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High-efficiency generation of fertile transplastomic Arabidopsis plants.
The development of technologies for the stable genetic transformation of plastid (chloroplast) genomes has been a boon to both basic and applied research. However, extension of the transplastomic technology to major crops and model plants has proven extremely challenging, and the species range of plastid transformation is still very much limited in that most species currently remain recalcitrant to plastid genome engineering. Here, we report an efficient plastid transformation technology for the model plant Arabidopsis thaliana that relies on root-derived microcalli as a source tissue for biolistic transformation. The method produces fertile transplastomic plants at high frequency when combined with a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-generated knockout allele of a nuclear locus that enhances sensitivity to the selection agent used for isolation of transplastomic events. Our work makes the model organism of plant biology amenable to routine engineering of the plastid genome, facilitates the combination of plastid engineering with the power of Arabidopsis nuclear genetics, and informs the future development of plastid transformation protocols for other recalcitrant species.
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Regulatory Considerations for Genome-Edited T-cell Therapies.
Methods to engineer the genomes of human cells for therapeutic intervention continue to advance at a remarkable pace. Chimeric antigen receptor-engineered T lymphocytes have pioneered the way for these therapies, initially beginning with insertions of chimeric antigen receptor transgenes into T-cell genomes using classical gene therapy vectors. The broad use of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies to edit endogenous genes has now opened the door to a new era of precision medicine. To add complexity, many engineered cellular therapies under development integrate gene therapy with genome editing to introduce novel biological functions and enhance therapeutic efficacy. Here, we review the current state of scientific, translational, and regulatory oversight of gene-edited cell products.
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Influence of gRNA efficiency and inversion size on the frequency of CRISPR/Cas9-induced chromosomal inversions in tomato protoplasts.
BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 enables induction of chromosomal inversions from hundreds of base pairs to millions of base pairs, but the factors influencing inversion frequency are not well understood. Prior reports differ in species, detection methods, and delivery strategies, making direct comparisons difficult. We addressed this by introducing a normalisation strategy based on a reference guide RNA (gRNA) as an internal standard and testing inversion sizes spanning kilobases to tens of megabases in tomato protoplasts. RESULTS: Tomato (Solanum lycopersicum) protoplasts were transfected with constructs encoding a fixed “reference” gRNA and a second “variable” gRNA positioned at increasing genomic distances, creating potential inversions from 1 kilobase to 37.5 megabases. Using the reference gRNA to normalise across samples, we found that up to ~ 1 megabase, inversion frequency tracked the cutting efficiency of the less efficient gRNA, consistent with gRNA activity being a major contributor within the chromosome tested. For these intervals, the inversion frequencies reached up to 1.24% when both gRNAs were efficient. Above ~ 1 megabase, inversion frequencies declined sharply despite efficient cutting, suggesting a size-dependent barrier to inversion formation; for example, 37.5 megabase inversions occurred at substantially lower frequency (up to 0.18%) despite efficient gRNAs. Because each interval corresponds to a distinct genomic location. Inversions were only observed when both gRNAs were active, and large deletions were more frequent than inversions when dual breaks were induced. CONCLUSIONS: In our experiments, the gRNA cutting efficiency was a major determinant of inversion frequency in our experiment up to ~ 1 megabase, while locus-specific genomic context may also contribute., Larger inversions may be limited by an additional, size-dependent constraint. These findings inform the design of edits aimed at reverting breeding-relevant inversions (for example, those linked to resistance loci) and suggest that achieving high efficiency for multi-megabase inversions will require strategies that overcome spatial or repair-related constraints. The internal reference gRNA normalises sample-to-sample variability in DNA delivery and Cas9 activity, enabling direct comparison of the performance of different gRNAs across samples on a shared, ratio-based scale. This ratio-to-reference strategy may likewise be used to benchmark gRNA performance and edit yields (inversions, deletions, translocations, and base/prime edits) across transfections in plant protoplasts, and may be extended to additional cell systems beyond plants.
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CRISPR-Cas9-mediated chromosome engineering in Arabidopsis thaliana.
The rise of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system has made it possible to induce double-strand breaks at almost any desired target site in the genome. In plant somatic cells, double-strand breaks are predominantly repaired by the error-prone nonhomologous end-joining pathway, which can lead to mutations at the break site upon repair. So far, it had only been possible to induce genomic changes of up to a few hundred kilobases in plants utilizing this mechanism. However, by combining the highly efficient Staphylococcus aureus Cas9 (SaCas9) with an egg-cell-specific promoter to facilitate heritable mutations, chromosomal rearrangements in the Mb range, such as inversion and translocations, were obtained in Arabidopsis thaliana recently. Here we describe the chromosome-engineering protocol used to generate these heritable chromosomal rearrangements in A. thaliana. The protocol is based on Agrobacterium-mediated transformation of A. thaliana with transfer DNA constructs containing SaCas9, which is driven by an egg-cell-specific promoter, and two guide RNAs that have been preselected based on their cutting efficiency. In the T1 generation, primary transformants are selected and, if required, analyzed by Droplet Digital PCR and propagated. In the following generations, junction-specific PCR screenings are carried out until plants that carry the rearrangement homozygously are identified. Using this protocol, overall rearrangement frequencies range between 0.03% and 0.5%, depending on the type of rearrangement. In total, it takes about 1 year to establish homozygous lines.
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Transcriptional enhancers: from prediction to functional assessment on a genome-wide scale.
Enhancers are cis-regulatory sequences located distally to target genes. These sequences consolidate developmental and environmental cues to coordinate gene expression in a tissue-specific manner. Enhancer function and tissue specificity depend on the expressed set of transcription factors, which recognize binding sites and recruit cofactors that regulate local chromatin organization and gene transcription. Unlike other genomic elements, enhancers are challenging to identify because they function independently of orientation, are often distant from their promoters, have poorly defined boundaries, and display no reading frame. In addition, there are no defined genetic or epigenetic features that are unambiguously associated with enhancer activity. Over recent years there have been developments in both empirical assays and computational methods for enhancer prediction. We review genome-wide tools, CRISPR advancements, and high-throughput screening approaches that have improved our ability to both observe and manipulate enhancers in vitro at the level of primary genetic sequences, chromatin states, and spatial interactions. We also highlight contemporary animal models and their importance to enhancer validation. Together, these experimental systems and techniques complement one another and broaden our understanding of enhancer function in development, evolution, and disease.
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A review on CRISPR/Cas-based epigenetic regulation in plants.
Epigenetic changes are the heritable modifications in genes without altering DNA sequences. The epigenetic changes occur in the plant genomes to regulate gene expression patterns, which were used to regulate different biological processes, including coping various environmental stresses. These changes, including DNA methylation, non-coding RNA regulation, and histone modification, play a vital role in the transcription and translation processes to regulate gene expression. Gene engineering for the development of stress-tolerant crops via the DNA methylation pathway initially needs a proper selection of genes and its promoter. Manipulating epigenetics requires genetic engineering tools such as Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas). However, CRISPR/Cas9 mediated epigenetic editing refers to transcriptional reprogramming at the targeted sites using epigenetic enzymes fused with decatalytical Cas9 (dCas9). This review focused on the different epigenetic mechanisms in plants and their potential contribution to developing epigenetic tools. The dCas9 endonuclease tethered with transcriptional repressor or activator domain leads to CRISPR inhibitor (CRISPRi) or activator (CRISPRa) for regulating gene expression. The dCas9 has been successfully fused with other various effector domains for constructing epigenetic tools, including the DNA methyltransferase 3A (DNMT3A), or the DNA demethylase TET. Multiple efforts have been made to improve epigenome editing in plants. Initially, incorporating SunTag into the dCas9-EpiEffector complex was used as an epigenetic tool; demethylation of target loci with dCas9-SunTag-TET1 futher increased its efficiency. Additionally, SunTag could also be fused with the dCas9-DNMT3A complex to augment CpG methylation at a targeted loci.
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CRISPR-READI: Efficient Generation of Knockin Mice by CRISPR RNP Electroporation and AAV Donor Infection.
Genetically engineered mouse models harboring large sequence insertions or modifications are critical for a wide range of applications including endogenous gene tagging, conditional knockout, site-specific transgene insertion, and gene replacement; however, existing methods to generate such animals remain laborious and costly. To address this, we developed an approach called CRISPR-READI (CRISPR RNP electroporation and AAV donor infection), combining adeno-associated virus (AAV)-mediated HDR donor delivery with Cas9/sgRNA RNP electroporation to engineer large site-specific modifications in the mouse genome with high efficiency and throughput. We successfully targeted a 774 bp fluorescent reporter, a 2.1 kb CreERT2 driver, and a 3.3 kb expression cassette into endogenous loci in both embryos and live mice. CRISPR-READI is applicable to most widely used knockin schemes requiring donor lengths within the 4.9 kb AAV packaging capacity. Altogether, CRISPR-READI is an efficient, high-throughput, microinjection-free approach for sophisticated mouse genome engineering with potential applications in other mammalian species.
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An improved Agrobacterium-mediated transformation method for genome editing using CRISPR/Cas9 in elite indica rice (Oryza sativa L.).
Rice feeds nearly half of the world’s population and underpins global food security. Climate change now poses a major threat to rice productivity worldwide. Genome editing has reshaped crop improvement strategies. Among these tools, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein (CRISPR/Cas) system stands out for its precision, efficiency, and scalability. However, Agrobacterium-mediated transformation efficiency is often low, particularly in indica rice varieties. Here, we optimized an Agrobacterium-mediated transformation protocol for indica rice cultivars. The method was established in Lalat and MTU-1010. Seed-derived embryogenic calli were used to introduce the thermosensitive genic male sterile (OsTMS5) gene. A CRISPR/Cas9 vector carrying a gRNA and the selectable marker hptII was used for transformation. Callus induction reached 96.87% in MTU-1010 and 93.30% in Lalat MS medium supplemented with 3 mg/L 2,4-D and 0.5 mg/L BAP. In contrast, regeneration efficiency was higher in Lalat (90.28%) than in MTU-1010 (87.51%) on MS medium supplemented with 0.25 mg/L NAA, 0.5 mg/L kinetin, and 2 mg/L BAP. In addition, PCR analysis further verifies the integration of the transgene. Subsequently, the transformation efficiency was 37.20% in Lalat and 29.62% in MTU-1010. Therefore, this protocol provides a robust platform for gene function analysis and trait editing in rice. Its application may accelerate yield improvement and enhance stress tolerance under changing climatic conditions.
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Rapid generation of homozygous fluorescent knock-in human cells using CRISPR-Cas9 genome editing and validation by automated imaging and digital PCR screening.
We previously described a protocol for genome engineering of mammalian cultured cells with clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR-Cas9) to generate homozygous knock-ins of fluorescent tags into endogenous genes. Here we are updating this former protocol to reflect major improvements in the workflow regarding efficiency and throughput. In brief, we have improved our method by combining high-efficiency electroporation of optimized CRISPR-Cas9 reagents, screening of single cell-derived clones by automated bright-field and fluorescence imaging, rapidly assessing the number of tagged alleles and potential off-targets using digital polymerase chain reaction (PCR) and automated data analysis. Compared with the original protocol, our current procedure (1) substantially increases the efficiency of tag integration, (2) automates the identification of clones derived from single cells with correct subcellular localization of the tagged protein and (3) provides a quantitative and high throughput assay to measure the number of on- and off-target integrations with digital PCR. The increased efficiency of the new procedure reduces the number of clones that need to be analyzed in-depth by more than tenfold and yields to more than 26% of homozygous clones in polyploid cancer cell lines in a single genome engineering round. Overall, we were able to dramatically reduce the hands-on time from 30 d to 10 d during the overall ~10 week procedure, allowing a single person to process up to five genes in parallel, assuming that validated reagents-for example, PCR primers, digital PCR assays and western blot antibodies-are available.
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Customizing the genome as therapy for the β-hemoglobinopathies.
Despite nearly complete understanding of the genetics of the β-hemoglobinopathies for several decades, definitive treatment options have lagged behind. Recent developments in technologies for facile manipulation of the genome (zinc finger nucleases, transcription activator-like effector nucleases, or clustered regularly interspaced short palindromic repeats-based nucleases) raise prospects for their clinical application. The use of genome-editing technologies in autologous CD34(+) hematopoietic stem and progenitor cells represents a promising therapeutic avenue for the β-globin disorders. Genetic correction strategies relying on the homology-directed repair pathway may repair genetic defects, whereas genetic disruption strategies relying on the nonhomologous end joining pathway may induce compensatory fetal hemoglobin expression. Harnessing the power of genome editing may usher in a second-generation form of gene therapy for the β-globin disorders.
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