cells and mouse embryonic stem cells reveals two principles of nucleosome organization: first, nucleosomes in heterochromatin regions, or that surround the transcription start sites of silent genes, show large variation in positioning across different cells but are highly uniformly spaced along the nucleosome array; and second, nucleosomes that surround the transcription start sites of active genes and DNase I hypersensitive sites show little variation in positioning across different cells but are relatively heterogeneously spaced along the nucleosome array. We found a bimodal distribution of nucleosome spacing at DNase I hypersensitive sites, which corresponds to inaccessible and accessible states and is associated with nucleosome variation and variation in accessibility across cells. Nucleosome variation is smaller within single cells than across cells, and smaller within the same cell type than across cell types. A large fraction of naive CD4 T cells and mouse embryonic stem cells shows depleted nucleosome occupancy at the de novo enhancers detected in their respective differentiated lineages, revealing the existence of cells primed for differentiation to specific lineages in undifferentiated cell populations.Single-cell micrococcal nuclease sequencing simultaneously measures chromatin accessibility and genome-wide nucleosome positioning in single cells to reveal principles of nucleosome organization., Author(s): Binbin Lai [sup.1] , Weiwu Gao [sup.1] [sup.2] , Kairong Cui [sup.1] , Wanli Xie [sup.1] [sup.3] , Qingsong Tang [sup.1] , Wenfei Jin [sup.4] , Gangqing Hu [sup.1] [...]

CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications.sup.1-6 but identifying unwanted off-target mutations is important for clinical translation.sup.7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.A strategy developed to define off-target effects of gene-editing nucleases in whole organisms is validated and leveraged to show that CRISPR-Cas9 nucleases can be used effectively in vivo without inducing detectable off-target mutations., Author(s): Pinar Akcakaya [sup.1] , Maggie L. Bobbin [sup.2] [sup.3] [sup.4] , Jimmy A. Guo [sup.2] [sup.3] , Jose Malagon-Lopez [sup.2] [sup.3] [sup.4] , Kendell Clement [sup.2] [sup.3] [sup.4] , [...]