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Posted ContentDOI

In vitro transposon targeting using a catalytically inactive Cas9

08 Mar 2019-bioRxiv (Cold Spring Harbor Laboratory)-pp 571653
TL;DR: In this paper, the Hsmar1 transposase was fused to a catalytically inactive Cas9 to bias transposon insertions into the vicinity of the target site bound by a guide RNA-dCas9 ribonucleoprotein complex.
Abstract: Transposases are attractive tools for the integration of therapeutic transgenes into the chromosome for gene therapy applications. Typically, transgenes can be flanked with inverted-terminal repeat sequences, which are recognised by the transposase and integrated at random sites. Minimising detrimental insertions of transgenes is a key goal in the development of gene delivery vectors for gene therapy. We fused the Hsmar1 transposase to a catalytically inactive Cas9. Our aim was to bias transposon insertions into the vicinity of the target site bound by a guide RNA-dCas9 ribonucleoprotein complex. Although we could not detect any targeted transposition events in vivo, we achieved a 15-fold enrichment of transposon insertions into a 600-bp target site in an in vitro plasmid-to-plasmid assay. Additionally, we show that among those integrations that were successfully targeted, the location is tightly constrained to a site immediately to one side of the guide RNA target site. We present an in vitro proof-of-concept study demonstrating that the transposase insertion profile can be biased using a catalytically inactive Cas9 variant as a programmable DNA-binding module. One factor that limits the utility of this approach is that the transposon continues to integrate randomly. Although the dCas9 domain can be targeted to chromosomal lacZ, as evidenced by transcriptional repression, we were unable to detect any targeted insertions in the vicinity of the target site. Any targeted insertions that did occur were masked be a much larger number of random insertions. It is therefore necessary to develop a method for the temporal control of the transposase to allow Cas9 time to locate its target site.
Citations
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Journal Article
TL;DR: In this article, the authors show that egfl7 mutants do not show any obvious phenotypes while animals injected with egfl 7 morpholino (morphants) exhibit severe vascular defects, indicating that the activation of a compensatory network to buffer against deleterious mutations was not observed after translational or transcriptional knockdown.
Abstract: Cells sense their environment and adapt to it by fine-tuning their transcriptome. Wired into this network of gene expression control are mechanisms to compensate for gene dosage. The increasing use of reverse genetics in zebrafish, and other model systems, has revealed profound differences between the phenotypes caused by genetic mutations and those caused by gene knockdowns at many loci, an observation previously reported in mouse and Arabidopsis. To identify the reasons underlying the phenotypic differences between mutants and knockdowns, we generated mutations in zebrafish egfl7, an endothelial extracellular matrix gene of therapeutic interest, as well as in vegfaa. Here we show that egfl7 mutants do not show any obvious phenotypes while animals injected with egfl7 morpholino (morphants) exhibit severe vascular defects. We further observe that egfl7 mutants are less sensitive than their wild-type siblings to Egfl7 knockdown, arguing against residual protein function in the mutants or significant off-target effects of the morpholinos when used at a moderate dose. Comparing egfl7 mutant and morphant proteomes and transcriptomes, we identify a set of proteins and genes that are upregulated in mutants but not in morphants. Among them are extracellular matrix genes that can rescue egfl7 morphants, indicating that they could be compensating for the loss of Egfl7 function in the phenotypically wild-type egfl7 mutants. Moreover, egfl7 CRISPR interference, which obstructs transcript elongation and causes severe vascular defects, does not cause the upregulation of these genes. Similarly, vegfaa mutants but not morphants show an upregulation of vegfab. Taken together, these data reveal the activation of a compensatory network to buffer against deleterious mutations, which was not observed after translational or transcriptional knockdown.

774 citations

Journal Article
TL;DR: The mechanism by which mariner, a eukaryotic transposable element, performs DNA cleavage is examined and it is shown that the nontransferred strand is cleaved initially, unlike prokaryotictransposons which cleave the transferred strand first.

80 citations

Posted ContentDOI
01 Nov 2021-bioRxiv
TL;DR: In this article, the authors present Programmable Addition via Site-specific Targeting Elements (PASTE), which achieves efficient and versatile gene integration at diverse loci by directing insertion with a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase.
Abstract: Programmable and multiplexed genome integration of large, diverse DNA cargo independent of DNA repair remains an unsolved challenge of genome editing. Current gene integration approaches require double-strand breaks that evoke DNA damage responses and rely on repair pathways that are inactive in terminally differentiated cells. Furthermore, CRISPR-based approaches that bypass double stranded breaks, such as Prime editing, are limited to modification or insertion of short sequences. We present Programmable Addition via Site-specific Targeting Elements, or PASTE, which achieves efficient and versatile gene integration at diverse loci by directing insertion with a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase. Without generating double stranded breaks, we demonstrate integration of sequences as large as ~36 kb with rates between 10-50% at multiple genomic loci across three human cell lines, primary T cells, and quiescent non-dividing primary human hepatocytes. To further improve PASTE, we discover thousands of novel serine integrases and cognate attachment sites from metagenomes and engineer active orthologs for high-efficiency integration using PASTE. We apply PASTE to fluorescent tagging of proteins, integration of therapeutically relevant genes, and production and secretion of transgenes. Leveraging the orthogonality of serine integrases, we engineer PASTE for multiplexed gene integration, simultaneously integrating three different genes at three genomic loci. PASTE has editing efficiencies comparable to or better than those of homology directed repair or non-homologous end joining based integration, with activity in non-dividing cells and fewer detectable off-target events. For therapeutic applications, PASTE can be delivered as mRNA with synthetically modified guides to programmably direct insertion of DNA templates carried by AAV or adenoviral vectors. PASTE expands the capabilities of genome editing via drag-and-drop gene integration, offering a platform with wide applicability for research, cell engineering, and gene therapy.

34 citations

References
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Book
01 Jan 1972
TL;DR: Molecular Genetics (Biology): An Overview | Sciencing Experimental in Molecular Genetics Experiments in molecular genetics (1972 edition) | Open ...
Abstract: Molecular Genetics (Biology): An Overview | Sciencing Experiments in Molecular Genetics Experiments in molecular genetics (1972 edition) | Open ... Experimental Molecular Genetics | Biology | MIT OpenCourseWare DNA experiments you can perform at home | SBS Science Experiments in molecular genetics Jeffrey H. Miller ... DNA and Molecular Genetics Experiments in Molecular Biology: Biochemical Applications ... Molecular Genetics Biology Experiment Please help ... Molecular genetics | biology | Britannica Molecular Genetic Experiment : Biology Lab 1793 Words ... Miller, J.H. (1972) Experiments in Molecular Genetics ... Griffith's experiment Wikipedia DNA as genetic material: Revisiting classic experiments ... Experiments in molecular genetics (Book, 1972) [WorldCat.org] Measuring βGalactosidase Activity in Bacteria: Cell ... Classic Experiments in

26,898 citations

Journal ArticleDOI
TL;DR: A set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies are described.
Abstract: Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.

8,663 citations

Journal ArticleDOI
TL;DR: With the development of a leukaemia-like syndrome in two patients cured of a disease by gene therapy, it is timely to contemplate how far this technology has come, and how far it still has to go.
Abstract: Gene therapy has a history of controversy. Encouraging results are starting to emerge from the clinic, but questions are still being asked about the safety of this new molecular medicine. With the development of a leukaemia-like syndrome in two of the small number of patients that have been cured of a disease by gene therapy, it is timely to contemplate how far this technology has come, and how far it still has to go.

2,451 citations

Journal ArticleDOI
31 Mar 2011-Nature
TL;DR: In this article, tracrRNA, a trans-encoded small RNA with 24-nucleotide complementarity to the repeat regions of crRNA precursor transcripts, is shown to direct the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein.
Abstract: CRISPR/Cas systems constitute a widespread class of immunity systems that protect bacteria and archaea against phages and plasmids, and commonly use repeat/spacer-derived short crRNAs to silence foreign nucleic acids in a sequence-specific manner. Although the maturation of crRNAs represents a key event in CRISPR activation, the responsible endoribonucleases (CasE, Cas6, Csy4) are missing in many CRISPR/Cas subtypes. Here, differential RNA sequencing of the human pathogen Streptococcus pyogenes uncovered tracrRNA, a trans-encoded small RNA with 24-nucleotide complementarity to the repeat regions of crRNA precursor transcripts. We show that tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein; all these components are essential to protect S. pyogenes against prophage-derived DNA. Our study reveals a novel pathway of small guide RNA maturation and the first example of a host factor (RNase III) required for bacterial RNA-mediated immunity against invaders.

2,336 citations

Book ChapterDOI
Le Cong1, Feng Zhang
TL;DR: This chapter presents all relevant methods including the initial site selection, molecular cloning, delivery of guide RNAs and Cas9 into mammalian cells, verification of target cleavage, and assays for detecting genomic modification including indels and homologous recombination.
Abstract: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is an adaptive immune system that exists in a variety of microbes. It could be engineered to function in eukaryotic cells as a fast, low-cost, efficient, and scalable tool for manipulating genomic sequences. In this chapter, detailed protocols are described for harnessing the CRISPR-Cas9 system from Streptococcus pyogenes to enable RNA-guided genome engineering applications in mammalian cells. We present all relevant methods including the initial site selection, molecular cloning, delivery of guide RNAs (gRNAs) and Cas9 into mammalian cells, verification of target cleavage, and assays for detecting genomic modification including indels and homologous recombination. These tools provide researchers with new instruments that accelerate both forward and reverse genetics efforts.

1,902 citations