Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing.
ABSTRACT: Simultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.
Project description:<h4>Background</h4>Genome editing using the CRISPR/Cas9 system is now well documented in basic studies and is expected to be applied to gene therapy. Simultaneous expression of multiplex guide RNA (gRNA) and Cas9/Cas9 derivative is attractive for the efficient knockout of genes and a safe double-nicking strategy. However, such use is limited because highly multiplex gRNA-expressing units are difficult to maintain stably in plasmids as a result of deletion via homologous recombination.<h4>Methods</h4>Lambda in vitro packaging was used instead of transformation for the construction and preparation of large, cos-containing plasmid (cosmid). Polymerase chain reaction fragments containing multiplex gRNA units were obtained using the Four-guide Tandem method. Transfection was performed by lipofection.<h4>Results</h4>We constructed novel cosmids consisting of linearized plasmid-DNA fragments containing up to 16 copies of multiplex gRNA-expressing units as trimer or tetramer (polygonal cosmids). These cosmids behaved as if they were monomer plasmids, and multiplex units could stably be maintained and amplified with a lack of deletion. Surprisingly, the deleted cosmid was removed out simply by amplifying the cosmid stock using lambda packaging. The DNA fragments containing multiplex gRNA-units and Cas9 were transfected to 293 cells and were found to disrupt the X gene of hepatitis B virus by deleting a large region between the predicted sites.<h4>Conclusions</h4>We present a simple method for overcoming the problem of constructing plasmids stably containing multiplex gRNA-expressing units. The method may enable the production of very large amounts of DNA fragments expressing intact, highly-multiplex gRNAs and Cas9/Cas9 derivatives for safe and efficient genome-editing therapy using non-viral vectors.
Project description:Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.
Project description:Clustered regularly interspaced short palindromic repeats (CRISPR) machineries are prokaryotic immune systems that have been adapted as versatile gene editing and manipulation tools. We found that CRISPR nucleases from two families, Cpf1 (also known as Cas12a) and Cas9, exhibit differential guide RNA (gRNA) sequence requirements for cleavage of the two strands of target DNA in vitro. As a consequence of the differential gRNA requirements, both Cas9 and Cpf1 enzymes can exhibit potent nickase activities on an extensive class of mismatched double-stranded DNA (dsDNA) targets. These properties allow the production of efficient nickases for a chosen dsDNA target sequence, without modification of the nuclease protein, using gRNAs with a variety of patterns of mismatch to the intended DNA target. In parallel to the nicking activities observed with purified Cas9 in vitro, we observed sequence-dependent nicking for both perfectly matched and partially mismatched target sequences in a Saccharomyces cerevisiae system. Our findings have implications for CRISPR spacer acquisition, off-target potential of CRISPR gene editing/manipulation, and tool development using homology-directed nicking.
Project description:Even for the genetically accessible yeast <i>Saccharomyces cerevisiae</i>, the CRISPR-Cas DNA editing technology has strongly accelerated and facilitated strain construction. Several methods have been validated for fast and highly efficient single editing events, and diverse approaches for multiplex genome editing have been described in the literature by means of <i>Sp</i>Cas9 or <i>Fn</i>Cas12a endonucleases and their associated guide RNAs (gRNAs). The gRNAs used to guide the Cas endonuclease to the editing site are typically expressed from plasmids using native Pol II or Pol III RNA polymerases. These gRNA expression plasmids require laborious, time-consuming cloning steps, which hampers their implementation for academic and applied purposes. In this study, we explore the potential of expressing gRNA from linear DNA fragments using the T7 RNA polymerase (T7RNAP) for single and multiplex genome editing in <i>Saccharomyces cerevisiae</i>. Using <i>Fn</i>Cas12a, this work demonstrates that transforming short, linear DNA fragments encoding gRNAs in yeast strains expressing T7RNAP promotes highly efficient single and duplex DNA editing. These DNA fragments can be custom ordered, which makes this approach highly suitable for high-throughput strain construction. This work expands the CRISPR toolbox for large-scale strain construction programs in <i>S. cerevisiae</i> and promises to be relevant for other less genetically accessible yeast species.
Project description:Background:The Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome. Results:We introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning the annealed products of two single-stranded oligonucleotide fragments harboring a complimentary target-binding sequence on each strand between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites. Conclusions:This multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.
Project description:The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA-gRNA architecture were efficiently and precisely processed into gRNAs with desired 5' targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits.
Project description:Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) are cancer-causing viruses that establish lifelong infections in humans. Gene editing using the Cas9-guideRNA (gRNA) CRISPR system has been applied to decrease the latent load of EBV in human Burkitt lymphoma cells. Validating the efficacy of Cas9-gRNA system in eradicating infection in vivo without off-target effects to the host genome will require animal model systems. To this end, we evaluated a series of gRNAs against individual genes and functional genomic elements of murine gammaherpesvirus 68 (MHV68) that are both conserved with KSHV and important for the establishment of latency or reactivation from latency in the host. gRNA sequences against ORF50, ORF72 and ORF73 led to insertion, deletion and substitution mutations in these target regions of the genome in cell culture. Murine NIH3T3 fibroblast cells that stably express Cas9 and gRNAs to ORF50 were most resistant to replication upon de novo infection. Latent murine A20 B cell lines that stably express Cas9 and gRNAs against MHV68 were reduced in their reactivation by approximately 50%, regardless of the viral gene target. Lastly, co-transfection of HEK293T cells with the vector expressing the Cas9-MHV68 gRNA components along with the viral genome provided a rapid read-out of gene editing and biological impact. Combinatorial, multiplex MHV68 gRNA transfections in HEK293T cells led to near complete ablation of infectious particle production. Our findings indicate that Cas9-gRNA editing of the murine gammaherpesvirus genome has a deleterious impact on productive replication in three independent infection systems.
Project description:We herein describe rapid and accurate clinical testing for COVID-19 by nicking and extension chain reaction system-based amplification (NESBA), an ultrasensitive version of NASBA. The primers to identify SARS-CoV-2 viral RNA were designed to additionally contain the nicking recognition sequence at the 5'-end of conventional NASBA primers, which would enable nicking enzyme-aided exponential amplification of T7 RNA promoter-containing double-stranded DNA (T7DNA). As a consequence of this substantially enhanced amplification power, the NESBA technique was able to ultrasensitively detect SARS-CoV-2 genomic RNA (gRNA) down to 0.5 copies/μL (= 10 copies/reaction) for both envelope (E) and nucleocapsid (N) genes within 30 min under isothermal temperature (41 °C). When the NESBA was applied to test a large cohort of clinical samples (n = 98), the results fully agreed with those from qRT-PCR and showed the excellent accuracy by yielding 100% clinical sensitivity and specificity. By employing multiple molecular beacons with different fluorophore labels, the NESBA was further modulated to achieve multiplex molecular diagnostics, so that the E and N genes of SARS-CoV-2 gRNA were simultaneously assayed in one-pot. By offering the superior analytical performances over the current qRT-PCR, the isothermal NESBA technique could serve as very powerful platform technology to realize the point-of-care (POC) diagnosis for COVID-19.
Project description:The CRISPR/Cas9 system has been used for spatio-temporal gene modification through the ubiquitous expression of gRNA by an RNA polymerase III promoter and the controlled expression of Cas9 using a tissue-specific or inducible promoter. However, unexpected gene disruptions indicate the necessity of a tissue-specific or inducible expression of not only Cas9 but also gRNA. In the present study, we attempted to develop a CRISPR/Cas9 system that could express functional gRNAs and Cas9 by a single RNA polymerase II promoter and induce multi-loci disruptions in specific cells. To this end, we designed vectors expressing ribozyme-flanked gRNAs (RGRs) and Cas9 mRNAs simultaneously. We showed that the mono-promoter-driven vector induces gene disruptions at the target loci in HEK 293 cells after transfection. In addition, two target loci were disrupted simultaneously by the transfection of a mono-promoter-driven vector expressing two RGRs and Cas9 mRNA. Finally, we constructed a universal vector for use in the construction of plasmids to be applied to the present mono-promoter-driven CRISPR/Cas9 system. We have thus provided a versatile tool for generating gene disruptions by the CRISPR/Cas9 system; this system should contribute to a wide range of investigations, including studies on spatio-temporal gene functions.
Project description:CRISPR-Cas9 is a powerful tool for genome engineering, but its efficiency largely depends on guide RNA (gRNA). There are multiple methods available to evaluate the efficiency of gRNAs, including the T7E1 assay, surveyor nuclease assay, deep sequencing, and surrogate reporter systems. In the present study, we developed a cleavage-based surrogate that we have named the LacI-reporter to evaluate gRNA cleavage efficiency. The LacI repressor, under the control of the EF-1α promoter, represses luciferase or EGFP reporter expression by binding to the lac operator. Upon CRISPR-Cas9 cleavage at a target site located between the EF-1α promoter and the lacI gene, repressor expression is disrupted, thereby triggering luciferase or EGFP expression. Using this system, we can quantitate gRNA cleavage efficiency by assessing luciferase activity or EGFP expression. We found a strong positive correlation between the cleavage efficiency of gRNAs measured using this reporter and mutation frequency, measured using surveyor and deep sequencing. The genome-editing efficiency of gRNAs was validated in human liver organoids. Our LacI-reporter system provides a useful tool to select efficient gRNAs for genome editing.