Gene targeting in adult organs using in vivo cleavable donor plasmids for CRISPR-Cas9 and CRISPR-Cas12a.

The CRISPR-Cas system for in vivo genome editing is a powerful tool for gene therapy against several diseases. We have previously developed the pCriMGET_9-12a system, an in vivo cleavable donor plasmid for precise targeted knock-in of exogenous DNA by both Cas9 and Cas12a. Here, we show that the pCriMGET_9-12a system can be applied for in vivo in-frame knock-in of exogenous DNA in adult mouse liver by hydrodynamic delivery of the targeting plasmids. The in vivo cleavable pCriMGET_9-12a donor plasmids significantly increased the knock-in efficiency of both CRISPR-Cas9 and CRISPR-Cas12a in the adult mouse liver compared to uncleavable donor plasmids. This strategy also achieved in-frame reporter gene knock-in without indel mutations. Therefore, in vivo gene targeting using the pCriMGET_9-12a system may contribute to the establishment of safer, more precise, versatile and efficient gene therapy methods in adult organs.

Donor template delivery by recombinant adeno-associated virus for the production of knock-in mice.

BACKGROUND: The ability of recombinant adeno-associated virus to transduce preimplantation mouse embryos has led to the use of this delivery method for the production of genetically altered knock-in mice via CRISPR-Cas9. The potential exists for this method to simplify the production and extend the types of alleles that can be generated directly in the zygote, obviating the need for manipulations of the mouse genome via the embryonic stem cell route. RESULTS: We present the production data from a total of 13 genetically altered knock-in mouse models generated using CRISPR-Cas9 electroporation of zygotes and delivery of donor repair templates via transduction with recombinant adeno-associated virus. We explore the efficiency of gene targeting at a total of 12 independent genetic loci and explore the effects of allele complexity and introduce strategies for efficient identification of founder animals. In addition, we investigate the reliability of germline transmission of the engineered allele from founder mice generated using this methodology. By comparing our production data against genetically altered knock-in mice generated via gene targeting in embryonic stem cells and their microinjection into blastocysts, we assess the animal cost of the two methods. CONCLUSIONS: Our results confirm that recombinant adeno-associated virus transduction of zygotes provides a robust and effective delivery route for donor templates for the production of knock-in mice, across a range of insertion sizes (0.9-4.7 kb). We find that the animal cost of this method is considerably less than generating knock-in models via embryonic stem cells and thus constitutes a considerable 3Rs reduction.

Molecular analysis of the role of polymerase theta in gene targeting in Arabidopsis thaliana.

Precise genetic modification can be achieved via a sequence homology-mediated process known as gene targeting (GT). Whilst established for genome engineering purposes, the application of GT in plants still suffers from a low efficiency for which an explanation is currently lacking. Recently reported reduced rates of GT in A. thaliana deficient in polymerase theta (Polθ), a core component of theta-mediated end joining (TMEJ) of DNA breaks, have led to the suggestion of a direct involvement of this enzyme in the homology-directed process. Here, by monitoring homology-driven gene conversion in plants with CRISPR reagent and donor sequences pre-integrated at random sites in the genome (in planta GT), we demonstrate that Polθ action is not required for GT, but instead suppresses the process, likely by promoting the repair of the DNA break by end-joining. This finding indicates that lack of donor integration explains the previously established reduced GT rates seen upon transformation of Polθ-deficient plants. Our study additionally provides insight into ectopic gene targeting (EGT), recombination events between donor and target that do not map to the target locus. EGT, which occurs at similar frequencies as "true" GT during transformation, was rare in our in planta GT experiments arguing that EGT predominantly results from target locus recombination with nonintegrated T-DNA molecules. By describing mechanistic features of GT our study provides directions for the improvement of precise genetic modification of plants.

Deficiency of both classical and alternative end-joining pathways leads to a synergistic defect in double-strand break repair but not to an increase in homology-dependent gene targeting in Arabidopsis.

In eukaryotes, double-strand breaks (DSBs) are either repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). In somatic plant cells, HR is very inefficient. Therefore, the vast majority of DSBs are repaired by two different pathways of NHEJ. The classical (cNHEJ) pathway depends on the heterodimer KU70/KU80, while polymerase theta (POLQ) is central to the alternative (aNHEJ) pathway. Surprisingly, Arabidopsis plants are viable, even when both pathways are impaired. However, they exhibit severe growth retardation and reduced fertility. Analysis of mitotic anaphases indicates that the double mutant is characterized by a dramatic increase in chromosome fragmentation due to defective DSB repair. In contrast to the single mutants, the double mutant was found to be highly sensitive to the DSB-inducing genotoxin bleomycin. Thus, both pathways can complement for each other efficiently in DSB repair. We speculated that in the absence of both NHEJ pathways, HR might be enhanced. This would be especially attractive for gene targeting (GT) in which predefined changes are introduced using a homologous template. Unexpectedly, the polq single mutant as well as the double mutant showed significantly lower GT frequencies in comparison to wildtype plants. Accordingly, we were able to show that elimination of both NHEJ pathways does not pose an attractive approach for Agrobacterium-mediated GT. However, our results clearly indicate that a loss of cNHEJ leads to an increase in GT frequency, which is especially drastic and attractive for practical applications, in which the in planta GT strategy is used.

Application of multiple sgRNAs boosts efficiency of CRISPR/Cas9-mediated gene targeting in Arabidopsis.

BACKGROUND: Precise gene targeting (GT) is a powerful tool for heritable precision genome engineering, enabling knock-in or replacement of the endogenous sequence via homologous recombination. We recently established a CRISPR/Cas9-mediated approach for heritable GT in Arabidopsis thaliana (Arabidopsis) and rice and reported that the double-strand breaks (DSBs) frequency of Cas9 influences the GT efficiency. However, the relationship between DSBs and GT at the same locus was not examined. Furthermore, it has never been investigated whether an increase in the number of copies of sgRNAs or the use of multiple sgRNAs would improve the efficiency of GT. RESULTS: Here, we achieved precise GT at endogenous loci Embryo Defective 2410 (EMB2410) and Repressor of Silencing 1 (ROS1) using the sequential transformation strategy and the combination of sgRNAs. We show that increasing of sgRNAs copy number elevates both DSBs and GT efficiency. On the other hand, application of multiple sgRNAs does not always enhance GT efficiency. Our results also suggested that some inefficient sgRNAs would play a role as a helper to facilitate other sgRNAs DSBs activity. CONCLUSIONS: The results of this study clearly show that DSB efficiency, rather than mutation pattern, is one of the most important key factors determining GT efficiency. This study provides new insights into the relationship between sgRNAs, DSBs, and GTs and the molecular mechanisms of CRISPR/Cas9-mediated GTs in plants.

CRISPR/Cas9 system is a suitable gene targeting editing tool to filamentous fungus Monascus pilosus.

Monascus pilosus has been used to produce lipid-lowering drugs rich in monacolin K (MK) for a long period. Genome mining reveals there are still many potential genes worth to be explored in this fungus. Thereby, efficient genetic manipulation tools will greatly accelerate this progress. In this study, we firstly developed the protocol to prepare protoplasts for recipient of CRISPR/Cas9 system. Subsequently, the vector and donor DNA were co-transformed into recipients (106 protoplasts/mL) to produce 60-80 transformants for one test. Three genes (mpclr4, mpdot1, and mplig4) related to DNA damage response (DDR) were selected to compare the gene replacement frequencies (GRFs) of Agrobacterium tumefaciens-mediated transformation (ATMT) and CRISPR/Cas9 gene editing system (CGES) in M. pilosus MS-1. The results revealed that GRF of CGES was approximately five times greater than that of ATMT, suggesting that CGES was superior to ATMT as a targeting gene editing tool in M. pilosus MS-1. The inactivation of mpclr4 promoted DDR via the non-homologous end-joining (NHEJ) and increased the tolerances to DNA damaging agents. The inactivation of mpdot1 blocked DDR and led to the reduced tolerances to DNA damaging agents. The inactivation of mplig4 mainly blocked the NHEJ pathway and led to obviously reduced tolerances to DNA damaging agents. The submerged fermentation showed that the ability to produce MK in strain Δmpclr4 was improved by 52.6% compared to the wild type. This study provides an idea for more effective exploration of gene functions in Monascus strains. KEY POINTS: • A protocol of high-quality protoplasts for CGES has been developed in M. pilosus. • The GRF of CGES was about five times that of ATMT in M. pilosus. • The yield of MK for Δmpclr4 was enhanced by 52.6% compared with the wild type.

GetPrimers: A generalized PCR-based genetic targeting primer designer enabling easy and standardized targeted gene modification across multiple systems.

Genetic targeting (e.g., gene knockout and tagging) based on polymerase chain reaction (PCR) is a simple yet powerful approach for studying gene functions. Although originally developed in classic budding and fission yeast models, the same principle applies to other eukaryotic systems with efficient homologous recombination. One-step PCR-based genetic targeting is conventionally used but the sizes of the homologous arms that it generates for recombination-mediated genetic targeting are usually limited. Alternatively, gene targeting can also be performed via fusion PCR, which can create homologous arms that are orders of magnitude larger, therefore substantially increasing the efficiency of recombination-mediated genetic targeting. Here, we present GetPrimers (https://www.evomicslab.org/app/getprimers/), a generalized computational framework and web tool to assist automatic targeting and verification primer design for both one-step PCR-based and fusion PCR-based genetic targeting experiments. Moreover, GetPrimers by design runs for any given genetic background of any species with full genome scalability. Therefore, GetPrimers is capable of empowering high-throughput functional genomic assays at multipopulation and multispecies levels. Comprehensive experimental validations have been performed for targeting and verification primers designed by GetPrimers across multiple organism systems and experimental setups. We anticipate GetPrimers to become a highly useful and popular tool to facilitate easy and standardized gene modification across multiple systems.

KnockTF 2.0: a comprehensive gene expression profile database with knockdown/knockout of transcription (co-)factors in multiple species.

Transcription factors (TFs), transcription co-factors (TcoFs) and their target genes perform essential functions in diseases and biological processes. KnockTF 2.0 (http://www.licpathway.net/KnockTF/index.html) aims to provide comprehensive gene expression profile datasets before/after T(co)F knockdown/knockout across multiple tissue/cell types of different species. Compared with KnockTF 1.0, KnockTF 2.0 has the following improvements: (i) Newly added T(co)F knockdown/knockout datasets in mice, Arabidopsis thaliana and Zea mays and also an expanded scale of datasets in humans. Currently, KnockTF 2.0 stores 1468 manually curated RNA-seq and microarray datasets associated with 612 TFs and 172 TcoFs disrupted by different knockdown/knockout techniques, which are 2.5 times larger than those of KnockTF 1.0. (ii) Newly added (epi)genetic annotations for T(co)F target genes in humans and mice, such as super-enhancers, common SNPs, methylation sites and chromatin interactions. (iii) Newly embedded and updated search and analysis tools, including T(co)F Enrichment (GSEA), Pathway Downstream Analysis and Search by Target Gene (BLAST). KnockTF 2.0 is a comprehensive update of KnockTF 1.0, which provides more T(co)F knockdown/knockout datasets and (epi)genetic annotations across multiple species than KnockTF 1.0. KnockTF 2.0 facilitates not only the identification of functional T(co)Fs and target genes but also the investigation of their roles in the physiological and pathological processes.

Mouse Gene-Targeting Strategies for Maximum Ease and Versatility.

Well-planned strategies are an essential prerequisite for any mutational analysis involving gene targeting. Consideration of the advantages or disadvantages of different methods will aid in the production of a final product that is both technically feasible and versatile. Strategies for gene-targeting experiments in the mouse are discussed, including the rationale behind some of the common elements of gene-targeting vectors, such as homologous DNA and the use of different site-specific recombinases. We detail positive and negative selection as well as screening strategies for homologous recombination events in embryonic stem (ES) cells. For the planning stages of making different types of alleles, we first consider general strategies and then provide details specific to either homologous recombination in ES cells or making alleles by gene editing with CRISPR-Cas in preimplantation embryos. The types of alleles considered are null or knockout alleles, reporter gene knock-in alleles, point mutations, and conditional null alleles.

Establishment of a stable transfection and gene targeting system in Babesia divergens.

Babesia divergens is an emerging tick-borne pathogen considered as the principal causative agent of bovine babesiosis in Europe with a notable zoonotic risk to human health. Despite its increasing impact, considerable gaps persist in our understanding of the molecular interactions between this parasite and its hosts. In this study, we address the current limitation of functional genomic tools in B. divergens and introduce a stable transfection system specific to this parasite. We define the parameters for a drug selection system hdhfr-WR99210 and evaluate different transfection protocols for highly efficient generation of transgenic parasites expressing GFP. We proved that plasmid delivery into bovine erythrocytes prior to their infection is the most optimal transfection approach for B. divergens, providing novel evidence of Babesia parasites' ability to spontaneously uptake external DNA from erythrocytes cytoplasm. Furthermore, we validated the bidirectional and symmetrical activity of ef-tgtp promoter, enabling simultaneous expression of external genes. Lastly, we generated a B. divergens knockout line by targeting a 6-cys-e gene locus. The observed dispensability of this gene in intraerythrocytic parasite development makes it a suitable recipient locus for further transgenic application. The platform for genetic manipulations presented herein serves as the initial step towards developing advanced functional genomic tools enabling the discovery of B. divergens molecules involved in host-vector-pathogen interactions.

Skeletal Muscle's Role in Prenatal Inter-organ Communication: A Phenogenomic Study with Qualitative Citation Analysis.

Gene targeting in mice allows for a complete elimination of skeletal (striated or voluntary) musculature in the body, from the beginning of its development, resulting in our ability to study the consequences of this ablation on other organs. Here I focus on the relationship between the muscle and lung, motor neurons, skeleton, and special senses. Since the inception of my independent laboratory, in 2000, with my team, we published more than 30 papers (and a book chapter), nearly 400 pages of data, on these specific relationships. Here I trace, using Web of Science, nearly 600 citations of this work, to understand its impact. The current report contains a summary of our work and its impact, NCBI's Gene Expression Omnibus accession numbers of all our microarray data, and three clear future directions doable by anyone using our publicly available data. Together, this effort furthers our understanding of inter-organ communication during prenatal development.

Unilateral ends-out gene targeting increases mistargeting through supporting extensive single-strand assimilation.

Ends-out gene targeting enables the swapping of endogenous alleles with exogenous ones through homologous recombination which bears great implications both fundamental and applicable. To address the recombination mechanism(s) behind it, an experimental system was designed to distinguish between a possible (but rarely active) unilateral and the expected bilateral targeting in the yeast Saccharomyces cerevisiae in which the proportions of the two alternative genetic outcomes are conceived to mirror the probabilities of the two scenarios. The quantitative analysis showed that the bilateral targeting was expectedly predominant. However, an analogous comparative analysis on a different experimental set suggested a prevalence of unilateral targeting unveiling an uncertainty whether the extensively resected targeting modules only mimic unilateral invasion. Based on this, a comprehensive qualitative analysis was conducted revealing a single basic ends-out gene targeting mechanism composed of two intertwined pathways differing in the way how the homologous invasion is initiated and/or the production of the intermediates is conducted. This study suggests that bilateral targeting lowers mistargeting plausibly by limiting strand assimilation, unlike unilateral targeting which may initiate extensive strand assimilation producing intermediates capable of supporting multiple genetic outcomes which leads to mistargeting. Some of these outcomes can also be produced by mimicking unilateral invasion.

Versatile generation of precise gene edits in bovines using SEGCPN.

BACKGROUND: Gene knockout and knock-in have been widely performed in large farm animals based on genome editing systems. However, many types of precise gene editing, including targeted deletion, gene tagging, and large gene fragment replacement, remain a challenge in large farm animals. RESULTS: Here, we established versatile self-excising gene-targeting technology in combination with programmable nucleases (SEGCPN) to efficiently generate various types of precise gene editing in bovine. First, we used this versatile method to successfully generate bovine embryos with point mutations and 11-bp deletions at the MSTN locus. Second, we successfully generated bulls with EGFP labeling at the SRY locus. Finally, we successfully generated humanized cows in which the endogenous 18-kb α-casein gene was replaced with a 2.6-kb human α-lactalbumin gene. CONCLUSIONS: In summary, our new SEGCPN method offers unlimited possibilities for various types of precise gene editing in large animals for application both in agriculture and disease models.

Efficient PCR-based gene targeting in isolates of the nonconventional yeast Debaryomyces hansenii.

Debaryomyces hansenii is a yeast with considerable biotechnological potential as an osmotolerant, stress-tolerant oleaginous microbe. However, targeted genome modification tools are limited and require a strain with auxotrophic markers. Gene targeting by homologous recombination has been reported to be inefficient, but here we describe a set of reagents and a method that allows gene targeting at high efficiency in wild-type isolates. It uses a simple polymerase chain reaction (PCR)-based amplification that extends a completely heterologous selectable marker with 50 bp flanks identical to the target site in the genome. Transformants integrate the PCR product through homologous recombination at high frequency (>75%). We illustrate the potential of this method by disrupting genes at high efficiency and by expressing a heterologous protein from a safe chromosomal harbour site. These methods should stimulate and facilitate further analysis of D. hansenii strains and open the way to engineer strains for biotechnology.

Shortened CRISPR-Cas9 arrays enable multiplexed gene targeting in bacteria from a smaller DNA footprint.

CRISPR technologies comprising a Cas nuclease and a guide RNA (gRNA) can utilize multiple gRNAs to enact multi-site editing or regulation in the same cell. Nature devised a highly compact means of encoding gRNAs in the form of CRISPR arrays composed of conserved repeats separated by targeting spacers. However, the capacity to acquire new spacers keeps the arrays longer than necessary for CRISPR technologies. Here, we show that CRISPR arrays utilized by the Cas9 nuclease can be shortened without compromising and sometimes even enhancing targeting activity. Using multiplexed gene repression in E. coli, we found that each region could be systematically shortened to varying degrees before severely compromising targeting activity. Surprisingly, shortening some spacers yielded enhanced targeting activity, which was linked to folding of the transcribed array prior to processing. Overall, shortened CRISPR-Cas9 arrays can facilitate multiplexed editing and gene regulation from a smaller DNA footprint across many bacterial applications of CRISPR technologies.

CRISPR/Cas9-Mediated Targeting of BPV-1-Transformed Primary Equine Sarcoid Fibroblasts.

Equine sarcoids (EqS) are fibroblast-derived skin tumors associated with bovine papillomavirus 1 and 2 (BPV-1 and -2). Based on Southern blotting, the BPV-1 genome was not found to be integrated in the host cell genome, suggesting that EqS pathogenesis does not result from insertional mutagenesis. Hence, CRISPR/Cas9 implies an interesting tool for selectively targeting BPV-1 episomes or genetically anchored suspected host factors. To address this in a proof-of-concept study, we confirmed the exclusive episomal persistence of BPV-1 in EqS using targeted locus amplification (TLA). To investigate the CRISPR/Cas9-mediated editing of BPV-1 episomes, primary equine fibroblast cultures were established and characterized. In the EqS fibroblast cultures, CRISPR-mediated targeting of the episomal E5 and E6 oncogenes as well as the BPV-1 long control region was successful and resulted in a pronounced reduction of the BPV-1 load. Moreover, the deletion of the equine Vimentin (VIM), which is highly expressed in EqS, considerably decreased the number of BPV-1 episomes. Our results suggest CRISPR/Cas9-based gene targeting may serve as a tool to help further unravel the biology of EqS pathogenesis.

Scarless Modification of the Drosophila Genome Near Any Mapped attP Sites.

Here we describe a Drosophila genome engineering technique that can scarlessly modify genomic sequences near any mapped attP attachment site previously integrated by transposon mobilization or gene targeting. This technique combines two highly efficient and robust procedures: phiC31 integrase-mediated site-specific integration and homing endonuclease-mediated resolution of local duplications. In this technique, a donor fragment containing the desired mutation(s) is first integrated into a selected attP site near the target locus by phiC31 integrase-mediated site-specific integration, which creates local duplications consisting of the mutant-containing donor fragment and the wild-type target locus. Next, homing endonuclease-induced double-stranded DNA breaks trigger recombination between the duplications and resolve the target locus to generate scarless mutant alleles. In every step, the desired flies can be easily identified by patterns of dominant markers, so no large-scale screens are needed. This technique is highly efficient and can be used to generate scarless point mutations, insertions, and deletions. The availability of large libraries of mapped attP site-containing transposon/CRISPR insertions in Drosophila allows the modification of more than half of the euchromatic Drosophila genome at a high efficiency. As more and more attP-containing insertions are generated and mapped, this technique will be able to modify larger portions of the Drosophila genome. The principles of this technique are applicable to other organisms where modifications to the genome are feasible. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Verifying attP-containing insertions Support Protocol: Extracting genomic DNA Basic Protocol 2: Generating the donor plasmid Basic Protocol 3: Injecting the donor plasmid and establishing transformant stocks Basic Protocol 4: Verifying the transformants Basic Protocol 5: Generating the final scarless alleles Basic Protocol 6: Verifying the final alleles.

PCR-based gene targeting in Hanseniaspora uvarum.

Lack of gene-function analyses tools limits studying the biology of Hanseniaspora uvarum, one of the most abundant yeasts on grapes and in must. We investigated a rapid PCR-based gene targeting approach for one-step gene replacement in this diploid yeast. To this end, we generated and validated two synthetic antibiotic resistance genes, pFA-hygXL and pFA-clnXL, providing resistance against hygromycin and nourseothricin, respectively, for use with H. uvarum. Addition of short flanking-homology regions of 56-80 bp to these selection markers via PCR was sufficient to promote gene targeting. We report here the deletion of the H. uvarum LEU2 and LYS2 genes with these marker genes via two rounds of consecutive transformations, each resulting in the generation of auxotrophic strains (leu2/leu2; lys2/lys2). The hereby constructed leucine auxotrophic leu2/leu2 strain was subsequently complemented in a targeted manner, thereby further validating this approach. PCR-based gene targeting in H. uvarum was less efficient than in Saccharomyces cerevisiae. However, this approach, combined with the availability of two marker genes, provides essential tools for directed gene manipulations in H. uvarum.

Selecting Therapeutic Antisense Oligonucleotides with Gene Targeting and TLR8 Potentiating Bifunctionality.

A growing body of preclinical evidence has led to the hypothesis that human Toll-like receptor 8 (hTLR8) activation in the tumor microenvironment (TME) could have potent anticancer effects through its action on monocytes, myeloid dendritic cells (mDCs), and natural killer (NK) cells. This has motivated the initiation of several clinical trials for chemical hTLR8 agonists in a variety of cancers. Concurrently, a growing number of synthetic antisense oligonucleotides (ASOs) are being developed as cancer therapeutics. We have recently reported that 2'-O-methyl (2'OMe)-modified ASOs can potentiate sensing of hTLR8 chemical agonists in a sequence-dependent manner. This suggests that select gene-targeting ASOs with anticancer activity may synergize with low-dose hTLR8 agonists in the TME. Here, we provide a detailed protocol to rapidly screen and identify such synthetic bifunctional oligonucleotides with synergistic activity on hTLR8 sensing.

CRISPR/Cas9-based split homing gene drive targeting doublesex for population suppression of the global fruit pest Drosophila suzukii.

Genetic-based methods offer environmentally friendly species-specific approaches for control of insect pests. One method, CRISPR homing gene drive that target genes essential for development, could provide very efficient and cost-effective control. While significant progress has been made in developing homing gene drives for mosquito disease vectors, little progress has been made with agricultural insect pests. Here, we report the development and evaluation of split homing drives that target the doublesex (dsx) gene in Drosophila suzukii, an invasive pest of soft-skinned fruits. The drive component, consisting of dsx single guide RNA and DsRed genes, was introduced into the female-specific exon of dsx, which is essential for function in females but not males. However, in most strains, hemizygous females were sterile and produced the male dsx transcript. With a modified homing drive that included an optimal splice acceptor site, hemizygous females from each of the four independent lines were fertile. High transmission rates of the DsRed gene (94 to 99%) were observed with a line that expressed Cas9 with two nuclear localization sequences from the D. suzukii nanos promoter. Mutant alleles of dsx with small in-frame deletions near the Cas9 cut site were not functional and thus would not provide resistance to drive. Finally, mathematical modeling showed that the strains could be used for suppression of lab cage populations of D. suzukii with repeated releases at relatively low release ratios (1:4). Our results indicate that the split CRISPR homing gene drive strains could potentially provide an effective means for control of D. suzukii populations.