Establishment of an Efficient Genome Editing System in Lettuce Without Sacrificing Specificity

Pan, Wenbo; Li, Xue; Li, Dayong; Zhang, Huawei

doi:10.3389/fpls.2022.930592

Cited by 6 publications

(5 citation statements)

References 45 publications

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“…Drawing from recent reports highlighting the success of including scattered introns throughout the Cas9 transgene to boost or sustain expression levels, 20,27,28 we tested installing 11 short human introns within the mScarlet-dCas9 cassette, drawn from essential housekeeping, cell cycle, tumor suppressor, or T cell-enriched genes. Cells manufactured using this candidate vector expressed greater dCas9 levels (+1.8-fold) with only a slight reduction in overall positivity (92%) relative to the non-intronized equivalent.…”

Section: Resultsmentioning

confidence: 99%

Genome-wide CRISPRa screens nominate modulators of CAR T cell survival within distinct tumor cytokine milieus

Curtis,

Spurrell,

Flint

et al. 2024

Preprint

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Chimeric Antigen Receptor (CAR) T cell therapy has revolutionized the treatment of B cell malignancies and translating this success to other cancers remains an ongoing clinical objective. Next-generation T cell products in development aim to genetically modulate many facets of cell behavior, for which gene-nominating platforms provide a useful framework for prioritization. Among competing screening approaches, CRISPR activation (CRISPRa) technology permits gain-of-function (GoF) gene surveys at genome-wide scale, but routine implementation in primary T cells has been stymied by high cell requirements (~107- 108) and abbreviated activity. Here, we describe a novel cell manufacturing schema using an all-in-one transposon-based gene delivery system coupled with CAR-restricted cell expansion to generate yields (109) of primary T cells bearing CAR and CRISPRa transgenes that are well above the threshold needed for genome-scale screening. CRISPRa activity is sustained via the inclusion of divergent, duplicate Elongation Factor 1α core/human T-cell leukemia virus (EF1α-HTLV) hybrid promoters; while guide RNA representation is preserved through late lentiviral transduction, thus preventing bottlenecking and premature candidate pruning. CRISPRa-CAR T cells manufactured via this pipeline retain potent on-target gene-overexpression (85% target+) across varied cell subsets (e.g. Tim-3+Lag3+or serial-challenge) and timescales (>14 days). When deployed to survival-based genome-wide selection landscapes, CRISPRa-CAR pools nominate known and novel endogenous genes capable of enhancing CD8+CAR T survival in cytokine-rich (e.g.MYC,FUT6,IRF4,GSE1) and cytokine-depleted (e.g.CSF2RB,STAT6,IRF4,GSE1) settings of tumor challenge. This system will have broad utility for therapy-enhancing gene discovery.

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Section: Resultsmentioning

confidence: 99%

Genome-wide CRISPRa screens nominate modulators of CAR T cell survival within distinct tumor cytokine milieus

Curtis,

Spurrell,

Flint

et al. 2024

Preprint

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“…If individual genes are edited too inefficiently, it makes later screening and identification doubly difficult and time-consuming, so it is also important not to express too many gRNAs when constructing editing vectors [ 137 ]. Although it is difficult to obtain higher-order mutants using a single editing vector, we can use crosses between stably inherited mutants of different genes to obtain multiple mutants [ 138 , 139 , 140 ]. In 2022, Mu et al created two different double mutants by targeting two genes with a single sgRNA and then obtained GmBIC quadruple mutants by hybridization.…”

Section: Strategies and Methods For Optimizing Crispr/cas9 Gene-editi...mentioning

confidence: 99%

Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

Zhou

Luan

Liu

et al. 2023

Plants

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Following recent developments and refinement, CRISPR-Cas9 gene-editing technology has become increasingly mature and is being widely used for crop improvement. The application of CRISPR/Cas9 enables the generation of transgene-free genome-edited plants in a short period and has the advantages of simplicity, high efficiency, high specificity, and low production costs, which greatly facilitate the study of gene functions. In plant molecular breeding, the gene-editing efficiency of the CRISPR-Cas9 system has proven to be a key step in influencing the effectiveness of molecular breeding, with improvements in gene-editing efficiency recently becoming a focus of reported scientific research. This review details strategies and methods for improving the efficiency of CRISPR/Cas9 gene editing in plant molecular breeding, including Cas9 variant enzyme engineering, the effect of multiple promoter driven Cas9, and gRNA efficient optimization and expression strategies. It also briefly introduces the optimization strategies of the CRISPR/Cas12a system and the application of BE and PE precision editing. These strategies are beneficial for the further development and optimization of gene editing systems in the field of plant molecular breeding.

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“…However, the efficiency is far from satisfactory even with the traditional delivery method [ 109 ]. To ensure the efficient and successful editing of desired sites, strong, constitutive, and sometimes virus promoters have often been used to boost the expression level of Cas and sgRNA [ 110 , 111 , 112 , 113 ]. These methods could be used in nanomaterial-mediated genome editing, and also in other nanomaterial-based plant genetic engineering.…”

Section: Future Prospects For Plant Genetic Engineering With Nanomate...mentioning

confidence: 99%

The Promising Nanovectors for Gene Delivery in Plant Genome Engineering

Zhi

Zhou

Pan

et al. 2022

IJMS

Self Cite

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Highly efficient gene delivery systems are essential for genetic engineering in plants. Traditional delivery methods have been widely used, such as Agrobacterium-mediated transformation, polyethylene glycol (PEG)-mediated delivery, biolistic particle bombardment, and viral transfection. However, genotype dependence and other drawbacks of these techniques limit the application of genetic engineering, particularly genome editing in many crop plants. There is a great need to develop newer gene delivery vectors or methods. Recently, nanomaterials such as mesoporous silica particles (MSNs), AuNPs, carbon nanotubes (CNTs), and layer double hydroxides (LDHs), have emerged as promising vectors for the delivery of genome engineering tools (DNA, RNA, proteins, and RNPs) to plants in a species-independent manner with high efficiency. Some exciting results have been reported, such as the successful delivery of cargo genes into plants and the generation of genome stable transgenic cotton and maize plants, which have provided some new routines for genome engineering in plants. Thus, in this review, we summarized recent progress in the utilization of nanomaterials for plant genetic transformation and discussed the advantages and limitations of different methods. Furthermore, we emphasized the advantages and potential broad applications of nanomaterials in plant genome editing, which provides guidance for future applications of nanomaterials in plant genetic engineering and crop breeding.

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Establishment of an Efficient Genome Editing System in Lettuce Without Sacrificing Specificity

Cited by 6 publications

References 45 publications

Genome-wide CRISPRa screens nominate modulators of CAR T cell survival within distinct tumor cytokine milieus

Genome-wide CRISPRa screens nominate modulators of CAR T cell survival within distinct tumor cytokine milieus

Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

The Promising Nanovectors for Gene Delivery in Plant Genome Engineering

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