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CRISPR-based non-nucleic acid detection.
Characterization of clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) trans-cleavage activities has initiated the era of next-generation CRISPR diagnostics. By using the trans-cleavage reaction for signal output, CRISPR systems have been engineered to detect non-nucleic acids (NNAs), including ions, inorganic small molecules, organic compounds, proteins, and bacteria. Diverse strategies are being used to specifically recognize NNAs and regulate Cas trans-cleavage activities, via generation or depletion of output signals. In this review, we introduce the principles and advantages of CRISPR-based NNA detection. We then classify CRISPR-based NNA detection strategies into three classes: the generation or depletion of free activators, synthesis of crRNAs, and reconstruction of active Cas effectors. Finally, we discuss the challenges and potential strategies to advance both clinical and nonclinical applications of CRISPR-based NNA detection.
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Gene Editing Thumbs a Ride with Oncolytic Virotherapy.
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Gene editing to facilitate hybrid crop production.
Capturing heterosis (hybrid vigor) is a promising way to increase productivity in many crops; hybrid crops often have superior yields, disease resistance, and stress tolerance compared with their parental inbred lines. The full utilization of heterosis faces a number of technical problems related to the specifics of crop reproductive biology, such as difficulties with generating and maintaining male-sterile lines and the low efficiency of natural cross-pollination for some genetic combinations. Innovative technologies, such as development of artificial in vitro systems for hybrid production and apomixis-based systems for maintenance of the resulting heterotic progeny, may substantially facilitate the production of hybrids. Genome editing using specifically targeted nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (CRISPR/Cas9) systems, which recognize targets by RNA:DNA complementarity, has recently become an integral part of research and development in life science. In this review, we summarize the progress of genome editing technologies for facilitating the generation of mutant male sterile lines, applications of haploids for hybrid production, and the use of apomixis for the clonal propagation of elite hybrid lines.
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Efficient CRISPR/Cas9 genome editing with Citrus embryogenic cell cultures.
BACKGROUND: Development of precise genome editing strategies is a prerequisite for producing edited plants that can aid in the study of gene function and help understand the genetic traits in a cultivar. Citrus embryogenic cell cultures can be used to rapidly produce a large population of genome edited transformed citrus lines. The ability to introduce specific mutations in the genome of these cells using two constructs (pC-PDS1 and pC-PDS2) was evaluated in this study. RESULTS: Citrus sinensis 'EV2' embryogenic cell cultures are amenable to Agrobacterium-mediated CRISPR/Cas9-based genome editing. Guide RNAs (gRNAs) targeting two locations in the phytoene desaturase (PDS) gene were either driven by the Arabidopsis U6-26 promoter (pC-PDS1) or assembled as a Csy4 array under the control of the CmYLCV promoter (pC-PDS2). All transgenic embryos were completely albino and no variegated phenotype was observed. We evaluated 12 lines from each construct in this study and the majority contain either insertion (1-2 bp), substitution (1 bp), or deletion (1-3 bp) mutations that occurred close to the protospacer adjacent motif. CONCLUSIONS: Both the pC-PDS1 and pC-PDS2 could successfully edit the citrus embryogenic cell cultures. However, the editing efficiency was dependent on the gRNA, confirming that the selection of a proper gRNA is essential for successful genome editing using the CRISPR/Cas9 technique. Also, utilization of embryogenic cell cultures offers another option for successful genome editing in citrus.
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CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway.
With its high efficiency for site-specific genome editing and easy manipulation, the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (CAS9) system has become the most widely used gene editing technology in biomedical research. In addition, significant progress has been made for the clinical development of CRISPR/CAS9 based gene therapies of human diseases, several of which are entering clinical trials. Here we report that CAS9 protein can function as a genome mutator independent of any exogenous guide RNA (gRNA) in human cells, promoting genomic DNA double-stranded break (DSB) damage and genomic instability. CAS9 interacts with the KU86 subunit of the DNA-dependent protein kinase (DNA-PK) complex and disrupts the interaction between KU86 and its kinase subunit, leading to defective DNA-PK-dependent repair of DNA DSB damage via non-homologous end-joining (NHEJ) pathway. XCAS9 is a CAS9 variant with potentially higher fidelity and broader compatibility, and dCAS9 is a CAS9 variant without nuclease activity. We show that XCAS9 and dCAS9 also interact with KU86 and disrupt DNA DSB repair. Considering the critical roles of DNA-PK in maintaining genomic stability and the pleiotropic impact of DNA DSB damage responses on cellular proliferation and survival, our findings caution the interpretation of data involving CRISPR/CAS9-based gene editing and raise serious safety concerns of CRISPR/CAS9 system in clinical application.
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A Survey of Genome Editing Activity for 16 Cas12a Orthologs.
The class 2 CRISPR-Cas endonuclease Cas12a (previously known as Cpf1) offers several advantages over Cas9, including the ability to process its own array and the requirement for just a single RNA guide. These attributes make Cas12a promising for many genome engineering applications. To further expand the suite of Cas12a tools available, we tested 16 Cas12a orthologs for activity in eukaryotic cells. Four of these new enzymes demonstrated targeted activity, one of which, from Moraxella bovoculi AAX11_00205 (Mb3Cas12a), exhibited robust indel formation. We also showed that Mb3Cas12a displays some tolerance for a shortened PAM (TTN versus the canonical Cas12a PAM TTTV). The addition of these enzymes to the genome editing toolbox will further expand the utility of this powerful technology.
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Application of CRISPR-Cas9-Mediated Genome Editing for the Treatment of Myotonic Dystrophy Type 1.
Myotonic dystrophy type 1 (DM1) is a debilitating multisystemic disorder, caused by expansion of a CTG microsatellite repeat in the 3' untranslated region of the DMPK (dystrophia myotonica protein kinase) gene. To date, novel therapeutic approaches have focused on transient suppression of the mutant, repeat-expanded RNA. However, recent developments in the field of genome editing have raised the exciting possibility of inducing permanent correction of the DM1 genetic defect. Specifically, repurposing of the prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) system has enabled programmable, site-specific, and multiplex genome editing. CRISPR-based strategies for the treatment of DM1 can be applied either directly to patients, or indirectly through the ex vivo modification of patient-derived cells, and they include excision of the repeat expansion, insertion of synthetic polyadenylation signals upstream of the repeat, steric interference with RNA polymerase II procession through the repeat leading to transcriptional downregulation of DMPK, and direct RNA targeting of the mutant RNA species. Potential obstacles to such therapies are discussed, including the major challenge of Cas9 and guide RNA transgene/ribonuclear protein delivery, off-target gene editing, vector genome insertion at cut sites, on-target unintended mutagenesis (e.g., repeat inversion), pre-existing immunity to Cas9 or AAV antigens, immunogenicity, and Cas9 persistence.
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Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9.
BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology has rapidly emerged as a very effective tool for gene editing. Although great advances on gene editing in the medical entomology field have arisen, no attempts of gene editing have been reported in sand flies, the vectors of Leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we described a detailed protocol for sand fly embryo microinjection taking into consideration the sand fly life cycle, and manipulation and oviposition requirements of this non-model organism. Following our microinjection protocol, a hatching rate of injected embryos of 11.90%-14.22% was achieved, a rate consistent with other non-model organism dipterans such as mosquitoes. Essential factors for the adaptation of CRISPR/Cas9 technology to the sand fly field were addressed including the selection of a target gene and the design and production of sgRNA. An in vitro cleavage assay was optimized to test the activity of each sgRNA and a protocol for Streptococcus pyogenes Cas9 (spCas9) protein expression and purification was described. Relevant considerations for a successful gene editing in the sand fly such as specifics of embryology and double-stranded break DNA repair mechanisms were discussed. CONCLUSION AND SIGNIFICANCE: The step-by-step methodology reported in this article will be of significant use for setting up a sand fly embryo microinjection station for the incorporation of CRISPR/Cas9 technology in the sand fly field. Gene editing strategies used in mosquitoes and other model insects have been adapted to work with sand flies, providing the tools and relevant information for adapting gene editing techniques to the vectors of Leishmaniasis. Gene editing in sand flies will provide essential information on the biology of these vectors of medical and veterinary relevance and will rise a better understanding of vector-parasite-host interactions.
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Enhancing in planta gene targeting efficiencies in Arabidopsis using temperature-tolerant CRISPR/LbCas12a.
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Multiplexed conditional genome editing with Cas12a in Drosophila.
CRISPR-Cas genome engineering has revolutionized biomedical research by enabling targeted genome modification with unprecedented ease. In the popular model organism Drosophila melanogaster, gene editing has so far relied exclusively on the prototypical CRISPR nuclease Cas9. Additional CRISPR systems could expand the genomic target space, offer additional modes of regulation, and enable the independent manipulation of genes in different cells of the same animal. Here we describe a platform for efficient Cas12a gene editing in Drosophila We show that Cas12a from Lachnospiraceae bacterium, but not Acidaminococcus spec., can mediate robust gene editing in vivo. In combination with most CRISPR RNAs (crRNAs), LbCas12a activity is high at 29 °C, but low at 18 °C, enabling modulation of gene editing by temperature. LbCas12a can directly utilize compact crRNA arrays that are substantially easier to construct than Cas9 single-guide RNA arrays, facilitating multiplex genome engineering. Furthermore, we show that conditional expression of LbCas12a is sufficient to mediate tightly controlled gene editing in a variety of tissues, allowing detailed analysis of gene function in a multicellular organism. We also test a variant of LbCas12a with a D156R point mutation and show that it has substantially higher activity and outperforms a state-of-the-art Cas9 system in identifying essential genes. Cas12a gene editing expands the genome-engineering toolbox in Drosophila and will be a powerful method for the functional annotation of the genome. This work also presents a fully genetically encoded Cas12a system in an animal, laying out principles for the development of similar systems in other genetically tractable organisms for multiplexed conditional genome engineering.
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