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Successful genome editing in primary human islets could reveal features of the genetic regulatory landscape underlying β cell function and diabetes risk. Here, we describe a CRISPR-based strategy to interrogate functions of predicted regulatory DNA elements using electroporation of a complex of Cas9 ribonucleoprotein (Cas9 RNP) and guide RNAs into primary human islet cells. We successfully targeted coding regions including the PDX1 exon 1, and non-coding DNA linked to diabetes susceptibility. CRISPR-Cas9 RNP approaches revealed genetic targets of regulation by DNA elements containing candidate diabetes risk SNPs, including an in vivo enhancer of the MPHOSPH9 gene. CRISPR-Cas9 RNP multiplexed targeting of two cis-regulatory elements linked to diabetes risk in PCSK1, which encodes an endoprotease crucial for Insulin processing, also demonstrated efficient simultaneous editing of PCSK1 regulatory elements, resulting in impaired β cell PCSK1 regulation and Insulin secretion. Multiplex CRISPR-Cas9 RNP provides powerful approaches to investigate and elucidate human islet cell gene regulation in health and diabetes.

Summary: In this study, we assessed the quality of de novo genome assemblies for three species of parasitic nematodes (Brugia malayi, Trichuris trichiura, and Ancylostoma caninum) generated using only Oxford Nanopore Technologies MinION data. Assemblies were compared to current reference genomes and against additional assemblies that were supplemented with short-read Illumina data through polishing or hybrid assembly approaches. For each species, assemblies generated using only MinION data had similar or superior measures of contiguity, completeness, and gene content. In terms of gene composition, depending on the species, between 88.9 and 97.6% of complete coding sequences predicted in MinION data only assemblies were identical to those predicted in assemblies polished with Illumina data. Polishing MinION data only assemblies with Illumina data therefore improved gene-level accuracy to a degree. Furthermore, modified DNA extraction and library preparation protocols produced sufficient genomic DNA from B. malayi and T. trichiura to generate de novo assemblies from individual specimens.

Summary: Kaspar Hauser’s parentage has been the subject of research and debate for nearly 200 years. As for his possible aristocratic descent through the House of Baden, there is suspicion that he was swapped as a baby, kidnapped, and kept in isolation to bring a collateral lineage to the throne. In the last 28 years, various genetic analyses have been carried out to investigate this possible aristocratic origin. Previous results using less sensitive Sanger and electrophoresis-based methods were contradictory, and moreover, the authenticity of some samples was disputed, thus leaving the question open. Our analyses using modern capture- and whole genome-based massively parallel sequencing techniques reveal that the mitochondrial DNA haplotypes in different samples attributed to Kaspar Hauser were identical, demonstrating authenticity for the first time, and clearly different from the mitochondrial lineage of the House of Baden, which rules out a maternal relationship and thus the widely believed “Prince theory”.

Summary: Atrial fibrillation (AF) is the most common arrhythmia in the world. Human genetics can provide strong AF therapeutic candidates, but the identification of the causal genes and their functions remains challenging. Here, we applied an AF fine-mapping strategy that leverages results from a previously published cross-ancestry genome-wide association study (GWAS), expression quantitative trait loci (eQTLs) from left atrial appendages (LAAs) obtained from two cohorts with distinct ancestry, and a paired RNA sequencing (RNA-seq) and ATAC sequencing (ATAC-seq) LAA single-nucleus assay (sn-multiome). At nine AF loci, our co-localization and fine-mapping analyses implicated 14 genes. Data integration identified several candidate causal AF variants, including rs7612445 at GNB4 and rs242557 at MAPT. Finally, we showed that the repression of the strongest AF-associated eQTL gene, LINC01629, in human embryonic stem cell-derived cardiomyocytes using CRISPR inhibition results in the dysregulation of pathways linked to genes involved in the development of atrial tissue and the cardiac conduction system.

Summary: It is a significant challenge to assess the functions of many uncharacterized genes in human malaria parasites. Here, we present a genetic screening tool to assess the contribution of essential genes from Plasmodium falciparum by the conditional CRISPR-/deadCas9-based interference and activation (i/a) systems. We screened both CRISPRi and CRISPRa sets, consisting of nine parasite lines per set targeting nine genes via their respective gRNAs. By conducting amplicon sequencing of gRNA loci, we identified the contribution of each targeted gene to parasite fitness upon drug (artemisinin, chloroquine) and stress (starvation, heat shock) treatment. The screening was highly reproducible, and the screening libraries were easily generated by transfection of mixed plasmids expressing different gRNAs. We demonstrated that this screening is straightforward, robust, and can provide a fast and efficient tool to study essential genes that have long presented a bottleneck in assessing their functions using existing genetic tools.

Summary: Chromosomal instability (CIN) is a hallmark of cancers, and CIN-promoting mutations are not fully understood. Here, we report 141 chromosomal instability aiding variant (CIVa) candidates by assessing the prevalence of loss-of-function (LoF) variants in 135 chromosome segregation genes from over 150,000 humans. Unexpectedly, we observe both heterozygous and homozygous CIVa in Astrin and SKA3, two evolutionarily conserved kinetochore and microtubule-associated proteins essential for chromosome segregation. To stratify harmful versus harmless variants, we combine live-cell microscopy and controlled protein expression. We find the naturally occurring Astrin p.Q1012∗ variant is harmful as it fails to localize normally and induces chromosome misalignment and missegregation, in a dominant negative manner. In contrast, the Astrin p.L7Qfs∗21 variant generates a shorter isoform that localizes and functions normally, and the SKA3 p.Q70Kfs∗7 variant allows wild-type SKA complex localisation and function, revealing distinct resilience mechanisms that render these variants harmless. Thus, we present a scalable framework to predict and stratify naturally occurring CIVa, and provide insight into resilience mechanisms that compensate for naturally occurring CIVa.

Summary: Rhizosphere dwelling microorganism such as Bacillus spp. are helpful for crop growth. However, these functions are adversely affected by long-term synthetic fertilizer application. We developed a modified CRISPR/Cas9 system using non-specific single-guide RNAs to disrupt the genome-wide cis-acting catabolite-responsive elements (cres) in a wild-type Bacillus pumilus strain, which conferred dual plant-benefit properties. Most of the mutations occurred around imperfectly matched cis-acting elements (cre-like sites) in genes that are mainly involved in carbon and secondary metabolism pathways. The comparative metabolomics and transcriptome results revealed that carbon is likely transferred to some pigments, such as riboflavin, carotenoid, and lycopene, or non-ribosomal peptides, such as siderophore, surfactin, myxochelin, and bacilysin, through the pentose phosphate and amino acid metabolism pathways. Collectively, these findings suggested that the mutation of global cre-like sequences in the genome might alter carbon flow, thereby allowing beneficial biological interactions between the rhizobacteria and plants.

Summary: Successful genome editing in primary human islets could reveal features of the genetic regulatory landscape underlying β cell function and diabetes risk. Here, we describe a CRISPR-based strategy to interrogate functions of predicted regulatory DNA elements using electroporation of a complex of Cas9 ribonucleoprotein (Cas9 RNP) and guide RNAs into primary human islet cells. We successfully targeted coding regions including the PDX1 exon 1, and non-coding DNA linked to diabetes susceptibility. CRISPR-Cas9 RNP approaches revealed genetic targets of regulation by DNA elements containing candidate diabetes risk SNPs, including an in vivo enhancer of the MPHOSPH9 gene. CRISPR-Cas9 RNP multiplexed targeting of two cis-regulatory elements linked to diabetes risk in PCSK1, which encodes an endoprotease crucial for Insulin processing, also demonstrated efficient simultaneous editing of PCSK1 regulatory elements, resulting in impaired β cell PCSK1 regulation and Insulin secretion. Multiplex CRISPR-Cas9 RNP provides powerful approaches to investigate and elucidate human islet cell gene regulation in health and diabetes.

Summary: There is considerable potential for nuclear genomic material in environmental DNA (eDNA) to inform us of population genetic structure within aquatic species. We tested if nuclear allelic composition data sourced from eDNA can resolve fine scale spatial genetic structure of the cichlid fish Astatotilapia calliptera in Lake Masoko, Tanzania. In this ∼35 m deep crater lake the species is diverging into two genetically distinguishable ecomorphs, separated by a thermo-oxycline at ∼15 m that divides biologically distinct water masses. We quantified population genetic structure along a depth transect using single nucleotide polymorphisms (SNPs) derived from genome sequencing of 530 individuals. This population genetic structure was reflected in a focal set of SNPs that were also reliably amplified from eDNA — with allele frequencies derived from eDNA reflecting those of fish within each depth zone. Thus, by targeting known genetic variation between populations within aquatic eDNA, we measured genetic structure within the focal species.

Summary: Optogenetic manipulation with single-cell resolution can be achieved by two-photon excitation. However, this frequently requires relatively high laser powers. Here, we developed a novel strategy that can improve the efficiency of current two-photon stimulation technologies by positioning fluorescent proteins or small fluorescent molecules with high two-photon cross-sections in the vicinity of opsins. This generates a highly localized source of endogenous single-photon illumination that can be tailored to match the optimal opsin absorbance. Through neuronal and vascular stimulation in the live mouse brain, we demonstrate the utility of this technique to achieve efficient opsin stimulation, without loss of cellular resolution. We also provide a theoretical framework for understanding the potential advantages and constrains of this methodology, with directions for future improvements. Altogether, this fluorescence transfer illumination method opens new possibilities for experiments difficult to implement in the live brain such as all-optical neural interrogation and control of regional cerebral blood flow.

Summary: Neovascular age-related macular degeneration AMD (nAMD) is characterized by choroidal neovascularization (CNV) and could lead to irreversible blindness. However, anti-vascular endothelial growth factor (VEGF) therapy has limited efficacy. Therefore, we generated a chimpanzee adenoviral vector (AdC68-PFC) containing three genes, pigment endothelial-derived factor (PEDF), soluble fms-like tyrosine kinase-1 (sFlt-1), and soluble forms of CD59 (sCD59), to treat nAMD. The results showed that AdC68-PFC mediated a strong onset of PEDF, sFlt-1, and sCD59 expression both in vivo and in vitro. AdC68-PFC showed preventive and therapeutic effects following intravitreal (IVT) injection in the laser-induced CNV model and very low-density lipoprotein receptor-deficient (Vldlr−/−) mouse model. In vitro assessment indicated that AdC68-PFC had a strong inhibitory effect on endothelial cells. Importantly, the safety test showed no evidence of in vivo toxicity of adenovirus in murine eyes. Our findings suggest that AdC68-PFC may be a long-acting and safe gene therapy vector for future nAMD treatments.

Summary: The development of biohydrogen as an alternative energy source has had great economic and environmental benefits. Hydrogen production from microalgae is considered a clean and sustainable energy production method that can both alleviate fuel shortages and recycle waste. Although algal hydrogen production has low energy consumption and requires only simple pretreatment, it has not been commercialized because of low product yields. To increase microalgal biohydrogen production several technologies have been developed, although they struggle with the oxygen sensitivity of the hydrogenases responsible for hydrogen production and the complexity of the metabolic network. In this review, several genetic and metabolic engineering studies on enhancing microalgal biohydrogen production are discussed, and the economic feasibility and future direction of microalgal biohydrogen commercialization are also proposed.

Summary: The human genome comprises approximately 3% of tandem repeats with variable length (VNTR), a few of which have been linked to human rare diseases. Autosomal dominant tubulointerstitial kidney disease—MUC1 (ADTKD-MUC1) is caused by specific frameshift variants in the coding VNTR of the MUC1 gene. Calling variants from VNTR using short-read sequencing (SRS) is challenging due to poor read mappability. We developed a computational pipeline, VNtyper, for reliable detection of MUC1 VNTR pathogenic variants and demonstrated its clinical utility in two distinct cohorts: (1) a historical cohort including 108 families with ADTKD and (2) a replication naive cohort comprising 2,910 patients previously tested on a panel of genes involved in monogenic renal diseases. In the historical cohort all cases known to carry pathogenic MUC1 variants were re-identified, and a new 25bp-frameshift insertion in an additional mislaid family was detected. In the replication cohort, we discovered and validated 30 new patients.

Summary: Hypospadias results from the impaired urethral development, which is influenced by androgens, but its genetic etiology is still unknown. Through whole exome sequencing analysis, we identified NR5A1, SRD5A2, and AR as mutational hotspots in the etiology of severe hypospadias, as these genes are related to androgen signaling. Additionally, rare damaging variants in cilia-related outer dynein arm heavy chain (ODNAH) genes (DNAH5, DNAH8, DNAH9, DNAH11, and DNAH17) (p = 8.5 × 10−47) were significantly enriched in hypospadias cases. The Dnah8 KO mice exhibited significantly decreased testosterone levels, which had an impact on urethral development and disrupted steroid biosynthesis. Combined with trios data, transcriptomic, and phenotypical and proteomic characterization of a mouse model, our work links ciliary genes with hypospadias. Overall, a panel of ODNAH genes with rare damaging variants was identified in 24% of hypospadias patients, providing significant insights into the underlying pathogenesis of hypospadias as well as genetic counseling.

Summary: The pantry moth Plodia interpunctella is a worldwide pest of stored food products and a promising laboratory model system for lepidopteran functional genomics. Here we describe efficient methods for precise genome editing in this insect. A spontaneous recessive white-eyed phenotype maps to a frameshift deletion (c.737delC) in the white gene. CRISPR NHEJ mutagenesis of white replicates this phenotype with high rates of somatic biallelic knockout. G0 individuals with mutant clones on both eyes produced 100% mutant progeny, making white an ideal marker for co-conversion when targeting other genes. CRISPR HDR experiments corrected c.737delC and reverted white eyes to a pigmented state in 37% of G0 mosaic adults. These repaired alleles showed practical rates of germline transmission in backcrosses, demonstrating the potential of the technique for precise genome editing. Plodia offers a promising avenue for research in this taxon because of its lab-ready features, egg injectability, and editability.

Summary: A first-in-human clinical trial of gene therapy in Leber congenital amaurosis due to mutations in the GUCY2D gene is underway, and early results are summarized. A recombinant adeno-associated virus serotype 5 (rAAV5) vector carrying the human GUCY2D gene was delivered by subretinal injection to one eye in three adult patients with severe visual loss, nystagmus, but preserved retinal structure. Safety and efficacy parameters were monitored for 9 months post-operatively. No systemic toxicity was detected; there were no serious adverse events, and ocular adverse events resolved. P1 and P2 showed statistically significant rod photoreceptor vision improvement by full-field stimulus testing in the treated eye. P1 also showed improvement in pupillary responses. Visual acuity remained stable from baseline in P1 and P2. P3, however, showed a gain of 0.3 logMAR in the treated eye, indicating greater cone-photoreceptor function. The results show safety and both rod- and cone-mediated efficacy of this therapy.

Borrelia miyamotoi is an emerging Ixodes tick-borne human pathogen in the Northern hemisphere. The aim of the current study was to compare whole genome sequences of B. miyamotoi isolates from different continents. Using a combination of Illumina and PacBio platforms and a novel genome assembly and plasmid typing pipeline, we reveal that the 21 sequenced B. miyamotoi isolates and publically available B. miyamotoi genomes from North America, Asia, and Europe form genetically distinct populations and cluster according to their geographical origin, where distinct Ixodes species are endemic. We identified 20 linear and 17 circular plasmid types and the presence of specific plasmids for isolates originating from different continents. Linear plasmids lp12, lp23, lp41, and lp72 were core plasmids found in all isolates, with lp41 consistently containing the vmp expression site. Our data provide insights into the genetic basis of vector competence, virulence, and pathogenesis of B. miyamotoi ., Competing Interests: All authors declare no competing interests., (© 2024 The Authors.)

Summary: Secreted chemokines are critical mediators of cellular communication that elicit intracellular signaling by binding membrane-bound receptors. Here we demonstrate the development and use of a sensitive real-time approach to quantify secretion and receptor binding of native chemokines in live cells to better understand their molecular interactions and function. CRISPR/Cas9 genome editing was used to tag the chemokine CXCL12 with the nanoluciferase fragment HiBiT. CXCL12 secretion was subsequently monitored and quantified by luminescence output. Binding of tagged CXCL12 to either chemokine receptors or membrane glycosaminoglycans could be monitored due to the steric constraints of nanoluciferase complementation. Furthermore, binding of native CXCL12-HiBiT to AlexaFluor488-tagged CXCR4 chemokine receptors could also be distinguished from glycosaminoglycan binding and pharmacologically analyzed using BRET. These live cell approaches combine the sensitivity of nanoluciferase with CRISPR/Cas9 genome editing to detect, quantify, and monitor binding of low levels of native secreted proteins in real time.

Summary: An important viromics challenge is associating bacteriophages to hosts. To address this, we developed adsorption sequencing (AdsorpSeq), a readily implementable method to measure phages that are preferentially adsorbed to specific host cell envelopes. AdsorpSeq thus captures the key initial infection cycle step. Phages are added to cell envelopes, adsorbed phages are isolated through gel electrophoresis, after which adsorbed phage DNA is sequenced and compared with the full virome. Here, we show that AdsorpSeq allows for separation of phages based on receptor-adsorbing capabilities. Next, we applied AdsorpSeq to identify phages in a wastewater virome that adsorb to cell envelopes of nine bacteria, including important pathogens. We detected 26 adsorbed phages including common and rare members of the virome, a minority being related to previously characterized phages. We conclude that AdsorpSeq is an effective new tool for rapid characterization of environmental phage adsorption, with a proof-of-principle application to Gram-negative host cell envelopes.

CRISPR/Cas9 technology is revolutionizing the field of gene editing. While this technology enables the targeting of any gene, it may also target unplanned loci, termed off-target sites (OTS), which are a few mismatches, insertions, and deletions from the target. While existing methods for finding OTS up to a given mismatch threshold are efficient, other methods considering insertions and deletions are limited by long runtimes, incomplete OTS lists, and partial support of versatile thresholds. Here, we developed SWOffinder, an efficient method based on Smith-Waterman alignment to find all OTS up to some edit distance. We implemented an original trace-back approach to find OTS under versatile criteria, such as separate limits on the number of insertions, deletions, and mismatches. Compared to state-of-the-art methods, only SWOffinder finds all OTS in the genome in just a few minutes. SWOffinder enables accurate and efficient genomic search of OTS, which will lead to safer gene editing., Competing Interests: The authors declare that they have no competing interests., (© 2023 The Author(s).)

The world faces significant challenges in preserving the diversity of vertebrate species due to wildlife crimes. DNA barcoding, an effective molecular marker for insufficient nuclear DNA, is an authentic and quick identification technique to trace the origin of seized samples in forensic investigations. Here, we present a multiplex assay capable of identifying twenty vertebrate wildlife species utilizing twenty species-specific primers that target short fragments of the mitochondrial Cyt b , COI , 16S rRNA , and 12S rRNA genes. The assay achieved strong species specificity and sensitivity with a detection limit as low as 5 pg of DNA input. Additionally, it effectively discriminated a minor contributor (≥1%) from binary mixtures and successfully identified of noninvasive samples, inhibited DNA samples, artificially degraded DNA samples, and case samples, demonstrating a sensitive, robust, practical and easily interpretable tool in screening, and investigating forensic wildlife crimes., Competing Interests: The authors have a patent related to this work. All other authors declare no competing interests., (© 2023 The Authors.)

Complexity metrics and machine learning (ML) models have been utilized to analyze the lengths of segmental genomic entities of DNA sequences (exonic, intronic, intergenic, repeat, unique) with the purpose to ask questions regarding the segmental organization of the human genome within the size distribution of these sequences. For this we developed an integrated methodology that is based upon the reconstructed phase space theorem, the non-extensive statistical theory of Tsallis, ML techniques, and a technical index, integrating the generated information, which we introduce and named complexity factor (COFA). Our analysis revealed that the size distribution of the genomic regions within chromosomes are not random but follow patterns with characteristic features that have been seen through its complexity character, and it is part of the dynamics of the whole genome. Finally, this picture of dynamics in DNA is recognized using ML tools for clustering, classification, and prediction with high accuracy., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Adeno-associated virus (AAV)-based vectors are considered efficient and safe gene delivery systems in gene therapy. We combined two guide RNA genes, Cas9, and a self-linearizing repair template in one vector (AIO-SL) to correct fumarylacetoacetate hydrolase (FAH) deficiency in mice. The vector genome of 5.73 kb was packaged into VP2-depleted AAV particles (AAV2/8 ΔVP2 ), which, however, did not improve cargo capacity. Reprogrammed hepatocytes were treated with AIO-SL.AAV2 ΔVP2 and subsequently transplanted, resulting in large clusters of FAH-positive hepatocytes. Direct injection of AIO-SL.AAV8 ΔVP2 likewise led to FAH expression and long-term survival. The AIO-SL vector achieved an ∼6-fold higher degree of template integration than vectors without template self-linearization. Subsequent analysis revealed that AAV8 particles, in contrast to AAV2, incorporate oversized genomes distinctly greater than 5.2 kb. Finally, our AAV8-based vector represents a promising tool for gene editing strategies to correct monogenic liver diseases requiring (large) fragment removal and/or simultaneous sequence replacement., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

A major challenge in using human pluripotent stem cells (hPSCs) in therapy is the risk of teratoma formation due to contaminating undifferentiated stem cells. We used CRISPR-Cas9 for in-frame insertion of a suicide gene, iC9, into the endogenous SOX2 locus in human embryonic stem cell (ESC) line H1 for specific eradication of undifferentiated cells without affecting differentiated cells. This locus was chosen over NANOG and OCT4, two other well-characterized stem cell loci, due to significantly reduced off-target effect. We showed that undifferentiated H1-iC9 cells were induced to apoptosis by iC9 inducer AP1903, whereas differentiated cell lineages including hematopoietic cells, neurons, and islet beta-like cells were not affected. We also showed that AP1903 selectively removed undifferentiated H1-iC9 cells from a mixed cell population. This strategy therefore provides a layer of safety control before transplantation of a stem-cell-derived product in therapy., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Binary gene regulatory tools such as the Tetracycline (Tet)-controlled transcription system have revolutionized genetic research in multiple organisms, but their applications to the worm remain very limited. Here we report that the canonical Tet system is largely inactive in the worm but can be adapted for the worm by introducing multiple modifications, a crucial one being the use of the transcription activation domain from the fungal Q binary system. The resultant Tet/Q hybrid system proves more robust and flexible than either of its precursors, enabling elaborate modes of transgene manipulation previously hard to achieve in the worm, including inducible intersectional regulation and, in combination with the Q system, independent control of distinct transgenes within the same cells. Furthermore, we demonstrated, as an example of its applications, that the hybrid system can tightly and efficiently control Cre expression. This study establishes Tet/Q as a premier binary system for worm genetic research., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intra-embryo genome editing by CRISPR/Cas9 enables easy generation of gene-modified animals by non-homologous end joining (NHEJ)-mediated frameshift mutations or homology-directed repair (HDR)-mediated point mutations. However, large modifications, such as gene replacement or gene fusions, are still difficult to introduce in embryos without costly micromanipulators. Moreover, micromanipulation techniques for intra-embryo genome editing have been established in only a small set of animals. To overcome these issues, we developed a method of large-fragment DNA knockin without micromanipulation. In this study, we successfully delivered the knockin donor DNA into zygotes by adeno-associated virus (AAV) without removing the zona pellucida, and we succeeded in both large-DNA fragment knockin and whole exon exchange with electroporation of CRISPR/Cas9 ribonucleoprotein. By this method, we can exchange large DNA fragments conveniently in various animal species without micromanipulation., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The role of maternal and embryonic leucine zipper kinase (MELK) in cancer cell proliferation has been contentious, with recent studies arriving at disparate conclusions. We investigated the in vitro dependency of cancer cells on MELK under a range of assay conditions. Abrogation of MELK expression has little effect under common culture conditions, in which cells are seeded at high densities and reach confluence in 3-5 days. However, MELK dependency becomes clearly apparent in clonogenic growth assays using either RNAi or CRISPR technologies to modulate MELK expression. This dependency is in sharp contrast to that of essential genes, such as those encoding classic mitotic kinases, but is similar to that of other oncogenes including MYC and KRAS. Our study provides an example demonstrating some of the challenges encountered in cancer target validation, and reveals how subtle, but important, technical variations can ultimately lead to divergent outcomes and conclusions., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Solving the structure of DNA in 1953 has unleashed a tour de force in molecular biology that has illuminated how the genetic information stored in DNA is copied and flows downstream into RNA and proteins. Currently, increasingly powerful technologies permit not only reading and writing DNA in vitro but also editing the genetic instructions in cells from virtually any organism. Editing specific genomic sequences in living cells has been particularly accelerated with the introduction of programmable RNA-guided nucleases (RGNs) based on prokaryotic CRISPR adaptive immune systems. The repair of chromosomal breaks made by RGNs with donor DNA patches results in targeted genome editing involving the introduction of specific genetic changes at predefined genomic positions. Hence, donor DNAs, guide RNAs, and nuclease proteins, each representing the molecular entities underlying the storage, transmission, and expression of genetic information, are, once delivered into cells, put to work as agents of change of that very same genetic text. Here, after providing an outline of the programmable nuclease-assisted genome editing field, we review the increasingly diverse range of DNA, RNA, and protein components (e.g., nucleases and "nickases") that, when brought together, underlie RGN-based genome editing in eukaryotic cells., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)