Multiplex Cas9-based excision of CLCuV betasatellite and DNA-A revealed reduction of viral load with asymptomatic cotton plants.

MAIN CONCLUSION: Multiplexed Cas9-based genome editing of cotton resulted in reduction of viral load with asymptomatic cotton plants. In depth imaging of proteomic dynamics of resulting CLCuV betasatellite and DNA-A protein was also performed. The notorious  cotton leaf curl virus (CLCuV), which is transmitted by the sap-sucking insect whitefly, continuously damages cotton crops. Although the application of various toxins and RNAi has shown some promise, sustained control has not been achieved. Consequently, CRISPR_Cas9 was applied by designing multiplex targets against DNA-A (AC2 and AC3) and betasatellite (ßC1) of CLCuV using CRISPR direct and ligating into the destination vector of the plant using gateway ligation method. The successful ligation of targets into the destination vector was confirmed by the amplification of 1049 bp using a primer created from the promoter and target, while restriction digestion using the AflII and Asc1 enzymes determined how compact the plasmid developed and the nucleotide specificity of the plasmid was achieved through Sanger sequencing. PCR confirmed the successful introduction of plasmid into CKC-1 cotton variety. Through Sanger sequencing and correlation with the mRNA expression of DNA-A and betasatellite in genome-edited cotton plants subjected to agroinfiltration of CLCuV infectious clone, the effectiveness of knockout was established. The genome-edited cotton plants demonstrated edited efficacy of 72% for AC2 and AC3 and 90% for the (ßC1) through amplicon sequencing, Molecular dynamics (MD) simulations were used to further validate the results. Higher RMSD values for the edited ßC1 and AC3 proteins indicated functional loss caused by denaturation. Thus, CRISPR_Cas9 constructs can be rationally designed using high-throughput MD simulation technique. The confidence in using this technology to control plant virus and its vector was determined by the knockout efficiency and the virus inoculation assay.

Different DNA repair pathways are involved in single-strand break-induced genomic changes in plants.

In nature, single-strand breaks (SSBs) in DNA occur more frequently (by orders of magnitude) than double-strand breaks (DSBs). SSBs induced by the CRISPR/Cas9 nickase at a distance of 50-100 bp on opposite strands are highly mutagenic, leading to insertions/deletions (InDels), with insertions mainly occurring as direct tandem duplications. As short tandem repeats are overrepresented in plant genomes, this mechanism seems to be important for genome evolution. We investigated the distance at which paired 5'-overhanging SSBs are mutagenic and which DNA repair pathways are essential for insertion formation in Arabidopsis thaliana. We were able to detect InDel formation up to a distance of 250 bp, although with much reduced efficiency. Surprisingly, the loss of the classical nonhomologous end joining (NHEJ) pathway factors KU70 or DNA ligase 4 completely abolished tandem repeat formation. The microhomology-mediated NHEJ factor POLQ was required only for patch-like insertions, which are well-known from DSB repair as templated insertions from ectopic sites. As SSBs can also be repaired using homology, we furthermore asked whether the classical homologous recombination (HR) pathway is involved in this process in plants. The fact that RAD54 is not required for homology-mediated SSB repair demonstrates that the mechanisms for DSB- and SSB-induced HR differ in plants.

CRISPR Guide RNA Design Guidelines for Efficient Genome Editing.

The simple applicability and facile target programming of the CRISPR/Cas9-system abolish the major boundaries of previous genome editing tools, making it the tool of choice for generating site-specific genome alterations. Its versatility and efficacy have been demonstrated in various organisms; however, accurately predicting guide RNA efficiencies remains an organism-independent challenge. Thus, designing optimal guide RNAs is essential to maximize the experimental outcome. Here, we summarize the current knowledge for guide RNA design and highlight discrepancies between different experimental systems.

Live-Cell CRISPR Imaging in Plant Cells with a Telomere-Specific Guide RNA.

Chromatin organization is highly dynamic in living cells. Therefore, it might have a regulatory role over biological mechanisms like transcription, replication, and DNA repair. To elucidate how these mechanisms are regulated, it is required to establish imaging methods to visualize the chromatin dynamic in living cells. Here, we provide a protocol for a live plant cell imaging technique based on application of two orthologs of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) from Streptococcus pyogenes and Staphylococcus aureus. This technique uses the inactive variants of Cas9 combined with different fluorescent proteins (GFP and mRuby) and telomere-specific guide RNA to target telomeric repeats in Nicotiana benthamiana. Our immuno-FISH data revealed that signals arising from the CRISPR/dCas9 method are specifically belonging to telomeric regions.

In planta gene targeting can be enhanced by the use of CRISPR/Cas12a.

The controlled change of plant genomes by homologous recombination (HR) is still difficult to achieve. We previously developed the in planta gene targeting (ipGT) technology which depends on the simultaneous activation of the target locus by a double-strand break and the excision of the target vector. Whereas the use of SpCas9 resulted in low ipGT frequencies in Arabidopsis, we were recently able to improve the efficiency by using egg cell-specific expression of the potent but less broadly applicable SaCas9 nuclease. In this study, we now tested whether we could improve ipGT further, by either performing it in cells with enhanced intrachromosomal HR efficiencies or by the use of Cas12a, a different kind of CRISPR/Cas nuclease with an alternative cutting mechanism. We could show before that plants possess three kinds of DNA ATPase complexes, which all lead to instabilities of homologous genomic repeats if lost by mutation. As these proteins act in independent pathways, we tested ipGT in double mutants in which intrachromosomal HR is enhanced 20-80-fold. However, we were not able to obtain higher ipGT frequencies, indicating that mechanisms for gene targeting (GT) and chromosomal repeat-induced HR differ. However, using LbCas12a, the GT frequencies were higher than with SaCas9, despite a lower non-homologous end-joining (NHEJ) induction efficiency, demonstrating the particular suitability of Cas12a to induce HR. As SaCas9 has substantial restrictions due to its longer GC rich PAM sequence, the use of LbCas12a with its AT-rich PAM broadens the range of ipGT drastically, particularly when targeting in CG-deserts like promoters and introns.

Plant breeding at the speed of light: the power of CRISPR/Cas to generate directed genetic diversity at multiple sites.

Classical plant breeding was extremely successful in generating high yielding crop varieties. Yet, in modern crops, the long domestication process has impoverished the genetic diversity available for breeding. This is limiting further improvements of elite germplasm by classical approaches. The CRISPR/Cas system now enables promising new opportunities to create genetic diversity for breeding in an unprecedented way. Due to its multiplexing ability, multiple targets can be modified simultaneously in an efficient way, enabling immediate pyramiding of multiple beneficial traits into an elite background within one generation. By targeting regulatory elements, a selectable range of transcriptional alleles can be generated, enabling precise fine-tuning of desirable traits. In addition, by targeting homologues of so-called domestication genes within one generation, it is now possible to catapult neglected, semi-domesticated and wild plants quickly into the focus of mainstream agriculture. This further enables the use of the enormous genetic diversity present in wild species or uncultured varieties of crops as a source of allele-mining, widely expanding the crop germplasm pool.

Application of CRISPR/Cas to Understand Cis- and Trans-Regulatory Elements in Plants.

The recent emergence of the CRISPR/Cas system as a genome editing tool enables simple, fast, and efficient induction of DNA double-strand breaks at precise positions in the genome. This has proven extremely useful for analysis and modification of protein-coding sequences. Regulatory sequences have received much less attention, but can now be quickly and easily disrupted as well. Editing of cis-regulatory elements (CRE) offers considerable potential for crop improvement via fine-tuning of gene expression that cannot be achieved by simple KO mutations, but its widespread application is still hampered by a lack of precise knowledge about functional motifs in CRE. As demonstrated for mammalian cells, CRISPR/Cas is also extremely useful for the identification and analysis of CRE in their native environment on a large scale using tiling screens. Transcriptional complexes are another promising target for crop genome editing, as demonstrated for pathogen resistance and regulation of flowering. The development of more diverse and sophisticated CRISPR/Cas tools for genome editing will allow even more efficient and powerful approaches for editing of regulatory sequences in the future.

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13.

Currently, biology is revolutionized by ever growing applications of the CRISPR/Cas system. As discussed in this Review, new avenues have opened up for plant research and breeding by the use of the sequence-specific DNases Cas9 and Cas12 (formerly named Cpf1) and, more recently, the RNase Cas13 (formerly named C2c2). Although double strand break-induced gene editing based on error-prone nonhomologous end joining has been applied to obtain new traits, such as powdery mildew resistance in wheat or improved pathogen resistance and increased yield in tomato, improved technologies based on CRISPR/Cas for programmed change in plant genomes via homologous recombination have recently been developed. Cas9- and Cas12- mediated DNA binding is used to develop tools for many useful applications, such as transcriptional regulation or fluorescence-based imaging of specific chromosomal loci in plant genomes. Cas13 has recently been applied to degrade mRNAs and combat viral RNA replication. By the possibility to address multiple sequences with different guide RNAs and by the simultaneous use of different Cas proteins in a single cell, we should soon be able to achieve complex changes of plant metabolism in a controlled way.

The CRISPR/Cas revolution reaches the RNA world: Cas13, a new Swiss Army knife for plant biologists.

Application of the bacterial CRISPR/Cas systems to eukaryotes is revolutionizing biology. Cas9 and Cas12 (previously called Cpf1) are widely used as DNA nucleases for inducing site-specific DNA breaks for different kinds of genome engineering applications, and in their mutated forms as DNA-binding proteins to modify gene expression. Moreover, histone modifications, as well as cytosine methylation or base editing, were achieved with these systems in plants. Recently, with the discovery of the nuclease Cas13a (previously called C2c2), molecular biologists have obtained a system that enables sequence-specific cleavage of single-stranded RNA molecules. The latest experiments with this and also the alternative Cas13b system demonstrate that these proteins can be used in a similar manner in eukaryotes for RNA manipulation as Cas9 and Cas12 for DNA manipulations. The first application of Cas13a for post-transcriptional regulation of gene expression in plants has been reported. Recent results show that the system is also applicable for combating viral infection in plants. As single-stranded RNA viruses are by far the most abundant class of viruses in plants, the application of this system is of special promise for crops. More interesting applications are imminent for plant biologists, with nuclease dead versions of Cas13 enabling the ability to visualize RNA molecules in vivo, as well as to edit different kinds of RNA molecules at specific bases by deamination or to modify them by conjugation. Moreover, by combining DNA- and RNA-directed systems, the most complex of changes in plant metabolism might be achievable.

Efficient in planta gene targeting in Arabidopsis using egg cell-specific expression of the Cas9 nuclease of Staphylococcus aureus.

Gene targeting (GT), the programmed change of genomic sequences by homologous recombination (HR), is still a major challenge in plants. We previously developed an in planta GT strategy by simultaneously releasing from the genome a dsDNA donor molecule and creating a double-stranded break (DSB) at a specific site within the targeted gene. Using Cas9 form Streptococcus pyogenes (SpCas9) under the control of a ubiquitin gene promoter, we obtained seeds harbouring GT events, although at a low frequency. In the present research we tested different developmentally controlled promotors and different kinds of DNA lesions for their ability to enhance GT of the acetolactate synthase (ALS) gene of Arabidopsis. For this purpose, we used Staphylococcus aureus Cas9 (SaCas9) nuclease and the SpCas9 nickase in various combinations. Thus, we analysed the effect of single-stranded break (SSB) activation of a targeted gene and/or the HR donor region. Moreover, we tested whether DSBs with 5' or 3' overhangs can improve in planta GT. Interestingly, the use of the SaCas9 nuclease controlled by an egg cell-specific promoter was the most efficient: depending on the line, in the very best case 6% of all seeds carried GT events. In a third of all lines, the targeting occurred around the 1% range of the tested seeds. Molecular analysis revealed that in about half of the cases perfect HR of both DSB ends occurred. Thus, using the improved technology, it should now be feasible to introduce any directed change into the Arabidopsis genome at will.

Towards CRISPR/Cas crops - bringing together genomics and genome editing.

Contents 682 I. 682 II. 683 III. 684 IV. 685 V. 685 VI. 688 VII. 690 VIII. 694 694 References 694 SUMMARY: With the rapid increase in the global population and the impact of climate change on agriculture, there is a need for crops with higher yields and greater tolerance to abiotic stress. However, traditional crop improvement via genetic recombination or random mutagenesis is a laborious process and cannot keep pace with increasing crop demand. Genome editing technologies such as clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (CRISPR/Cas) allow targeted modification of almost any crop genome sequence to generate novel variation and accelerate breeding efforts. We expect a gradual shift in crop improvement away from traditional breeding towards cycles of targeted genome editing. Crop improvement using genome editing is not constrained by limited existing variation or the requirement to select alleles over multiple breeding generations. However, current applications of crop genome editing are limited by the lack of complete reference genomes, the sparse knowledge of potential modification targets, and the unclear legal status of edited crops. We argue that overcoming technical and social barriers to the application of genome editing will allow this technology to produce a new generation of high-yielding, climate ready crops.

Knocking out consumer concerns and regulator's rules: efficient use of CRISPR/Cas ribonucleoprotein complexes for genome editing in cereals.

Selection-free genome editing using Cas9 ribonucleoprotein embryo bombardment has been achieved for maize and wheat. This is a breakthrough that should make new breeding technologies more acceptable for worldwide use.