A CRISPR-Cas9-integrase complex generates precise DNA fragments for genome integration.
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ABSTRACT:
SUBMITTER: Jakhanwal S
PROVIDER: S-EPMC8034620 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The CRISPR/Cas9 system provides a powerful tool for genome editing in various model organisms, including zebrafish. The establishment of targeted gene-disrupted zebrafish (knockouts) is readily achieved by CRISPR/Cas9-mediated genome modification. Recently, exogenous DNA integration into the zebrafish genome via homology-independent DNA repair was reported, but this integration contained various mutations at the junctions of genomic and integrated DNA. Thus, precise genome modification into targeted genomic loci remains to be achieved. Here, we describe efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences (10-40?bp) flanking the genomic target locus. We succeeded in integrating with high efficiency an exogenous mCherry or eGFP gene into targeted genes (tyrosinase and krtt1c19e) in frame. We found the precise in-frame integration of exogenous DNA without backbone vector sequences when Cas9 cleavage sites were introduced at both sides of the left homology arm, the eGFP sequence and the right homology arm. Furthermore, we confirmed that this precise genome modification was heritable. This simple method enables precise targeted gene knock-in in zebrafish.
| S-EPMC4350073 | biostudies-literature
Project description:CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in), by utilizing specially designed 3'-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3'-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.
| S-EPMC10235934 | biostudies-literature
Project description:The introduction of engineered site-specific DNA endonucleases has brought precise genome editing in many model organisms and human cells into the realm of possibility. In zebrafish, loss-of-function alleles have been successfully produced; however, germ line transmission of functional targeted knock-ins of protein tags or of SNP exchanges have not been reported. Here we show by phenotypic rescue that the CRISPR/Cas system can be used to target and repair a premature stop codon at the albino (alb) locus in zebrafish with high efficiency and precision. Using circular donor DNA containing CRISPR target sites we obtain close to 50% of larvae with precise homology-directed repair of the alb(b4) mutation, a small fraction of which transmitted the repaired allele in the germ line to the next generation (3/28 adult fish). The in vivo demonstration of germ line transmission of a precise SNP exchange in zebrafish underscores its suitability as a model for genetic research.
| S-EPMC4299274 | biostudies-other
Project description:The efficient knock-in of large DNA fragments to label endogenous proteins remains especially challenging in non-dividing cells such as neurons. We developed Targeted Knock-In with Two (TKIT) guides as a novel CRISPR/Cas9 based approach for efficient, and precise, genomic knock-in. Through targeting non-coding regions TKIT is resistant to INDEL mutations. We demonstrate TKIT labeling of endogenous synaptic proteins with various tags, with efficiencies up to 42% in mouse primary cultured neurons. Utilizing in utero electroporation or viral injections in mice TKIT can label AMPAR subunits with Super Ecliptic pHluorin, enabling visualization of endogenous AMPARs in vivo using two-photon microscopy. We further use TKIT to assess the mobility of endogenous AMPARs using fluorescence recovery after photobleaching. Finally, we show that TKIT can be used to tag AMPARs in rat neurons, demonstrating precise genome editing in another model organism and highlighting the broad potential of TKIT as a method to visualize endogenous proteins.
| S-EPMC7946428 | biostudies-literature
Project description:Germline manipulation using CRISPR/Cas9 genome editing has dramatically accelerated the generation of new mouse models. Nonetheless, many metabolic disease models still depend upon laborious germline targeting, and are further complicated by the need to avoid developmental phenotypes. We sought to address these experimental limitations by generating somatic mutations in the adult liver using CRISPR/Cas9, as a new strategy to model metabolic disorders. As proof-of-principle, we targeted the low-density lipoprotein receptor (Ldlr), which when deleted, leads to severe hypercholesterolemia and atherosclerosis. Here we show that hepatic disruption of Ldlr with AAV-CRISPR results in severe hypercholesterolemia and atherosclerosis. We further demonstrate that co-disruption of Apob, whose germline loss is embryonically lethal, completely prevented disease through compensatory inhibition of hepatic LDL production. This new concept of metabolic disease modeling by somatic genome editing could be applied to many other systemic as well as liver-restricted disorders which are difficult to study by germline manipulation.
| S-EPMC5353616 | biostudies-literature
Project description:Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) is a revolutionary technology that enables efficient genomic modification in many organisms. Currently, the wide use of Streptococcus pyogenes Cas9 (SpCas9) primarily recognizes sites harbouring a canonical NGG protospacer adjacent motif (PAM). The newly developed VQR (D1135V/R1335Q/T1337R) variant of Cas9 has been shown to cleave sites containing NGA PAM in rice, which greatly expanded the range of genome editing. However, the low editing efficiency of the VQR variant remains, which limits its wide application in genome editing. In this study, by modifying the single guide RNA (sgRNA) structure and strong endogenous promoters, we significantly increased the editing efficiency of the VQR variant. The modified CRISPR-Cas9-VQR system provides a robust toolbox for multiplex genome editing at sites containing noncanonical NGA PAM.
| S-EPMC5785341 | biostudies-literature
Project description:CRISPR-Cas systems depend on the Cas1-Cas2 integrase to capture and integrate short foreign DNA fragments into the CRISPR locus, enabling adaptation to new viruses. We present crystal structures of Cas1-Cas2 bound to both donor and target DNA in intermediate and product integration complexes, as well as a cryo-electron microscopy structure of the full CRISPR locus integration complex, including the accessory protein IHF (integration host factor). The structures show unexpectedly that indirect sequence recognition dictates integration site selection by favoring deformation of the repeat and the flanking sequences. IHF binding bends the DNA sharply, bringing an upstream recognition motif into contact with Cas1 to increase both the specificity and efficiency of integration. These results explain how the Cas1-Cas2 CRISPR integrase recognizes a sequence-dependent DNA structure to ensure site-selective CRISPR array expansion during the initial step of bacterial adaptive immunity.
| S-EPMC5748385 | biostudies-literature
Project description:Targeted genome editing by CRISPR/Cas9 is extremely well fitted to generate gene disruptions, although precise sequence replacement by CRISPR/Cas9-mediated homology-directed repair (HDR) suffers from low efficiency, impeding its use for high-throughput knock-in disease modeling. In this study, we used next-generation sequencing (NGS) analysis to determine the efficiency and reliability of CRISPR/Cas9-mediated HDR using several types of single-stranded oligodeoxynucleotide (ssODN) repair templates for the introduction of disease-relevant point mutations in the zebrafish genome. Our results suggest that HDR rates are strongly determined by repair-template composition, with the most influential factor being homology-arm length. However, we found that repair using ssODNs does not only lead to precise sequence replacement but also induces integration of repair-template fragments at the Cas9 cut site. We observed that error-free repair occurs at a relatively constant rate of 1-4% when using different repair templates, which was sufficient for transmission of point mutations to the F1 generation. On the other hand, erroneous repair mainly accounts for the variability in repair rate between the different repair templates. To further improve error-free HDR rates, elucidating the mechanism behind this erroneous repair is essential. We show that the error-prone nature of ssODN-mediated repair, believed to act via synthesis-dependent strand annealing (SDSA), is most likely due to DNA synthesis errors. In conclusion, caution is warranted when using ssODNs for the generation of knock-in models or for therapeutic applications. We recommend the application of in-depth NGS analysis to examine both the efficiency and error-free nature of HDR events.This article has an associated First Person interview with the first author of the paper.
| S-EPMC6215429 | biostudies-literature
Project description:Klebsiella pneumoniae is a promising industrial microorganism as well as a major human pathogen. The recent emergence of carbapenem-resistant K. pneumoniae has posed a serious threat to public health worldwide, emphasizing a dire need for novel therapeutic means against drug-resistant K. pneumoniae Despite the critical importance of genetics in bioengineering, physiology studies, and therapeutic-means development, genome editing, in particular, the highly desirable scarless genetic manipulation in K. pneumoniae, is often time-consuming and laborious. Here, we report a two-plasmid system, pCasKP-pSGKP, used for precise and iterative genome editing in K. pneumoniae By harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome cleavage system and the lambda Red recombination system, pCasKP-pSGKP enabled highly efficient genome editing in K. pneumoniae using a short repair template. Moreover, we developed a cytidine base-editing system, pBECKP, for precise C?T conversion in both the chromosomal and plasmid-borne genes by engineering the fusion of the cytidine deaminase APOBEC1 and a Cas9 nickase. By using both the pCasKP-pSGKP and the pBECKP tools, the bla KPC-2 gene was confirmed to be the major factor that contributed to the carbapenem resistance of a hypermucoviscous carbapenem-resistant K. pneumoniae strain. The development of the two editing tools will significantly facilitate the genetic engineering of K. pneumoniae IMPORTANCE Genetics is a key means to study bacterial physiology. However, the highly desirable scarless genetic manipulation is often time-consuming and laborious for the major human pathogen K. pneumoniae We developed a CRISPR-Cas9-mediated genome-editing method and a cytidine base-editing system, enabling rapid, highly efficient, and iterative genome editing in both industrial and clinically isolated K. pneumoniae strains. We applied both tools in dissecting the drug resistance mechanism of a hypermucoviscous carbapenem-resistant K. pneumoniae strain, elucidating that the bla KPC-2 gene was the major factor that contributed to the carbapenem resistance of the hypermucoviscous carbapenem-resistant K. pneumoniae strain. Utilization of the two tools will dramatically accelerate a wide variety of investigations in diverse K. pneumoniae strains and relevant Enterobacteriaceae species, such as gene characterization, drug discovery, and metabolic engineering.
| S-EPMC6238054 | biostudies-literature
Project description:BACKGROUND:Gene targeting by homology-directed repair (HDR) can precisely edit the genome and is a versatile tool for biomedical research. However, the efficiency of HDR-based modification is still low in many model organisms including zebrafish. Recently, long single-stranded DNA (lssDNA) molecules have been developed as efficient alternative donor templates to mediate HDR for the generation of conditional mouse alleles. Here we report a method, zLOST (zebrafish long single-stranded DNA template), which utilises HDR with a long single-stranded DNA template to produce more efficient and precise mutations in zebrafish. RESULTS:The efficiency of knock-ins was assessed by phenotypic rescue at the tyrosinase (tyr) locus and confirmed by sequencing. zLOST was found to be a successful optimised rescue strategy: using zLOST containing a tyr repair site, we restored pigmentation in at least one melanocyte in close to 98% of albino tyr25del/25del embryos, although more than half of the larvae had only a small number of pigmented cells. Sequence analysis showed that there was precise HDR dependent repair of the tyr locus in these rescued pigmented embryos. Furthermore, quantification of zLOST knock-in efficiency at the rps14, nop56 and th loci by next generation sequencing demonstrated that zLOST showed a clear improvement. We utilised the HDR efficiency of zLOST to precisely model specific human disease mutations in zebrafish with ease. Finally, we determined that this method can achieve a germline transmission rate of up to 31.8%. CONCLUSIONS:In summary, these results show that zLOST is a useful method of zebrafish genome editing, particularly for generating desired mutations by targeted DNA knock-in through HDR.
| S-EPMC6974980 | biostudies-literature