The generation and characterization of a transgenic zebrafish line with lens-specific Cre expression.

Purpose: Danio rerio zebrafish constitute a popular model for studying lens development and congenital cataracts. However, the specific deletion of a gene with a Cre/LoxP system in the zebrafish lens is unavailable because of the lack of a lens-Cre-transgenic zebrafish. This study aimed to generate a transgenic zebrafish line in which Cre recombinase was specifically expressed in the lens. Methods: The pTol2 cryaa:Cre-polyA-cryaa:EGFP (enhanced green fluorescent protein) plasmid was constructed and co-injected with Tol2-transposase into one-to-two-cell-stage wild-type (WT) zebrafish embryos. Whole-mount in situ hybridization (ISH), tissue section, hematoxylin and eosin staining, a Western blot, a split-lamp observation, and a grid transmission assay were used to analyze the Cre expression, lens structure, and lens transparency of the transgenic zebrafish. Results: In this study, we generated a transgenic zebrafish line, zTg(cryaa:Cre-cryaa:EGFP), in which Cre recombinase and EGFP were driven by the lens-specific cryaa promoter. zTg(cryaa:Cre-cryaa:EGFP) began to express Cre and EGFP specifically in the lens at the 22 hpf stage, and this ectopic Cre could efficiently and specifically delete the red fluorescent protein (RFP) signal from the lens when zTg(cryaa:Cre-cryaa:EGFP) embryos were injected with the loxP-flanked RFP plasmid. The overexpression of Cre and EGFP did not impair zebrafish development or lens transparency. Accordingly, this zTg(cryaa:Cre-cryaa:EGFP) zebrafish line is a useful tool for gene editing, specifically with zebrafish lenses. Conclusions: We established a zTg(cryaa:Cre-cryaa:EGFP) zebrafish line that can specifically express an active Cre recombinase in lens tissues. This transgenic zebrafish line can be used as a tool to specifically manipulate a gene in zebrafish lenses.

Belt and braces: Two escape ways to maintain the cassette reservoir of large chromosomal integrons.

Integrons are adaptive devices that capture, stockpile, shuffle and express gene cassettes thereby sampling combinatorial phenotypic diversity. Some integrons called sedentary chromosomal integrons (SCIs) can be massive structures containing hundreds of cassettes. Since most of these cassettes are non-expressed, it is not clear how they remain stable over long evolutionary timescales. Recently, it was found that the experimental inversion of the SCI of Vibrio cholerae led to a dramatic increase of the cassette excision rate associated with a fitness defect. Here, we question the evolutionary sustainability of this apparently counter selected genetic context. Through experimental evolution, we find that the integrase is rapidly inactivated and that the inverted SCI can recover its original orientation by homologous recombination between two insertion sequences (ISs) present in the array. These two outcomes of SCI inversion restore the normal growth and prevent the loss of cassettes, enabling SCIs to retain their roles as reservoirs of functions. These results illustrate a nice interplay between gene orientation, genome rearrangement, bacterial fitness and demonstrate how integrons can benefit from their embedded ISs.

Glycosylation as a tracer of off-target Cre-lox activation in development.

The Cre-lox system is one of the most widely used methods for lineage-specific and inducible genome editing in vivo. However, incomplete penetrance and off-target effects due to transient promoter expression in a stem or pluripotent precursor cell can be problematic and difficult to detect, especially if the target gene is not normally present in the fully differentiated but off-target cells. Yet, the loss of the target gene through the transient expression of Cre may impact the differentiation of those cells by virtue of transient expression in a precursor population. In these situations, off-target effects in an unknown precursor cell can, at best, complicate conclusions drawn from the model, and at worst, invalidate all data generated from that knockout strain. Thus, identifying Cre-driver promoter expression along entire cell lineages is crucial to improve rigor and reproducibility. As an example, transient expression in an early precursor cell has been documented in a variety of Cre strains such as the Tie2-based Cre-driver system that is used as an "endothelial cell-specific" model 1. Yet, Tie2 is now known to be transiently expressed in a stem cell upstream of both hematopoietic and endothelial cell lineages. Here, we use the Tie2 Cre-driver strain to demonstrate that due to its ubiquitous nature, plasma membrane glycans are a useful marker of both penetrance and specificity of a Cre-based knockout.

Albumin promoter-driven FlpO expression induces efficient genetic recombination in mouse liver.

Tissue-specific gene manipulations are widely used in genetically engineered mouse models. A single recombinase system, such as the one using Alb-Cre, has been commonly used for liver-specific genetic manipulations. However, most diseases are complex, involving multiple genetic changes and various cell types. A dual recombinase system is required for conditionally modifying different genes sequentially in the same cell or inducing genetic changes in different cell types within the same organism. A FlpO cDNA was inserted between the last exon and 3'-UTR of the mouse albumin gene in a bacterial artificial chromosome (BAC-Alb-FlpO). The founders were crossed with various reporter mice to examine the efficiency of recombination. Liver cancer tumorigenesis was investigated by crossing the FlpO mice with FSF-KrasG12D mice and p53frt mice (KPF mice). BAC-Alb-FlpO mice exhibited highly efficient recombination capability in both hepatocytes and intrahepatic cholangiocytes. No recombination was observed in the duodenum and pancreatic cells. BAC-Alb-FlpO-mediated liver-specific expression of mutant KrasG12D and conditional deletion of p53 gene caused the development of liver cancer. Remarkably, liver cancer in these KPF mice manifested a distinctive mixed hepatocellular carcinoma and cholangiocarcinoma phenotype. A highly efficient and liver-specific BAC-Alb-FlpO mouse model was developed. In combination with other Cre lines, different genes can be manipulated sequentially in the same cell, or distinct genetic changes can be induced in different cell types of the same organism.NEW & NOTEWORTHY A liver-specific Alb-FlpO mouse line was generated. By coupling it with other existing CreERT or Cre lines, the dual recombinase approach can enable sequential gene modifications within the same cell or across various cell types in an organism for liver research through temporal and spatial gene manipulations.

Expanding the DNA editing toolbox: Novel lambda integrase variants targeting microalgal and human genome sequences.

Recombinase enzymes are extremely efficient at integrating very large DNA fragments into target genomes. However, intrinsic sequence specificities curtail their use to DNA sequences with sufficient homology to endogenous target motifs. Extensive engineering is therefore required to broaden applicability and robustness. Here, we describe the directed evolution of novel lambda integrase variants capable of editing exogenous target sequences identified in the diatom Phaeodactylum tricornutum and the algae Nannochloropsis oceanica. These microorganisms hold great promise as conduits for green biomanufacturing and carbon sequestration. The evolved enzyme variants show >1000-fold switch in specificity towards the non-natural target sites when assayed in vitro. A single-copy target motif in the human genome with homology to the Nannochloropsis oceanica site can also be efficiently targeted using an engineered integrase, both in vitro and in human cells. The developed integrase variants represent useful additions to the DNA editing toolbox, with particular application for targeted genomic insertion of large DNA cargos.

Orthogonal LoxPsym sites allow multiplexed site-specific recombination in prokaryotic and eukaryotic hosts.

Site-specific recombinases such as the Cre-LoxP system are routinely used for genome engineering in both prokaryotes and eukaryotes. Importantly, recombinases complement the CRISPR-Cas toolbox and provide the additional benefit of high-efficiency DNA editing without generating toxic DNA double-strand breaks, allowing multiple recombination events at the same time. However, only a handful of independent, orthogonal recombination systems are available, limiting their use in more complex applications that require multiple specific recombination events, such as metabolic engineering and genetic circuits. To address this shortcoming, we develop 63 symmetrical LoxP variants and test 1192 pairwise combinations to determine their cross-reactivity and specificity upon Cre activation. Ultimately, we establish a set of 16 orthogonal LoxPsym variants and demonstrate their use for multiplexed genome engineering in both prokaryotes (E. coli) and eukaryotes (S. cerevisiae and Z. mays). Together, this work yields a significant expansion of the Cre-LoxP toolbox for genome editing, metabolic engineering and other controlled recombination events, and provides insights into the Cre-LoxP recombination process.

The effect of site-specific recombinases XerCD on the removal of over-replicated chromosomal DNA through outer membrane vesicles in bacteria.

Outer membrane vesicles (OMVs) are universally produced by Gram-negative bacteria and play important roles in symbiotic and pathogenic interactions. The DNA from the lumen of OMVs from the Alphaproteobacterium Dinoroseobacter shibae was previously shown to be enriched for the region around the terminus of replication ter and specifically for the recognition sequence dif of the two site-specific recombinases XerCD. These enzymes are highly conserved in bacteria and play an important role in the last phase of cell division. Here, we show that a similar enrichment of ter and dif is found in the DNA inside OMVs from Prochlorococcus marinus, Pseudomonas aeruginosa, Vibrio cholerae, and Escherichia coli. The deletion of xerC or xerD in E. coli reduced the enrichment peak directly at the dif sequence, while the enriched DNA region around ter became broader, demonstrating that either enzyme influences the DNA content inside the lumen of OMVs. We propose that the intra-vesicle DNA originated from over-replication repair and the XerCD enzymes might play a role in this process, providing them with a new function in addition to resolving chromosome dimers.IMPORTANCEImprecise termination of replication can lead to over-replicated parts of bacterial chromosomes that have to be excised and removed from the dividing cell. The underlying mechanism is poorly understood. Our data show that outer membrane vesicles (OMVs) from diverse Gram-negative bacteria are enriched for DNA around the terminus of replication ter and the site-specific XerCD recombinases influence this enrichment. Clearing the divisome from over-replicated parts of the bacterial chromosome might be a so far unrecognized and conserved function of OMVs.

A Novel Cre Recombinase Mouse Strain for Cell-Specific Deletion of Floxed Genes in Ribbon Synapse-Forming Retinal Neurons.

We generated a novel Cre mouse strain for cell-specific deletion of floxed genes in ribbon synapse-forming retinal neurons. Previous studies have shown that the RIBEYE promotor targets the expression of recombinant proteins such as fluorescently tagged RIBEYE to photoreceptors and retinal bipolar cells and generates fluorescent synaptic ribbons in situ in these neurons. Here, we used the same promotor to generate a novel transgenic mouse strain in which the RIBEYE promotor controls the expression of a Cre-ER(T2) recombinase (RIBEYE-Cre). To visualize Cre expression, the RIBEYE-Cre animals were crossed with ROSA26 tau-GFP (R26-τGFP) reporter mice. In the resulting RIBEYE-Cre/R26 τGFP animals, Cre-mediated removal of a transcriptional STOP cassette results in the expression of green fluorescent tau protein (tau-GFP) that binds to cellular microtubules. We detected robust tau-GFP expression in retinal bipolar cells. Surprisingly, we did not find fluorescent tau-GFP expression in mouse photoreceptors. The lack of tau-GFP reporter protein in these cells could be based on the previously reported absence of tau protein in mouse photoreceptors which could lead to the degradation of the recombinant tau protein. Consistent with this, we detected Cre and tau-GFP mRNA in mouse photoreceptor slices by RT-PCR. The transgenic RIBEYE-Cre mouse strain provides a new tool to study the deletion of floxed genes in ribbon synapse-forming neurons of the retina and will also allow for analyzing gene deletions that are lethal if globally deleted in neurons.

Bacteriophage tRNA-dependent lysogeny: requirement of phage-encoded tRNA genes for establishment of lysogeny.

Bacteriophages are large and diverse components of the biosphere, and many phages are temperate. Upon infection, temperate phages can establish lysogeny in which a prophage is typically integrated into the bacterial chromosome. Here, we describe the phenomenon of tRNA-dependent lysogeny, a previously unrecognized behavior of some temperate phages. tRNA-dependent lysogeny is characterized by two unusual features. First, a phage-encoded tyrosine family integrase mediates site-specific recombination between a phage attP site and a bacterial attB site overlapping a host tRNA gene. However, attP and attB share only a short (~10 bp) common core such that a functional tRNA is not reconstructed upon integration. Second, the phage encodes a tRNA of the same isotype as the disrupted but essential host tRNA, complementing its loss, and consequently is required for the survival of lysogenic progeny. As expected, an integrase-defective phage mutant forms turbid plaques, and bacterial progeny are immune to superinfection, but they lack stability, and the prophage is rapidly lost. In contrast, a tRNA-defective phage mutant forms clear plaques and more closely resembles a repressor mutant, and lysogens are recovered only at very low frequency through the use of secondary attachment sites elsewhere in the host genome. Integration-proficient plasmids derived from these phages must also carry a cognate phage tRNA gene for efficient integration, and these may be useful tools for mycobacterial genetics. We show that tRNA-dependent lysogeny is used by phages within multiple different groups of related viruses and may be prevalent elsewhere in the broader phage community.IMPORTANCEBacteriophages are the most numerous biological entities in the biosphere, and a substantial proportion of phages are temperate, forming stable lysogens in which a prophage copy of the genome integrates into the bacterial chromosome. Many phages encode a variety of tRNA genes whose roles are poorly understood, although it has been proposed that they enhance translational efficiencies in lytic growth or that they counteract host defenses that degrade host tRNAs. Here, we show that phage-encoded tRNAs play key roles in the establishment of lysogeny of some temperate phages. They do so by compensating for the loss of tRNA function when phages integrate at an attB site overlapping a tRNA gene but fail to reconstruct the tRNA at the attachment junction. In this system of tRNA-dependent lysogeny, the phage-encoded tRNA is required for lysogeny, and deletion of the phage tRNA gives rise to a clear plaque phenotype and obligate lytic growth.

Integron cassettes integrate into bacterial genomes via widespread non-classical attG sites.

Integrons are genetic elements involved in bacterial adaptation which capture, shuffle and express genes encoding adaptive functions embedded in cassettes. These events are governed by the integron integrase through site-specific recombination between attC and attI integron sites. Using computational and molecular genetic approaches, here we demonstrate that the integrase also catalyses cassette integration into bacterial genomes outside of its known att sites. Once integrated, these cassettes can be expressed if located near bacterial promoters and can be excised at the integration point or outside, inducing chromosomal modifications in the latter case. Analysis of more than 5 × 105 independent integration events revealed a very large genomic integration landscape. We identified consensus recombination sequences, named attG sites, which differ greatly in sequence and structure from classical att sites. These results unveil an alternative route for dissemination of adaptive functions in bacteria and expand the role of integrons in bacterial evolution.

Transfer and accumulation of antibiotic resistance genes and bacterial pathogens in the mice gut due to consumption of organic foods.

Over the last few decades, organic food demand has grown largely because of increasing personal health concerns. Organic farming introduces antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) into foods. However, potential effects of organic foods on the gut microbiome and ARGs have been overlooked. Using high-throughput quantitative PCR and 16S rRNA high-throughput sequencing technology, we examined 132 ARGs from major classes, eight transposase genes, universal class I integron-integrase gene (intI), clinical class I integron-integrase gene (cintI), and the bacterial community in mouse gut after 8 weeks with an either organic or inorganic lettuce and wheat diet. A total of 8 types of major ARGs and 10 mobile genetic elements (MGEs) were detected in mice gut, including tetracycline, multidrug, sulfonamide, aminoglycoside, beta-lactamase, chloramphenicol, MLSB and vancomycin resistance genes. We found that abundance and diversity of ARGs, mobile gene elements, and potential ARB in the gut increased with time after consumption of organic foods, whereas no significant changes were observed in inorganic treated groups. Moreover, MGEs, including IS613, Tp614 and tnpA_03 were found to play an important role in regulating ARG profiles in the gut microbiome following consumption of organic foods. Importantly, feeding organic food increased the relative abundance of the potentially antibiotic-resistant pathogens, Bacteroides and Streptococcus. Our results confirm that there is an increasing risk of ARGs and ARB in the gut microbiome, which highlights the importance of organic food industries taking into account the potential accumulation and transmission of ARGs as a risk factor.

Heterologous production of 3-hydroxypropionic acid in Methylorubrum extorquens by introducing the mcr gene via a multi-round chromosomal integration system based on cre-lox71/lox66 and transposon.

BACKGROUND AND AIM: Reprogramming microorganisms to enhance the production of metabolites is a part of contemporary synthetic biology, which relies on the availability of genetic tools to successfully manipulate the bacteria. Methylorubrum extorquens AM1 is a platform microorganism used to convert C1 compounds into various value-added products. However, the repertoire of available plasmids to conveniently and quickly fine-tune the expression of multiple genes in this strain is extremely limited compared with other model microorganisms such as Escherichia coli. Thus, this study aimed to integrate existing technologies, such as transposon-mediated chromosomal integration and cre-lox-mediated recombination, to achieve the diversified expression of target genes through multiple chromosomal insertions in M. extorquens AM1. RESULTS: A single plasmid toolkit, pSL-TP-cre-km, containing a miniHimar1 transposon and an inducible cre-lox71/lox66 system, was constructed and characterized for its multiple chromosomal integration capacity. A co-transcribed mcr-egfp cassette [for the production of 3-hydroxypropionic acid (3-HP) and a reporting green fluorescent protein] was added to construct pTP-cre-mcr-egfp for evaluating its utility in mediating the expression of heterologous genes, resulting in the production of 3-HP with a titer of 34.7-55.2 mg/L by two chromosomal integration copies. Furthermore, in association with the expression of plasmid-based mcr, 3-HP production increased to 65.5-92.4 mg/L. CONCLUSIONS: This study used a multi-round chromosomal integration system based on cre-lox71/lox66 and a transposon to construct a single constructed vector. A heterologous mcr gene was introduced through this vector, and high expression of 3-hydroxypropionic acid was achieved in M. extorquens. This study provided an efficient genetic tool for manipulating M. extorquens, which not only help increase the expression of heterologous genes in M. extorquens but also provide a reference for strains lacking genetic manipulation vectors.

Viral vector-mediated transgene delivery with novel recombinase systems for targeting neuronal populations defined by multiple features.

A comprehensive understanding of neuronal diversity and connectivity is essential for understanding the anatomical and cellular mechanisms that underlie functional contributions. With the advent of single-cell analysis, growing information regarding molecular profiles leads to the identification of more heterogeneous cell types. Therefore, the need for additional orthogonal recombinase systems is increasingly apparent, as heterogeneous tissues can be further partitioned into increasing numbers of specific cell types defined by multiple features. Critically, new recombinase systems should work together with pre-existing systems without cross-reactivity in vivo. Here, we introduce novel site-specific recombinase systems based on ΦC31 bacteriophage recombinase for labeling multiple cell types simultaneously and a novel viral strategy for versatile and robust intersectional expression of any transgene. Together, our system will help researchers specifically target different cell types with multiple features in the same animal.

Harnessing CRISPR-Cas adaptation for RNA recording and beyond.

Prokaryotes encode clustered regularly interspaced short palindromic repeat (CRISPR) arrays and CRISPR-associated (Cas) genes as an adaptive immune machinery. CRISPR-Cas systems effectively protect hosts from the invasion of foreign enemies, such as bacteriophages and plasmids. During a process called 'adaptation', non-self-nucleic acid fragments are acquired as spacers between repeats in the host CRISPR array, to establish immunological memory. The highly conserved Cas1-Cas2 complexes function as molecular recorders to integrate spacers in a time course manner, which can subsequently be expressed as crRNAs complexed with Cas effector proteins for the RNAguided interference pathways. In some of the RNA-targeting type III systems, Cas1 proteins are fused with reverse transcriptase (RT), indicating that RT-Cas1-Cas2 complexes can acquire RNA transcripts for spacer acquisition. In this review, we summarize current studies that focus on the molecular structure and function of the RT-fused Cas1-Cas2 integrase, and its potential applications as a directional RNA-recording tool in cells. Furthermore, we highlight outstanding questions for RT-Cas1-Cas2 studies and future directions for RNA-recording CRISPR technologies. [BMB Reports 2024; 57(1): 40-49].

Development of a Cre-recombination-based color-switching reporter system for cell fusion detection.

Cell fusion plays a key role in the development and formation of tissues and organs in several organisms. Skeletal myogenesis is assessed in vitro by cell shape and gene and protein expression using immunofluorescence and immunoblotting assays. However, these conventional methods are complex and do not allow for easy time-course observation in living cells. Therefore, this study aimed to develop a Cre recombination-based fluorescent reporter system to monitor cell-cell fusion. We combined green and red fluorescent proteins with a Cre-loxP system to detect syncytium formation using a fluorescent binary switch. This allowed us to visualize mononucleated cells with green fluorescence before fusion and multinucleated syncytia with red fluorescence by conditional expression after cell fusion. The formation of multinuclear myotubes during myogenic differentiation was detected by the change in fluorescence from green to red after Cre-mediated recombination. The distribution of the fluorescence signal correlated with the expression of myogenic differentiation markers. Moreover, red reporter fluorescence intensity was correlated with the number of nuclei contained in the red fluorescent-positive myotubes. We also successfully demonstrated that our fusion monitoring system is applicable to the formation of skeletal muscle myotube and placental syncytiotrophoblast. These results suggest that the color-switching fluorescent reporter system, using Cre-mediated recombination, could be a robust tool used to facilitate the study of cell-to-cell fusion.

Variegate expression of Cre recombinase in hematopoietic cells in CD11c-cre transgenic mice.

In this study, we performed an in-depth analysis of Cre expression in the widely used CD11c-Cre transgenic mice generated by the group of Boris Reizis. In contrast to previous observation, using the highly sensitive Rosa-26-floxed-tdTomato reporter mouse line, we show variegated expression of Cre in multiple hematopoietic linage cells starting in hematopoietic stem cells. Indeed, we found that in the CD11c-Cre driver mice, Cre is expressed in cDC linage cells and pDC starting from the myeloid dendritic cell precursor, as expected, but also in a substantial fraction of hematopoietic stem cells and common lymphoid progenitors and, consequently, in >50% of all leukocytes. Hence, this study indicates that the reporter mice used to characterize Cre expression in Cre-driver mice should be selected with caution and considering the sensitivity of the reporter system. This study also suggests that the interpretation of some reports using this CD11c-Cre transgenic mice may need to be re-considered based on a careful evaluation of the cell type-specificity of Cre-mediated in their model.

The Prl3d1-Cre mouse line selectively induces the expression of Cre recombinase in parietal trophoblast giant cells.

The placenta plays a pivotal role in the maintenance of normal pregnancy, but how it forms, matures, and performs its function remains poorly understood. Here, we describe a novel mouse line (Prl3d1-iCre) that expresses iCre recombinase under the control of the endogenous prl3d1 promoter. Prl3d1 has been proposed as a marker for distinguishing trophoblast giant cells (TGCs) from other trophoblast cells in the placenta. The in vivo efficiency and specificity of the Cre line were analyzed by interbreeding Prl3d1-iCre mice with B6-G/R reporter mice. Through anatomical studies of the placenta and other tissues of Prl3d1-iCre/+; B6-G/R mouse mice, we found that the tdTomato signal was expressed in parietal trophoblast giant cells (P-TGCs). Thus, we report a mouse line with ectopic Cre expression in P-TGCs, which provides a valuable tool for studying human pathological pregnancies caused by implantation failure or abnormal trophoblast secretion due to aberrant gene regulation.

Towards a Light-mediated Gene Therapy for the Eye using Caged Ethinylestradiol and the Inducible Cre/lox System.

Increasingly, retinal pathologies are being treated with virus-mediated gene therapies. To be able to target viral transgene expression specifically to the pathological regions of the retina with light, we established an in vivo photoactivated gene expression paradigm for retinal tissue. Based on the inducible Cre/lox system, we discovered that ethinylestradiol is a suitable alternative to Tamoxifen as ethinylestradiol is more amenable to modification with photosensitive protecting compounds, i.e., "caging." Identification of ethinylestradiol as a ligand for the mutated human estradiol receptor was supported by in silico binding studies showing the reduced binding of caged ethinylestradiol. Caged ethinylestradiol was injected into the eyes of double transgenic GFAP-CreERT2 mice with a Cre-dependent tdTomato reporter transgene followed by irradiation with light of 450 nm. Photoactivation significantly increased retinal tdTomato expression compared to controls. We thus demonstrated a first step towards the development of a targeted, light-mediated gene therapy for the eyes.

Optically Controlled CRISPR-Cas9 and Cre Recombinase for Spatiotemporal Gene Editing: A Review.

CRISPR-Cas9 and Cre recombinase, two tools extensively used for genome interrogation, have catalyzed key breakthroughs in our understanding of complex biological processes and diseases. However, the immense complexity of biological systems and off-target effects hinder clinical applications, necessitating the development of platforms to control gene editing over spatial and temporal dimensions. Among the strategies developed for inducible control, light is particularly attractive as it is noninvasive and affords high spatiotemporal resolution. The principles for optical control of Cas9 and Cre recombinase are broadly similar and involve photocaged enzymes and small molecules, engineered split- and single-chain constructs, light-induced expression, and delivery by light-responsive nanocarriers. Few systems enable spatiotemporal control with a high dynamic range without loss of wild-type editing efficiencies. Such systems posit the promise of light-activatable systems in the clinic. While the prospect of clinical applications is palpably exciting, optimization and extensive preclinical validation are warranted. Judicious integration of optically activated CRISPR and Cre, tailored for the desired application, may help to bridge the "bench-to-bedside" gap in therapeutic gene editing.

Transmitted drug resistance to antiretroviral drugs in Spain during the period 2019-2021.

To evaluate the prevalence of transmitted drug resistance (TDR) to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTI, NNRTI), protease inhibitors (PI), and integrase strand transfer inhibitors (INSTI) in Spain during the period 2019-2021, as well as to evaluate transmitted clinically relevant resistance (TCRR) to antiretroviral drugs. Reverse transcriptase (RT), protease (Pro), and Integrase (IN) sequences from 1824 PLWH (people living with HIV) were studied. To evaluate TDR we investigated the prevalence of surveillance drug resistance mutations (SDRM). To evaluate TCRR (any resistance level ≥ 3), and for HIV subtyping we used the Stanford v.9.4.1 HIVDB Algorithm and an in-depth phylogenetic analysis. The prevalence of NRTI SDRMs was 3.8% (95% CI, 2.8%-4.6%), 6.1% (95% CI, 5.0%-7.3%) for NNRTI, 0.9% (95% CI, 0.5%-1.4%) for PI, and 0.2% (95% CI, 0.0%-0.9%) for INSTI. The prevalence of TCRR to NRTI was 2.1% (95% CI, 1.5%-2.9%), 11.8% for NNRTI, (95% CI, 10.3%-13.5%), 0.2% (95% CI, 0.1%-0.6%) for PI, and 2.5% (95% CI, 1.5%-4.1%) for INSTI. Most of the patients were infected by subtype B (79.8%), while the majority of non-Bs were CRF02_AG (n = 109, 6%). The prevalence of INSTI and PI resistance in Spain during the period 2019-2021 is low, while NRTI resistance is moderate, and NNRTI resistance is the highest. Our results support the use of integrase inhibitors as first-line treatment in Spain. Our findings highlight the importance of ongoing surveillance of TDR to antiretroviral drugs in PLWH particularly with regard to first-line antiretroviral therapy.