Development of a Highly Efficient Multiplex Genome Editing System in Outcrossing Tetraploid Alfalfa (Medicago sativa).
ABSTRACT: Alfalfa (Medicago sativa) is an outcrossing tetraploid legume species widely cultivated in the world. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system has been successfully used for genome editing in many plant species. However, the use of CRISPR/Cas9 for gene knockout in alfalfa is still very challenging. Our initial single gRNA-CRISPR/Cas9 system had very low mutagenesis efficiency in alfalfa with no mutant phenotype. In order to develop an optimized genome editing system in alfalfa, we constructed multiplex gRNA-CRISPR/Cas9 vectors by a polycistronic tRNA-gRNA approach targeting the Medicago sativa stay-green (MsSGR) gene. The replacement of CaMV35S promoter by the Arabidopsis ubiquitin promoter (AtUBQ10) to drive Cas9 expression in the multiplex gRNA system led to a significant improvement in genome editing efficiency, whereas modification of the gRNA scaffold resulted in lower editing efficiency. The most effective multiplex system exhibited 75% genotypic mutagenesis efficiency, which is 30-fold more efficient than the single gRNA vector. Importantly, phenotypic change was easily observed in the mutants, and the phenotypic mutation efficiency reached 68%. This highly efficient multiplex gRNA-CRISPR/Cas9 genome editing system allowed the generation of homozygous mutants with a complete knockout of the four allelic copies in the T0 generation. This optimized system offers an effective way of testing gene functions and overcomes a major barrier in the utilization of genome editing for alfalfa improvement.
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: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:Several expression systems for multiple guide RNA (gRNAs) have been developed in the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system to induce multiple-gene modifications in plants. Here, we evaluated mutation efficiencies in the tomato genome using multiplex CRISPR/Cas9 vectors consisting of various Cas9 expression promoters with multiple gRNA expression combinations. In transgenic tomato calli induced with these vectors, mutation patterns varied depending on the promoters used to express Cas9. By using the tomato ELONGATION FACTOR-1? (SlEF1?) promoter to drive Cas9, occurrence of various types of mutations with high efficiency was detected in the tomato genome. Furthermore, sequence analysis showed that the majority of mutations using the multiplex system with the SlEF1? promoter corresponded to specific mutation pattern of deletions produced by self-ligation at two target sites of CRISPR/Cas9 with low mosaic mutations. These results suggest that optimizing the Cas9 expression promoter used in CRISPR/Cas9-mediated mutation improves multiplex genome editing, and could be used effectively to disrupt functional domains precisely in the tomato genome.
Project description:As a powerful tool for fast and precise genome editing, the CRISPR/Cas9 system has been applied in filamentous fungi to improve the efficiency of genome alteration. However, the method of delivering guide RNA (gRNA) remains a bottleneck in performing CRISPR mutagenesis in Aspergillus species. Here we report a gRNA transcription driven by endogenous tRNA promoters which include a tRNA gene plus 100 base pairs of upstream sequence. Co-transformation of a cas9-expressing plasmid with a linear DNA coding for gRNA demonstrated that 36 of the 37 tRNA promoters tested were able to generate the intended mutation in A. niger. When gRNA and cas9 were expressed in a single extra-chromosomal plasmid, the efficiency of gene mutation was as high as 97%. Co-transformation with DNA template for homologous recombination, the CRISPR/Cas9 system resulted ~42% efficiency of gene replacement in a strain with a functioning non-homologous end joining machinery (kusA+), and an efficiency of >90% gene replacement in a kusA- background. Our results demonstrate that tRNA promoter-mediated gRNA expressions are reliable and efficient in genome editing in A. niger.
Project description:The CRISPR/Cas9 technology has greatly improved genome editing in Saccharomyces cerevisiae over recent years. However, several current CRISPR/Cas9 systems suffer from work-intensive cloning procedures and/or the requirement of co-transforming target cells with multiple system components simultaneously which can reduce the effectivity of such applications. Here, we present a new set of all-in-one CRISPR/Cas9 vectors that combine unique benefits of different already existent systems in order to further expand the technology's design possibilities. Our vectors mediate constitutive gRNA expression whereas Cas9 expression is either driven from a constitutive or an inducible promoter. The introduction of desired gRNA targeting sequences into our inducible single gRNA vector relies just on in vivo homologous recombination-mediated assembly of overlapping single-stranded oligonucleotides, thus reducing efforts of plasmid cloning to an absolute minimum. By employing the inducible system, yeast cells can be easily preloaded with plasmids encoding for a functional CRISPR/Cas9 system, thereby chronologically separating the cloning procedure from the genome editing step. Gene knockouts could be achieved with high efficiency and effectivity by simply transforming preloaded cells with a selectable disruption cassette without the need of co-introducing any CRISPR/Cas9 system component. We also show the feasibility of efficient gene knockouts even when multiple gene copies were present such as in non-haploid strain backgrounds as well as the simultaneous deletion of two different genes in a haploid genetic background by using a multiplex variant of our inducible vector. The versatile applicability of our inducible vector system was further demonstrated by CRISPR/Cas9-mediated mating type switching of yeast.
Project description:The genome editing tool Cas9-gRNA (guide RNA) has been successfully applied in different cell types and organisms with high efficiency. However, more efforts need to be made to enhance both efficiency and specificity. In the current study, we optimized the guide RNA structure of Streptococcus pyogenes CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system to improve its genome editing efficiency. Comparing with the original functional structure of guide RNA, which is composed of crRNA and tracrRNA, the widely used chimeric gRNA has shorter crRNA and tracrRNA sequence. The deleted RNA sequence could form extra loop structure, which might enhance the stability of the guide RNA structure and subsequently the genome editing efficiency. Thus the genome editing efficiency of different forms of guide RNA was tested. And we found that the chimeric structure of gRNA with original full length of crRNA and tracrRNA showed higher genome editing efficiency than the conventional chimeric structure or other types of gRNA we tested. Therefore our data here uncovered the new type of gRNA structure with higher genome editing efficiency.
Project description:The CRISPR/Cas9 system is a powerful, revolutionary tool for genome editing. However, it is not without limitations. There are PAM-free and CRISPR-tolerant regions that cannot be modified by the standard CRISPR/Cas9 system, and off-target activity impedes its broader applications. To avoid these drawbacks, we developed a very simple CRISPR/Cas9-assisted gRNA-free one-step (CAGO) genome editing technique which does not require the construction of a plasmid to express a specific gRNA. Instead, a universal N20 sequence with a very high targeting efficiency is inserted into the E. coli chromosome by homologous recombination, which in turn undergoes a double-stranded break by CRISPR/Cas9 and induces an intra-chromosomal recombination event to accomplish the editing process. This technique was shown to be able to edit PAM-free and CRISPR-tolerant regions with no off-target effects in Escherichia coli. When applied to multi-locus editing, CAGO was able to modify one locus in two days with a near 100% editing efficiency. Furthermore, modified CAGO was used to edit large regions of up to 100 kbp with at least 75% efficiency. Finally, genome editing by CAGO only requires a transformation procedure and the construction of a linear donor DNA cassette, which was further simplified by applying a modular design strategy. Although the technique was established in E. coli, it should be applicable to other organisms with only minor modifications.
Project description:BACKGROUND: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. METHODS: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. RESULTS: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. CONCLUSIONS: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:Since its initial application in mammalian cells, CRISPR-Cas9 has rapidly become a preferred method for genome engineering experiments. The Cas9 nuclease is targeted to genomic DNA using guide RNAs (gRNA), either as the native dual RNA system consisting of a DNA-targeting CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), or as a chimeric single guide RNA (sgRNA). Entirely DNA-free CRISPR-Cas9 systems using either Cas9 protein or Cas9 mRNA and chemically synthesized gRNAs allow for transient expression of CRISPR-Cas9 components, thereby reducing the potential for off-targeting, which is a significant advantage in therapeutic applications. In addition, the use of synthetic gRNA allows for the incorporation of chemical modifications for enhanced properties including improved stability. Previous studies have demonstrated the utility of chemically modified gRNAs, but have focused on one pattern with multiple modifications in co-electroporation with Cas9 mRNA or multiple modifications and patterns with Cas9 plasmid lipid co-transfections. Here we present gene editing results using a series of chemically modified synthetic sgRNA molecules and chemically modified crRNA:tracrRNA molecules in both electroporation and lipid transfection assessing indel formation and/or phenotypic gene knockout. We show that while modifications are required for co-electroporation with Cas9 mRNA, some modification patterns of the gRNA are toxic to cells compared to the unmodified gRNA and most modification patterns do not significantly improve gene editing efficiency. We also present modification patterns of the gRNA that can modestly improve Cas9 gene editing efficiency when co-transfected with Cas9 mRNA or Cas9 protein (> 1.5-fold difference). These results indicate that for certain applications, including those relevant to primary cells, the incorporation of some, but not all chemical modification patterns on synthetic crRNA:tracrRNA or sgRNA can be beneficial to CRISPR-Cas9 gene editing.
Project description:Editing the ?-globin locus in hematopoietic stem cells is an alternative therapeutic approach for gene therapy of ?-thalassemia and sickle cell disease. Using the CRISPR/Cas9 system, we genetically modified human hematopoietic stem and progenitor cells (HSPCs) to mimic the large rearrangements in the ?-globin locus associated with hereditary persistence of fetal hemoglobin (HPFH), a condition that mitigates the clinical phenotype of patients with ?-hemoglobinopathies. We optimized and compared the efficiency of plasmid-, lentiviral vector (LV)-, RNA-, and ribonucleoprotein complex (RNP)-based methods to deliver the CRISPR/Cas9 system into HSPCs. Plasmid delivery of Cas9 and gRNA pairs targeting two HPFH-like regions led to high frequency of genomic rearrangements and HbF reactivation in erythroblasts derived from sorted, Cas9<sup>+</sup> HSPCs but was associated with significant cell toxicity. RNA-mediated delivery of CRISPR/Cas9 was similarly toxic but much less efficient in editing the ?-globin locus. Transduction of HSPCs by LVs expressing Cas9 and gRNA pairs was robust and minimally toxic but resulted in poor genome-editing efficiency. Ribonucleoprotein (RNP)-based delivery of CRISPR/Cas9 exhibited a good balance between cytotoxicity and efficiency of genomic rearrangements as compared to the other delivery systems and resulted in HbF upregulation in erythroblasts derived from unselected edited HSPCs.