Homing of a group II intron from Lactococcus lactis subsp. lactis ML3.

Ll.ltrB is a functional group II intron located within a gene (ltrB) encoding a conjugative relaxase essential for transfer of the lactococcal element pRSO1. In this work, the Ll.ltrB intron was shown to be an independent mobile element capable of inserting into an intronless allele of the ltrB gene. Ll.ltrB was not observed to insert into a deletion derivative of the ltrB gene in which the intron splice site was removed. In contrast, a second vector containing a 271-nucleotide segment of ltrB spanning the Ll.ltrB splice site was shown to be a proficient recipient of intron insertion. Efficient homing was observed in the absence of a functional host homologous recombination system. This work demonstrates that the Ll.ltrB intron is a novel site-specific mobile element in lactococci and that group II intron self-transfer is a mechanism for intron dissemination among bacteria.     

A reliable lacZ expression reporter cassette for multipurpose, knockout-first alleles

Alteration of the mouse genome through homologous recombination in embryonic stem (ES) cells is the most accurate and versatile way to dissect gene function in a vertebrate model. Most often, a selectable marker is used to create a knockout allele by replacing an essential part of the gene. However, knockout strategies are limited because the mutation is present constitutively. Conditional approaches based on the Cre-loxP site-specific recombination (SSR) system address this limitation; however, it requires that all parts of the targeted gene remain in ES cells. Here we report success with a "knockout-first" strategy that ablates gene function by insertion of RNA processing signals without deletion of any of the target gene. Incorporation of site-specific recombination target sites creates a multipurpose allele for both knockout and conditional applications.     

Targeted gene conversion induced by triplex-directed psoralen interstrand crosslinks in mammalian cells

Correction of a defective gene is a promising approach for both basic research and clinical gene therapy. However, the absence of site-specific targeting and the low efficiency of homologous recombination in human cells present barriers to successful gene targeting. In an effort to overcome these barriers, we utilized triplex-forming oligonucleotides (TFOs) conjugated to a DNA interstrand crosslinking (ICL) agent, psoralen (pTFO-ICLs), to improve the gene targeting efficiency at a specific site in DNA. Gene targeting events were monitored by the correction of a deletion on a recipient plasmid with the homologous sequence from a donor plasmid in human cells. The mechanism underlying this event is stimulation of homologous recombination by the pTFO-ICL. We found that pTFO-ICLs are efficient in inducing targeted gene conversion (GC) events in human cells. The deletion size in the recipient plasmid influenced both the recombination frequency and spectrum of recombinants; i.e. plasmids with smaller deletions had a higher frequency and proportion of GC events. The polarity of the pTFO-ICL also had a prominent effect on recombination. Our results suggest that pTFO-ICL induced intermolecular recombination provides an efficient method for targeted gene correction in mammalian cells.     

Conjugation mediates transfer of the Ll.LtrB group II intron between different bacterial species

Some self-splicing group II introns (ribozymes) are mobile retroelements. These retroelements, which can insert themselves into cognate intronless alleles or ectopic sites by reverse splicing, are thought to be the evolutionary progenitors of the widely distributed eukaryotic spliceosomal introns. Lateral or horizontal transmission of introns (i.e. between species), although never experimentally demonstrated, is a well-accepted model for intron dispersal and evolution. Horizontal transfer of the ancestral bacterial group II introns may have contributed to the dispersal and wide distribution of spliceosomal introns present in modern eukaryotic genomes. Here, the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis was used as a model system to address the dissemination of introns in the bacterial kingdom. We report the first experimental demonstration of horizontal transfer of a group II intron. We show that the Ll.LtrB group II intron, originally discovered on an L. lactis conjugative plasmid (pRS01) and within a chromosomally located sex factor in L. lactis 712, invades new sites using both retrohoming and retrotransposition pathways after its transfer by conjugation. Ll.LtrB lateral transfer is shown among different L. lactis strains (intraspecies) (retrohoming and retrotransposition) and between L. lactis and Enterococcus faecalis (interspecies) (retrohoming). These results shed light on long-standing questions about intron evolution and propagation, and demonstrate that conjugation is one of the mechanisms by which group II introns are, and probably were, broadly disseminated between widely diverged organisms.     

Targeted Genome Editing by Recombinant Adeno-Associated Virus （rAAV） Vectors for Generating Genetically Modified Pigs

Recombinant adeno-associated virus(rAAV) vectors have been extensively used for experimental gene therapy of inherited human diseases.Several advantages,such as simple vector construction,high targeting frequency by homologous recombination,and applicability to many cell types,make rAAV an attractive approach for targeted genome editing.Combined with cloning by somatic cell nuclear transfer(SCNT),this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis, hereditary tyrosinemia type 1,and breast cancer.This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination.We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts,which are subsequently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.     

Distribution of the vanG-like gene cluster in Clostridium difficile clinical isolates

Treatment of Clostridium difficile infections generally requires cessation of their causative antibiotic and subsequent administration of metronidazole or vancomycin. Intriguingly, the genome of C. difficile 630 contains a cryptic gene cluster homologous to the vanG-type operon of Enterococcus faecalis BM4518. We detected this cluster by PCR in 35 out of 41 clinical isolates, confirming its large prevalence in this species. The cluster was found to be located in a unique locus. Comparison of this locus with that of strains devoid of the vanG-like cluster indicated that acquisition of the gene cluster occurred in a perfect 19-bp inverted repeat, in the absence of a detectable mobile structure.     

Efficient and simple generation of unmarked gene deletions in Mycobacterium smegmatis

Genetic research in molecular laboratories relies heavily on directed mutagenesis and gene deletion techniques. In mycobacteria, however, genetic analysis is often hindered by difficulties in the preparation of deletion mutants. Indeed, in comparison to the allelic exchange systems available for the study of other common model organisms, such as Saccharomyces cerevisiae and Escherichia coli, mycobacterial gene disruption systems suffer from low mutant isolation success rates, mostly due to inefficient homologous recombination and a high degree of non-specific recombination. Here, we present a gene deletion system that combines efficient homologous recombination with advanced screening of mutants. This novel methodology allows for gene disruption in three consecutive steps. The first step relies on the use of phage Che9c recombineering proteins for directed insertion into the chromosome of a linear DNA fragment that encodes GFP and confers hygromycin resistance. In the second step, GFP positive and hygromycin resistant colonies are selected, and in the last step, the gfp-hyg cassette is excised from the chromosome, thus resulting in the formation of an unmarked deletion. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the prcSBA operon of Mycobacterium smegmatis.     

Use of two gRNAs for CRISPR/Cas9 improves bi‐allelic homologous recombination efficiency in mouse embryonic stem cells

Targeted genome editing in mouse embryonic stem cells (ESCs) is a powerful resource to functionally characterize genes and regulatory elements. The use of the CRISPR/Cas9 genome editing approach has remarkably improved the time and efficiency of targeted recombination. However, the efficiency of this protocol is still far from ideal when aiming for bi‐allelic homologous recombination, requiring at least two independent targeting recombination events. Here we describe an improved protocol that uses two gRNAs flanking the selected targeted region, leading to highly efficient homologous recombination in mouse ESCs. The bi‐allelic recombination targeting efficiency is over 90% when using two gRNAs together with the inhibition of non‐homologous end‐joint repair. Moreover, this technique is compatible with the generation of knocked‐in mice and the use of ESC‐derived differentiation protocols, therefore facilitating and accelerating the gene targeting in mice and ESCs.     

Size Of Gene Specific Inverted Repeat - Dependent Gene Deletion In Saccharomyces cerevisiae

We describe here an approach for rapidly producing scar-free and precise gene deletions in S. cerevisiae with high efficiency. Preparation of the disruption gene cassette in this approach was simply performed by overlap extension-PCR of an invert repeat of a partial or complete sequence of the targeted gene with URA3. Integration of the prepared disruption gene cassette to the designated position of a target gene leads to the formation of a mutagenesis cassette within the yeast genome, which consists of a URA3 gene flanked by the targeted gene and its inverted repeat between two short identical direct repeats. The inherent instability of the inverted sequences in close proximity facilitates the self-excision of the entire mutagenesis cassette deposited in the genome and promotes homologous recombination resulting in a seamless deletion via a single transformation. This rapid assembly circumvents the difficulty during preparation of disruption gene cassettes composed of two inverted repeats of the URA3, which requires the engineering of unique restriction sites for subsequent digestion and T4 DNA ligation in vitro. We further identified that the excision of the entire mutagenesis cassette flanked by two DRs in the transformed S. cerevisiae is dependent on the length of the inverted repeat of which a minimum of 800 bp is required for effective gene deletion. The deletion efficiency improves with the increase of the inverted repeat till 1.2 kb. Finally, the use of gene-specific inverted repeats of target genes enables simultaneous gene deletions. The procedure has the potential for application on other yeast strains to achieve precise and efficient removal of gene sequences.     

Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein^1,2. The Δku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The Δku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale^1-4.Here, we report methods for using type I and type II Δku80Δhxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).     

Elimination of a group II intron from a plastid gene causes a mutant phenotype

Group II introns are found in bacteria and cell organelles (plastids, mitochondria) and are thought to represent the evolutionary ancestors of spliceosomal introns. It is generally believed that group II introns are selfish genetic elements that do not have any function. Here, we have scrutinized this assumption by analyzing two group II introns that interrupt a plastid gene (ycf3) involved in photosystem assembly. Using stable transformation of the plastid genome, we have generated mutant plants that lack either intron 1 or intron 2 or both. Interestingly, the deletion of intron 1 caused a strong mutant phenotype. We show that the mutants are deficient in photosystem I and that this deficiency is directly related to impaired ycf3 function. We further show that, upon deletion of intron 1, the splicing of intron 2 is strongly inhibited. Our data demonstrate that (i) the loss of a group II intron is not necessarily phenotypically neutral and (ii) the splicing of one intron can depend on the presence of another.     

Homologous recombination between plasmid DNA molecules in maize protoplasts

Summary The requirements for homologous recombination between plasmid DNA molecules have been studied using the PEG (polyethylene glycol)-mediated transformation system of maize (Zea mays L.) protoplasts coupled with the transient expression assay for -glucuronidase (GUS). Two plasmids were introduced into maize protoplasts; one plasmid (pB×26) contained a genomic clone of the Adh1 maize gene; the other plasmid (piGUS) was a promoterless construction containing part of intron A of the Adhl gene fused to the gusA coding sequence. Thus, the two vectors shared an effective homologous region consisting of a 459 by (Hindlll—PvuII) fragment of the yAdh1 intron A sequence. An active gusA fusion gene would result upon homologous recombination between the plasmids within the intron A sequence, and indeed GUS activity was observed in extracts following co-transformation of maize protoplasts with the two plasmids. The presence of recombinant DNA molecules in protoplast DNA isolated 1 day after co-transformation was verified using polymerase chain reactions (PCR) and Southern blots. For efficient homologous recombination, both plasmids had to be linearized. The recombination reaction was induced by restriction of the plasmid molecules either inside the effective homologous region or at the borders of the intron sequence. However, the presence of even small, terminal, nonhomologous sequences at the 3 end of the pB×26 fragment inhibited the recombination reaction. Also, both ends of the linearized piGUS DNA molecules were involved in the recombination reaction. The results revealed some features of homologous recombination reactions occurring in plant cells which cannot be accommodated by mechanisms postulated for similar reactions in animal system and in lower eukaryotes.     

Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination

Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR–Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR–CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism.     

Efficient generation of gene knockout plasmids for Dictyostelium discoideum using one-step cloning

The amoeba Dictyostelium discoideum is a well-established model organism for studying numerous aspects of cellular and developmental functions. Its rather small (~34Mb) chromosomal genome and the high efficiency of gene disruption by homologous recombination have enabled researchers to dissect various specific gene functions. We describe here the use of one-step cloning for the fast and efficient generation of deletion vectors that are produced in a one-step reaction by inserting two PCR products into an organism-specific, generic acceptor system. This worked efficiently for all 16 tested constructs directed against genes in the amoeba Dictyostelium discoideum. Saving cost and time, the used protocol represents a significant advancement in the generation of such plasmids compared to the conventionally applied restriction enzyme/ligation approach. Using appropriate selection markers, similar systems could also be useful in other organisms, where genes can be knocked out by homologous recombination.     

Directed genomic integration, gene replacement, and integrative gene expression in Streptococcus thermophilus.

Several pGEM5- and pUC19-derived plasmids containing a selectable erythromycin resistance marker were integrated into the chromosome of Streptococcus thermophilus at the loci of the lactose-metabolizing genes. Integration occurred via homologous recombination and resulted in cointegrates between plasmid and genome, flanked by the homologous DNA used for integration. Selective pressure on the plasmid-located erythromycin resistance gene resulted in multiple amplifications of the integrated plasmid. Release of this selective pressure, however, gave way to homologous resolution of the cointegrate structures. By integration and subsequent resolution, we were able to replace the chromosomal lacZ gene with a modified copy carrying an in vitro-generated deletion. In the same way, we integrated a promoterless chloramphenicol acetyltransferase (cat) gene between the chromosomal lacS and lacZ genes of the lactose operon. The inserted cat gene became a functional part of the operon and was expressed and regulated accordingly. Selective pressure on the essential lacS and lacZ genes under normal growth conditions in milk ensures the maintenance and expression of the integrated gene. As there are only minimal repeated DNA sequences (an NdeI site) flanking the inserted cat gene, it was stably maintained even in the absence of lactose, i.e., when grown on sucrose or glucose. The methodology represents a stable system in which to express and regulate foreign genes in S. thermophilus, which could qualify in the future for an application with food.     

Exon skipping caused by an intronic insertion of a young Alu Yb9 element leads to severe hemophilia A

Short interspersed elements, such as Alu elements, have propagated to more than one million copies in the human genome. They affect the genome in several ways, caused by retrotransposition, recombination between elements, gene conversion, and alterations in gene expression. These events, including novel insertions into active genes, have been associated with a number of human disorders. Hemophilia A is an X-linked severe bleeding disorder and is caused by mutations in the Factor VIII gene. The spectrum of mutations includes point mutations, rearrangements, insertions, and deletions. Recently, an Alu retrotransposition event in a coding exon has been reported in a family with a severe form of hemophilia A. This was the first report of an Alu insertion in the Factor VIII gene. Here, we report a second Alu insertion event that lies in an intron of the same gene that causes exon skipping and the complete disruption of gene expression.