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Transgenic and Genome Editing Approaches for Modifying Plant Oils.
Vegetable oils are important for human and animal nutrition and as renewable resources for chemical feedstocks. We provide an overview of transgenic and genome editing approaches for modifying plant oils, describing useful model and crop systems and different strategies for transgenic modifications. We also describe new genome editing approaches that are beginning to be applied to oilseed plants and crops. These approaches are illustrated with examples for modifying the nutritional quality of vegetable oils by altering fatty acid desaturation, producing non-native fatty acids in oilseeds, and enhancing the overall accumulation of oil in seeds and leaves.
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Intrinsic RNA-targeting activity limits the applicability of CRISPR-Cas13.
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Precision genome editing.
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A Novel White-to-Blue Colony Formation Assay to Select for Optimized sgRNAs.
CRISPR/Cas9-mediated genome editing technology consists of a single-guide RNA (sgRNA), and the Cas9 endonuclease has the potential to treat genetic diseases in most tissues and organisms. In this system, the Cas9 protein can be directed to target genomic DNA sequences as "molecular scissors" with the guidance of sgRNAs. However, the target-specific activities of different sgRNAs are highly variable; thus, it is crucial to search for a simple, quick and economical method to screen for optimized sgRNAs with high target specificity. We have adopted and verified a newly developed white-to-blue colony formation assay to quickly screen for sgRNAs optimized for the EphA2 gene, which is highly expressed in hormone-resistant prostate cancer (PC-3) cells. This assay promises to screen for optimized sgRNAs more simply, rapidly, and efficiently. Our results suggest that the white-to-blue colony formation assay might be a useful screening strategy to quickly select for optimized sgRNAs.
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Delivery of exogenous proteins into eggs by injection into the mother's ovary (IMO) in zebrafish.
Genome editing has had profound effects on biological experimentation and can now be applied to many organisms, including non-conventional models. However, the introduction of genome editing components is time- and labor-consuming and sometimes requires special skills for microinjection. In this study, we developed a technique to deliver exogenous proteins into eggs by injection into the mother's ovary (IMO), which leads to the delivery of CRISPR/Cas9 into the eggs of oviparous animals, including fish. To test this technique, we examined whether exogenous proteins tagged with GFP or luciferase (Luc), and fluorescent-labeled RNP (Cas9 and sgRNA complex), can be delivered into eggs by IMO. When GFP-Luc or Cas9-Luc was delivered by IMO, their incorporation into fertilized eggs was confirmed by GFP fluorescence or luciferase activity; proteins were accumulated in the yolk. Cas9-RNP (targeting tyrosinase) was also incorporated into the eggs. However, genome editing of the target gene, tyrosinase, was not observed yet. This is presumably because the RNP delivered by IMO was packed in the yolk granules and did not reach into the embryonic nuclei. Thus, this report shows that exogenous molecules including Cas9-RNP were successfully delivered into fertilized eggs by IMO. Transferring the delivered RNP into nuclei will be critical for successful genome editing via the IMO delivery system.
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[Establishment and validation of human cancer cell lines with stable Cas9 expression].
Objective: To establish human cancer cell strains with stable Cas9 expression, and to validate the gene editing activity of Cas9 for simple gene editing in future study. Methods: Fifteen cancer cell lines of different tissue origins were infected with pLv-EF1α-Cas9-Flag-Neo or pLv-EF1α-Cas9-Flag-Puro by lentivirus and clone selection was employed to screen Cas9 stably expressed cancer cell lines. Afterward designed guide RNA vectors targeting TSC22 gene were transiently transfected into 3 of cell lines, and subsequently the gene editing activity of Cas9 was evaluated by genomic PCR, sequencing and Western blot. Results: Sixty-nine human cancer cell strains with stable Cas9 expression from different cancers were established, and by transient transfection with designed guide RNA, long fragment deletion was detected in TSC22 gene. Conclusions: Sixty-nine human cancer cell strains are successfully established with stable expression of Cas9 protein and gene editing activity. These cell strains may be employed in large-scale drug screening, screening of new drug targets and gene function investigation.
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Enhancing the genome editing toolbox: genome wide CRISPR arrayed libraries.
CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome. For flexibility in generating stable cell lines the sgRNAs have been cloned in a lentivirus backbone containing PiggyBac transposase recognition elements together with fluorescent and drug selection markers. Over 95% of tested sgRNA induced specific DNA cleavage as measured by CEL-1 assays. Furthermore, sgRNA targeting GPI anchor protein pathway genes induced loss of function mutations in human and mouse cell lines measured by FLAER labelling. These arrayed libraries offer the prospect for performing screens on individual genes, combinations as well as larger gene sets. They also facilitate rapid deconvolution of signals from genome-wide screens. This set of vectors provide an organized comprehensive gene editing toolbox of considerable scientific value.
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Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing.
The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications.
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Retron Library Recombineering: Next Powerful Tool for Genome Editing after CRISPR/Cas.
Retron library recombineering (RLR) is a powerful tool in the field of genome editing that exceeds the scope and specificity of the CRISPR/Cas technique. In RLR, single-stranded DNA produced in vivo by harnessing the in-built potential of bacterial retrons is used for replication-dependent genome editing. RLR introduces several genomic variations at once, resulting in pooled and barcoded variant libraries, thus permitting multiplexed applications. Retron-generated RT-DNA has already shown promise for use in genome editing. Thus, this new tool will result in fresh, intriguing, and surprising developments in molecular biology and its juncture with other disciplines of research, including medicine, agriculture, and microbiology. In this review, we discuss the current state of this brand-new tool that could eventually boost genome editing research.
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Biased and Unbiased Methods for the Detection of Off-Target Cleavage by CRISPR/Cas9: An Overview.
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 endonuclease (Cas9) derived from bacterial adaptive immune systems is a revolutionary tool used in both basic and applied science. It is a versatile system that enables the genome of different species to be modified by generating double strand breaks (DSBs) at specific locations. However, all of the CRISPR/Cas9 systems can also produce DSBs at off-target sites that differ substantially from on-target sites. The generation of DSBs in locations outside the intended site can produce mutations that need to be carefully monitored, especially when using these tools for therapeutic purposes. However, off-target analyses of the CRISPR/Cas9 system have been very challenging, particularly when performed directly in cells. In this manuscript, we review the different strategies developed to identify off-targets generated by CRISPR/cas9 systems and other specific nucleases (ZFNs, TALENs) in real target cells.
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