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CRISPR/Cas9 Editing of the Plasmodium falciparum Genome.
The ability to interrogate gene function in Plasmodium parasites has been greatly enhanced by the advent of CRISPR/Cas9 systems. The breadth of genome manipulations ranges from single point mutations to large multigene deletions, however many of the technical considerations for designing CRISPR-based experiments are common to any editing approach. This review will discuss protocols for vector construction and donor design for genome editing P. falciparum, including pitfalls, variables, and validation methods.
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Optimized tRNA processing and TREX2-SpCas9 fusion enable high-efficiency multiplex genome editing in plants.
Multiplex genome editing is a powerful approach for dissecting gene networks and engineering complex traits in crops because it enables the simultaneous modification of multiple genomic loci. However, achieving high editing efficiency across multiple targets remains a significant challenge. To address this, we developed an optimized CRISPR system for rice that combines a monomeric TREX2-SpCas9 fusion with a novel array of tRNA-based gRNA processing elements. The TREX2-SpCas9 fusion significantly enhanced editing performance, resulting in higher editing efficiency, larger deletions, and increased mutation frequencies compared with wild-type SpCas9 and other exonuclease fusions. By systematically evaluating 38 endogenous rice tRNA genes, we identified 13 high-performing candidates, including tRNALeu-1 and tRNAPro-1, that outperformed the widely used tRNAGly and tRNAMet elements, enabling highly efficient processing of multiplexed gRNA arrays. Incorporating these top-performing tRNAs into our system enabled simultaneous editing of up to 29 OsCPK genes in a single rice plant. Furthermore, we demonstrated the cross-species applicability of this platform in the dicot Nicotiana benthamiana using transient expression, where rice-derived tRNA elements facilitated high-efficiency editing. This optimized multiplex gene-editing system provides a robust, scalable platform for accelerating plant functional genomics and engineering complex agronomic traits.
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CRISPR-Cas12a system in fission yeast for multiplex genomic editing and CRISPR interference.
The CRISPR-Cas12a is a class II, type V clustered regularly interspaced short palindromic repeat (CRISPR) system with both RNase and DNase activity. Compared to the CRISPR-Cas9 system, it recognizes T-rich PAM sequences and has the advantage of multiplex genomic editing. Here, in fission yeast Schizosaccharomyces pombe, we successfully implemented the CRISPR-Cas12a system for versatile genomic editing and manipulation. In addition to the rrk1 promoter, we used new pol II promoters from endogenous coding genes to express crRNA for Cas12a and obtained a much higher editing efficiency. This new design expands the promoter choices for potential applications in fission yeast and other organisms. In addition, we expressed a gRNA array using a strong constitutive pol II promoter. The array transcript is processed by Cas12a itself to release multiple mature crRNAs. With this construct, multiplex genomic editing of up to three loci was achieved from a single yeast transformation. We also built a CRISPR interference system using a DNase-dead Cas12a to significantly repress endogenous gene expression. Our study provides the first CRISPR-Cas12a toolkit for efficient and rapid genomic gene editing and regulation in fission yeast.
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CRISPR/Cas9-based multiplex genome editing of BCL11A and HBG efficiently induces fetal hemoglobin expression.
Beta-hemoglobinopathies are caused by mutations in the β-globin gene. One strategy to cure this disease relies on re-activating the γ-globin expression. BCL11A is an important transcription factor that suppresses the γ-globin expression, which makes it one of the most promising therapeutic targets in β-hemoglobinopathies. Here, we performed single-gene editing and multiplex gene editing via CRISPR/Cas9 technology to edit BCL11A erythroid-specific enhancer and BCL11A binding site on γ-globin gene promoter in HUDEP-2 cells and adult human CD34+ cells. Multiplex gene editing led to higher γ-globin expression than single-gene editing without inhibiting erythroid differentiation. By further optimizing the on-target DNA editing efficiency of multiplex gene editing, the percentage of F-cells exceeded 50% in HUDEP-2 cells. Amplicon deep sequencing and whole genome sequencing were used to detect the editing frequency of on- and potential off-target sites in CD34+ cells. No off-target mutations were detected, suggesting its accuracy in HSPCs. In summary, our study provides a new approach which can be used for the treatment of β-hemoglobinopathies in the future.
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New Gene-Editing Tool Slowly Advances Into the Clinic.
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Homology directed correction, a new pathway model for point mutation repair catalyzed by CRISPR-Cas.
Gene correction is often referred to as the gold standard for precise gene editing and while CRISPR-Cas systems continue to expand the toolbox for clinically relevant genetic repair, mechanistic hurdles still hinder widespread implementation. One of the most prominent challenges to precise CRISPR-directed point mutation repair centers on the prevalence of on-site mutagenesis, wherein insertions and deletions appear at the targeted site following correction. Here, we introduce a pathway model for Homology Directed Correction, specifically point mutation repair, which enables a foundational analysis of genetic tools and factors influencing precise gene editing. To do this, we modified an in vitro gene editing system which utilizes a cell-free extract, CRISPR-Cas RNP and donor DNA template to catalyze point mutation repair. We successfully direct correction of four unique point mutations which include two unique nucleotide mutations at two separate targeted sites and visualize the repair profiles resulting from these reactions. This extension of the cell-free gene editing system to model point mutation repair may provide insight for understanding the factors influencing precise point mutation correction.
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Gene Editing for HIV Cure at the Edge.
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Establishment of an efficient early flowering-assisted CRISPR/Cas9 gene-editing system in Arabidopsis.
Ectopic expression of the florigen FT gene along with the Cas9 cassette promotes fast reproduction of the T1 transgenic plants, and the late-flowering phenotype serves as a convenient marker for transgene-free plants screening in T2 segregation population.
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CRISPR/Cas9 encouraged CAR-T cell immunotherapy reporting efficient and safe clinical results towards cancer.
CRISPR is a customized genome-editing tool that snips DNA in a simpler, cheaper and more precise way than any other gene editing tool. In recent years CRISPR/Cas has completely transformed an existing discipline of genetic engineering. This 'review' focuses on the generations and modifications in CAR-T as an advanced cancer therapeutic tool and CAR-T-approved products. It also highlights three path-breaking successful autologous and allogenic ex vivo CAR-T clinical trials in treating cancer using CRISPR/Cas9 which reported successful results despite the controversies regarding the safety of this technique. Outcomes from the first successful clinical trial showed the beneficial long-term effect on genetically modified T-cells in targeting cancer cells which opens the door for CRISPR to be the most preferred technique to help treat cancer and other diseases in the future. We searched the MEDLINE, EMBASE and PUBMED databases for original studies and meta-analysis on the use of CRISPR/Cas9 to edit T-cells until 2021. We finally selected 15 pre-clinical and 26 clinical studies for the review.
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Effects of electroporation treatment using different concentrations of Cas9 protein with gRNA targeting Myostatin (MSTN) genes on the development and gene editing of porcine zygotes.
This study was conducted to investigate the effect of seven concentrations of Cas9 protein (0, 25, 50, 100, 200, 500, and 1,000 ng/µl) on the development and gene editing of porcine embryos. This included the target editing and off-target effect of embryos developed from zygotes that were edited via electroporation of the Cas9 protein with guide RNA targeting Myostatin genes. We found that the development to blastocysts of electroporated zygotes was not affected by the concentration of Cas9 protein. Although the editing rate, which was defined as the ratio of edited blastocysts to total examined blastocysts, did not differ with Cas9 protein concentration, the editing efficiency, which was defined as the frequency of indel mutations in each edited blastocyst, was significantly decreased in the edited blastocysts from zygotes electroporated with 25 ng/µl of Cas9 protein compared with that of blastocysts from zygotes electroporated with higher Cas9 protein concentrations. Moreover the frequency of indel events at the two possible off-target sites was not significantly different with different concentrations of Cas9 protein. These results indicate that the concentration of Cas9 protein affects gene editing efficiency in embryos but not the embryonic development, gene editing rate, and non-specific cleavage of off-target sites.
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