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Engineering Wax Ester Composition in Euglena gracilis Using Genome Editing.
Genome editing technologies have significantly expanded the potential for metabolic engineering in non-model organisms. In Euglena gracilis, genome editing methods using Cas9 and Cas12a were reported in 2019 and 2024, respectively, and are increasingly being applied to modify metabolic functions. This chapter provides a detailed protocol for CRISPR/Cas9-based genome editing that enables stable modification of wax ester composition under anaerobic conditions. By targeting key enzymes in the reversed β-oxidation pathway, the method allows the generation of knockout mutants with altered wax ester chain lengths. Beyond this application, the protocol supports reproducible and stable genetic modification of E. gracilis metabolism. It can be extended to the engineering of other biosynthetic pathways and is compatible with future integration of knock-in strategies. The approach offers a practical basis for the broader use of E. gracilis as a green chassis organism in synthetic biology and biomanufacturing.
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CRISPR meets AI-based robotics: Advancing sustainable agriculture.
In this issue of Cell, Xu and colleagues develop an approach integrating genome editing, artificial intelligence, and robotics to enhance crop improvement. By reconfiguring reproductive traits for automated pollination in crops such as tomatoes and soybeans, their approach accelerates hybrid seed production and yields crops with better stress tolerance, flavor, and resilience, supporting sustainable agriculture and crop diversity.
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'CAMERA' records cell action with new CRISPR tricks.
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Assessment of Cas12a-mediated gene editing efficiency in plants.
The CRISPR/Cas12a editing system opens new possibilities for plant genome engineering. To obtain a comparative assessment of RNA-guided endonuclease (RGEN) types in plants, we adapted the CRISPR/Cas12a system to the GoldenBraid (GB) modular cloning platform and compared the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described GB-assembled Streptococcus pyogenes Cas9 (SpCas9) constructs in eight Nicotiana benthamiana loci using transient expression. All three nucleases showed drastic target-dependent differences in efficiency, with LbCas12 producing higher mutagenesis rates in five of the eight loci assayed, as estimated with the T7E1 endonuclease assay. Attempts to engineer crRNA direct repeat (DR) had little effect improving on-target efficiency for AsCas12a and resulted deleterious in the case of LbCas12a. To complete the assessment of Cas12a activity, we carried out genome editing experiments in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, we also resequenced Cas12a-free segregating T2 lines to assess possible off-target effects. Our results showed that the mutagenesis footprint of Cas12a is enriched in deletions of -10 to -2 nucleotides and included in some instances complex rearrangements in the surroundings of the target sites. We found no evidence of off-target mutations neither in related sequences nor somewhere else in the genome. Collectively, this study shows that LbCas12a is a viable alternative to SpCas9 for plant genome engineering.
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Formation and nucleolytic processing of Cas9-induced DNA breaks in human cells quantified by droplet digital PCR.
Cas9 endonuclease from S. pyogenes is widely used to induce controlled double strand breaks (DSB) at desired genomic loci for gene editing. Here, we describe a droplet digital PCR (ddPCR) method to precisely quantify the kinetic of formation and 5'-end nucleolytic processing of Cas9-induced DSB in different human cells lines. Notably, DSB processing is a finely regulated process, which dictates the choice between non-homologous end joining (NHEJ) and homology directed repair (HDR). This step of DSB repair is also a relevant point to be taken into consideration to improve Cas9-mediated technology. Indeed, by this protocol, we show that processing of Cas9-induced DSB is impaired by CTIP or BRCA1 depletion, while it is accelerated after down-regulation of DNA-PKcs and 53BP1, two DSB repair key factors. In conclusion, the method we describe here can be used to study DSB repair mechanisms, with direct utility for molecularly optimising the knock-out/in outcomes in genome manipulation.
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[Progress of gene editing technologies and prospect in traditional Chinese medicine].
Gene editing is a kind of technologies that makes precise modification to the genome. It can be used to knock out/in and replace the specific DNA fragment, and make accurate gene editing on the genome level. The essence of the technique is the DNA sequence change with use of non homologous end link repair and homologous recombination repair, combined with specific DNA target recognition and endonuclease.This technology has wide range of development prospects and high application value in terms of scientific research, agriculture, medical treatment and other fields. In the field of gene therapy, gene editing technology has achieved cross-time success in cancers such as leukemia, genetic disorders such as hemophilia, thalassemia, multiple muscle nutritional disorders and retrovirus associated infectious diseases such as AIDS and other diseases. The preparation work for new experimental methods and animal models combined with gene editing technology is under rapid development and improvement. Laboratories around the world have also applied gene editing technique in prevention of malaria, organ transplantation, biological pharmaceuticals, agricultural breeding improvement, resurrection of extinct species, and other research areas. This paper summarizes the application and development status of gene editing technique in the above fields, and also preliminarily explores the potential application prospect of the technology in the field of traditional Chinese medicine, and discusses the present controversy and thoughts.
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Genome Editing in Soybean with CRISPR/Cas9.
CRISPR/Cas9 mediated genome editing technology has experienced rapid advances in recent years and has been applied to a wide variety of plant species, including soybean. Several platforms have been developed for designing and cloning of single CRISPR targets or multiple targets in a single destination vector. This chapter provides an updated working protocol for applying CRISPR/Cas9 technology to target a single gene or multiple genes simultaneously in soybean. We describe two platforms for cloning single CRISPR targets and multiplexing targets, respectively, and reagent delivery methodologies. The protocols address crucial limiting steps that can limit CRISPR editing in soybean hairy roots, composite plants, and tissue culture-based regenerated whole plants. To date, transgenic soybean plants with mutagenesis in up to three target genes have been obtained with this procedure.
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The Promise and Challenge of In Vivo Delivery for Genome Therapeutics.
CRISPR-based genome editing technologies are poised to enable countless new therapies to prevent, treat, or cure diseases with a genetic basis. However, the safe and effective delivery of genome editing enzymes represents a substantial challenge that must be tackled to enable the next generation of genetic therapies. In this Review, we summarize recent progress in developing enzymatic tools to combat genetic disease and examine current efforts to deliver these enzymes to the cells in need of correction. Viral vectors already in use for traditional gene therapy are being applied to enable in vivo CRISPR-based therapeutics, as are emerging technologies such as nanoparticle-based delivery of CRISPR components and direct delivery of preassembled RNA-protein complexes. Success in these areas will allow CRISPR-based genome editing therapeutics to reach their full potential.
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Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA.
Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research.
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Cas9 Protein Outperforms mRNA in Lipid Nanoparticle-Mediated CFTR Repair.
Lipid nanoparticles (LNPs) are currently one of the most clinically advanced delivery systems for nucleic acid cargo and hold great potential for clinical applications in gene editing and the treatment of genetic diseases. LNP-mediated delivery of Cas9 with single guide RNA (sgRNA) and homology-directed repair DNA template (ssDNA) enables efficient and precise editing in vitro and in vivo. Comparative analysis of LNP delivery of Cas9 as protein or mRNA for relevant clinical targets, such as cystic fibrosis (CF), which is caused by mutations in the CFTR gene, is imperative in the design of corrective therapeutics for genetic diseases. Here, we show that delivery of Cas9 protein LNPs outperforms Cas9 mRNA LNPs when evaluated for in vivo lung editing as well as corrective CRISPR/Cas9 editing and functional recovery of the CFTR protein. These results demonstrate the ability to optimize the use of CRISPR/Cas9 LNPs for cystic fibrosis applications.
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