Genome Editing Platform Research Articles

Genome Editing Technologies towards Tomato Improvement: Recent Advances and Future Perspectives

Tomato (Solanum lycopersicon L.) is the world's second major vegetable crop and a superior model plant for studies on fruit biology. However, the changing climatic conditions are hugely impacting the yield and quality of tomato. CRISPR/Cas9 technology has been widely used in tomato breeding for enhanced disease resistance, herbicide tolerance, domestication and urban farming of wild tomato, and improved fruit yield and quality. Furthermore, new and advanced editing systems like Cas12a, Cas12b, base editing, and prime editing have been recently applied for high-precision tomato improvement. CRISPR variants, PAM-less genome editing, advanced transformation protocols, and gene delivery systems have played a critical role in fast breeding. This review offers an informative summary of recent progress in various genome editing methods and applications for improving tomatoes. It also focuses on critical issues, regulatory concerns, and prospects of genome editing platforms to improve tomato and allied crops.

Haplotype-resolved assembly of the mule duck genome using high-fidelity sequencing technology.

Mule duck is vitally important to the production of global duck meat. Here, we present two high-quality haplotypes of a female mule duck (haplotype 1 (H1):1.28 Gb, haplotype 2 (H2): 1.40 Gb). The continuity (H1: contig N50 = 14.90 Mb, H2: contig N50 = 15.70 Mb) and completeness (BUSCO: H1 = 96.9%, H2 = 97.3%) are substantially better than those of other duck genomes. We detected the structural variations (SVs) in H1 and H2. We observed a positive correlation between autosome length and the number of SVs. Z chromosome was some deficient in deletions and insertions, but W chromosome was some excessive. A total of 1,451 genes were haplotype specific expression (HSEs). Among them, 737 specifically expressed in H1, and 714 specifically expressed in H2. We found that H1 and H2 HSEs tended to be involved in similar biological processes, such as myometrial relaxation and contraction pathways, muscle structure development and phosphorylation. Our haplotype-resolved genome assembly provides a powerful platform for future functional genomics, molecular breeding, and genome editing in mule duck.

CRISPR-Cas Genome Editing in Ex Vivo Human Lungs to Rewire the Translational Path of Genome-Targeting Therapeutics.

The ongoing advancements in CRISPR-Cas technologies can significantly accelerate the preclinical development of both invivo and ex vivo organ genome-editing therapeutics. One of the promising applications is to genetically modify donor organs prior to implantation. The implantation of optimized donor organs with long-lasting immunomodulatory capacity holds promise for reducing the need for lifelong potent whole-body immunosuppression in recipients. However, assessing genome-targeting interventions in a clinically relevant manner prior to clinical trials remains a major challenge owing to the limited modalities available. This study introduces a novel platform for testing genome editing in human lungs ex vivo, effectively simulating preimplantation genetic engineering of donor organs. We identified gene regulatory elements whose disruption via Cas nucleases led to the upregulation of the immunomodulatory gene interleukin 10 (IL-10). We combined this approach with adenoviral vector-mediated IL-10 delivery to create favorable kinetics for early (immediate postimplantation) graft immunomodulation. Using ex vivo organ machine perfusion and precision-cut tissue slice technology, we demonstrated the feasibility of evaluating CRISPR genome editing in human lungs. To overcome the assessment limitations in ex vivo perfused human organs, we conducted an invivo rodent study and demonstrated both early gene induction and sustained editing of the lung. Collectively, our findings lay the groundwork for a first-in-human-organ study to overcome the current translational barriers of genome-targeting therapeutics.

Magnetic Nanoparticle-Assisted Non-Viral CRISPR-Cas9 for Enhanced Genome Editing to Treat Rett Syndrome.

The CRISPR-Cas9 technology has the potential to revolutionize the treatment of various diseases, including Rett syndrome, by enabling the correction of genes or mutations in human patient cells. However, several challenges need to be addressed before its widespread clinical application. These challenges include the low delivery efficiencies to target cells, the actual efficiency of the genome-editing process, and the precision with which the CRISPR-Cas system operates. Herein, the study presents a Magnetic Nanoparticle-Assisted Genome Editing (MAGE) platform, which significantly improves the transfection efficiency, biocompatibility, and genome-editing accuracy of CRISPR-Cas9 technology. To demonstrate the feasibility of the developed technology, MAGE is applied to correct the mutated MeCP2 genein induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs) from a Rett syndrome patient. By combining magnetofection and magnetic-activated cell sorting, MAGE achieves higher multi-plasmid delivery (99.3%) and repairing efficiencies (42.95%) with significantly shorter incubation times than conventional transfection agents without size limitations onplasmids. The repaired iPSC-NPCs showed similar characteristics as wild-type neurons when they differentiated into neurons, further validating MAGE and its potential for future clinical applications. In short, the developed nanobio-combined CRISPR-Cas9 technology offers the potential for various clinical applications, particularly in stem cell therapies targeting different genetic diseases.

Allogeneic CAR T Cells: Complex Cellular Therapy Designs Test the Limits of Our Preclinical Models.

All chimeric antigen receptor (CAR) T-cell products currently approved by the FDA are autologous, which poses several challenges for widespread use. In this issue, Degagné and colleagues present their preclinical research on creating off-the-shelf CAR T cells for multiple myeloma. They utilized the CRISPR/Cas12a genome editing platform and gene knock-in techniques to eliminate alloreactivity and decrease susceptibility to natural killer (NK)-cell elimination. This work has led to an ongoing phase I trial of off-the-shelf CAR T cells for multiple myeloma treatment. See related article by Degagné et al., p. 462 (2).

Abstract 1673: KRAS dependency, a gene editing approach

Abstract The RAS family genes encode small GTP-binding cytoplasmic proteins that are important signaling molecules. They regulate cell growth, survival and differentiation by coupling receptor activation to downstream effector pathways. Activating mutations of oncogenic RAS pathway genes are frequently detected in human cancers. The role of KRAS in tumor formation is not questionable. It’s implication in tumor maintenance is less well validated. The KRAS dependency is a key question to answer before to develop KRAS inhibitor. Targeted genome editing using CRISPR/Cas9 is a relatively new, revolutionary technology allowing for efficient and directed alterations of the genome. CRISPR is a genome-editing platform that makes use of the bacterially-derived endonuclease Cas9 to introduce DNA double-strand breaks at precise locations in the genome using complementary guide RNAs. These double strand breaks can be repaired by homologous recombination DNA repair mechanisms thank to a donor DNA template. By this way mutations can be introduced at desired loci, DNA fragment can be deleted or introduce at the desired location. We aimed at investigating the KRAS dependence and we applied the CRISPR-Cas9 technology to engineer isogenic cancer cells harboring a “conditional KRAS gene knockout" by exchanging the KRAS endogenous promoter. This promoter replacement approach can be used also to validate new targets. Citation Format: Christophe Marcireau, Fréderic Lacroix, Dietmar Hoffmann, May Cindhuchao, Loreley Calvet, Yvette Ruffin, Laurent Debussche. KRAS dependency, a gene editing approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1673.

Abstract 3382: Unveiling gastric premalignancy: A comprehensive study of tumor initiation and evolution using organoid models

Abstract Tumor initiation and evolution is still a poorly characterized process. Organoid models recapitulate three-dimensional tissue structure, differentiation, and disease pathogenesis. These models can help to better characterize pre-neoplastic processes and tumor evolution, with implications for early cancer detection and personalized therapeutic strategies for patients with gastric cancer (GC). In our research, we performed whole exome sequencing (WES) on 24 GC patient tumors, uncovering distinct mutation frequencies in well-known tumor suppressor genes -APC, TP53, and ARID1A (18.75%, 37.5%, 43.75%, respectively) -compared to The Cancer Genome Atlas (TCGA) PanCancer Altas Cohort (n=440) (13.48%, 50.56%, 22.47% respectively). To gain a deeper understanding of how these mutated genes promote gastric premalignancy, we generated multiple isogenic gastric organoid lines with mutant APC (AT-dmut) or ARID1A (ArT-dmut) on a TP53 mutant background as well as TP53 mutant-only lines (T-mut) using our established CRISPR/Cas9 genome-editing platform and two HLA patient-derived normal gastric organoid lines (HL-PDOs). After confirmation of functional gene knockouts, we focused on elucidating carcinogenic changes via a two-pronged approach: (1) a comparative analysis of growth and morphology between these mutant organoid models and the parental normal gastric organoids and (2) an in-depth examination of mutational and transcriptional changes in our edited AT-mut, ArT-mut and T-mut organoid models. We cultured each edited organoid line for up to 350 days and collected histological and molecular data at multiple time points. Preliminary analysis shows an increased growth rate of all edited cell lines compared to parental normal lines. The ArT-mut and AT-mut cell lines exhibit distinct morphological and 3D structural alterations, while T-mut cell lines do not. We expect to find additional mutation gains in these cells’ lines compared to the parental normal gastric organoids that promote gastric phenotype with the greatest gain of alternations at the 350-day timepoint. These additional mutations and transcriptional changes will help us gain insights into the pathways altered in gastric cancer initiation and early progression that may be specific to HLAs. Our overall goal is to enhance our understanding of the molecular mechanisms underlying gastric premalignancy among HLAs, ultimately contributing to advancements in early detection and tailored therapeutic interventions for HLA patients with GC and advancing cancer health equity. Citation Format: Alexa Morales Arana, Nicole B. Halmai, Hongyong Zhang, Paul Lott, Ana Estrada, University of California Minority Patient-DerivedXenograft Development and Trial (UCaMP) Center, Luis Carvajal-Carmona. Unveiling gastric premalignancy: A comprehensive study of tumor initiation and evolution using organoid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3382.

HyCas9-12aGEP: an efficient genome editing platform for Corynebacterium glutamicum.

Corynebacterium glutamicum plays a crucial role as a significant industrial producer of metabolites. Despite the successful development of CRISPR-Cas9 and CRISPR-Cas12a-assisted genome editing technologies in C. glutamicum, their editing resolution and efficiency are hampered by the diverse on-target activities of guide RNAs (gRNAs). To address this problem, a hybrid CRISPR-Cas9-Cas12a genome editing platform (HyCas9-12aGEP) was developed in C. glutamicum in this study to co-express sgRNA (corresponding to SpCas9 guide RNA), crRNA (corresponding to FnCas12a guide RNA), or hfgRNA (formed by the fusion of sgRNA and crRNA). HyCas9-12aGEP improves the efficiency of mapping active gRNAs and outperforms both CRISPR-Cas9 and CRISPR-Cas12a in genome editing resolution and efficiency. In the experiment involving the deletion of the cg0697-0740 gene segment, an unexpected phenotype was observed, and HyCas9-12aGEP efficiently identified the responsible genotype from more than 40 genes. Here, HyCas9-12aGEP greatly improve our capability in terms of genome reprogramming in C. glutamicum.

Engineering self-deliverable ribonucleoproteins for genome editing in the brain

The delivery of CRISPR ribonucleoproteins (RNPs) for genome editing in vitro and in vivo has important advantages over other delivery methods, including reduced off-target and immunogenic effects. However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineer self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules. Screening of cell-penetrating peptides (CPPs) fused to CRISPR-Cas9 protein identifies potent constructs capable of efficient genome editing of neural progenitor cells. Further engineering of these fusion proteins establishes a C-terminal Cas9 fusion with three copies of A22p, a peptide derived from human semaphorin-3a, that exhibits substantially improved editing efficacy compared to other constructs. We find that self-deliverable Cas9 RNPs generate robust genome edits in clinically relevant genes when injected directly into the mouse striatum. Overall, self-deliverable Cas9 proteins provide a facile and effective platform for genome editing in vitro and in vivo.

Domain-inlaid Nme2Cas9 adenine base editors with improved activity and targeting scope

Nme2Cas9 has been established as a genome editing platform with compact size, high accuracy, and broad targeting range, including single-AAV-deliverable adenine base editors. Here, we engineer Nme2Cas9 to further increase the activity and targeting scope of compact Nme2Cas9 base editors. We first use domain insertion to position the deaminase domain nearer the displaced DNA strand in the target-bound complex. These domain-inlaid Nme2Cas9 variants exhibit shifted editing windows and increased activity in comparison to the N-terminally fused Nme2-ABE. We next expand the editing scope by swapping the Nme2Cas9 PAM-interacting domain with that of SmuCas9, which we had previously defined as recognizing a single-cytidine PAM. We then use these enhancements to introduce therapeutically relevant edits in a variety of cell types. Finally, we validate domain-inlaid Nme2-ABEs for single-AAV delivery in vivo.

CRISPR-Cas12a for Highly Efficient and Marker-Free Targeted Integration in Human Pluripotent Stem Cells.

The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated, for the first time, the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a GFP marker gene into the AAVS1 safe harbor site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.

DNA-free CRISPR/Cas9 genome editing system for oil palm protoplasts using multiple ribonucleoproteins (RNPs) complexes

CRISPR/Cas9 is a powerful genome editing tool applicable to diverse plant species. However, the application of CRISPR/Cas9-ribonucleoproteins (RNPs) in oil palm (Elaeis guineensis) has not yet been reported. In this paper, the specificity, efficiency, and sensitivity of CRISPR/Cas9-RNPs in oil palm were evaluated by assessing the effect of different amounts of Cas9 and heat stress using E. guineensis phytoene desaturase (EgPDS) as the target gene. Seven gRNAs were designed and combined with Cas9 protein to form three combinations of RNPs complexes; RNP1/RNP2, RNP3/RNP4, and RNP5/RNP6/RNP7. Assessment of optimal amount of Cas9 and heat stress using RNP5/RNP6/RNP7 demonstrated that 40 µg of Cas9 and heat treatment at 39ºC increased the genome editing efficiency up to 95% in oil palm protoplasts. The use of both optimal conditions produced targeted mutations at frequencies of up to 63.6% in protoplasts transformed with RNP1/RNP2 and RNP3/RNP4 and up to 100% with RNP5/RNP6/RNP7 combinations. Multiple site mutagenesis at two to three target sites with fragment removal up to 179 bp, 69 bp, and 816 bp occurred at high frequencies producing editing efficiency and a Knockout Score (KO) of 100%. Altogether, this study demonstrates a promising, highly efficient, and precise transgene-free CRISPR/Cas9-RNPs genome editing platform in oil palm that also holds enormous potential for application in other transformation-recalcitrant species.

Usefulness of current sgRNA design guidelines and in vitro cleavage assays for plant CRISPR/Cas genome editing: a case targeting the polyphenol oxidase gene family in eggplant (Solanum melongena L.).

The advent of genome editing platforms such as the CRISPR/Cas9 system ushers an unprecedented speed in the development of new crop varieties that can withstand the agricultural challenges of the 21st century. The CRISPR/Cas9 system depends on the specificity of engineered single guide RNAs (sgRNAs). However, sgRNA design in plants can be challenging due to the multitude of design tools to choose from, many of which use guidelines that are based on animal experiments yet allow the use of plant genomes. Upon choosing sgRNAs, it is also unclear whether an in vitro assay is needed to validate the targeting efficiency of a particular sgRNA before in vivo delivery of the CRISPR/Cas9 system. Here, we demonstrate the in vitro and in vivo activity of four different sgRNAs that we selected based on their ability to target multiple members of the eggplant polyphenol oxidase gene family. Some sgRNAs that have high in vitro cleavage activity did not produce edits in vivo, suggesting that an in vitro assay may not be a reliable basis to predict sgRNAs with highly efficient in vivo cleavage activity. Further analysis of our sgRNAs using other design algorithms suggest that plant-validated criteria such as the presence of necessary secondary structures and appropriate base-pairing may be the reason for the discrepancy between our observed in vitro and in vivo cleavage efficiencies. However, recent reports and our data suggests that there is no guaranteed way to ensure the in vivo cleavage of chosen sgRNAs.

Efficient CRISPR/Cas9-mediated genome editing in sheepgrass (Leymus chinensis).

The lack of genome editing platforms has hampered efforts to study and improve forage crops that can be grown on lands not suited to other crops. Here, we established efficient Agrobacterium-mediated clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) genome editing in a perennial, stress-tolerant forage grass, sheepgrass (Leymus chinensis). By screening for active single-guide RNAs (sgRNAs), accessions that regenerate well, suitable Agrobacterium strains, and optimal culture media, and co-expressing the morphogenic factor TaWOX5, we achieved 11% transformation and 5.83% editing efficiency in sheepgrass. Knocking out Teosinte Branched1 (TB1) significantly increased tiller number and biomass. This study opens avenues for studying gene function and breeding in sheepgrass.

An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis

SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for invivo gene therapy.

Opportunities and Challenges of In Vitro Tissue Culture Systems in the Era of Crop Genome Editing.

Currently, the development of genome editing (GE) tools has provided a wide platform for targeted modification of plant genomes. However, the lack of versatile DNA delivery systems for a large variety of crop species has been the main bottleneck for improving crops with beneficial traits. Currently, the generation of plants with heritable mutations induced by GE tools mostly goes through tissue culture. Unfortunately, current tissue culture systems restrict successful results to only a limited number of plant species and genotypes. In order to release the full potential of the GE tools, procedures need to be species and genotype independent. This review provides an in-depth summary and insights into the various in vitro tissue culture systems used for GE in the economically important crops barley, wheat, rice, sorghum, soybean, maize, potatoes, cassava, and millet and uncovers new opportunities and challenges of already-established tissue culture platforms for GE in the crops.

Developing an efficient and visible prime editing system to restore tobacco 8-hydroxy-copalyl diphosphate gene for labdane diterpene Z-abienol biosynthesis.

Prime editing (PE) is a versatile CRISPR-Cas based precise genome-editing platform widely used to introduce a range of possible base conversions in various organisms. However, no PE systems have been shown to induce heritable mutations in tobacco, nor in any other dicot. In this study, we generated an efficient PE system in tobacco that not only introduced heritable mutations, but also enabled anthocyanin-based reporter selection of transgene-free T1 plants. This system was used to confer Z-abienol biosynthesis in the allotetraploid tobacco cultivar HHDJY by restoring a G>T conversion in the NtCPS2 gene. High levels of Z-abienol were detected in the leaves of homozygous T1 plants at two weeks after topping. This study describes an advance in PE systems and expands genome-editing toolbox in tobacco, even in dicots, for use in basic research and molecular breeding. And restoring biosynthesis of Z-abienol in tobacco might provide an efficient way to obtain Z-abienol in plants.

Interrogation of human microglial phagocytosis by CRISPR genome editing.

Microglia are an integral part of central nervous system, but our understanding of microglial biology is limited due to the challenges in obtaining and culturing primary human microglia. HMC3 is an important cell line for studying human microglia because it is readily accessible and straightforward to maintain in standard laboratories. Although HMC3 is widely used for microglial research, a robust genetic method has not been described. Here, we report a CRISPR genome editing platform, by the electroporation of Cas9 ribonucleoproteins (Cas9 RNP) and synthetic DNA repair templates, to enable rapid and precise genetic modifications of HMC3. For proof-of-concept demonstrations, we targeted the genes implicated in the regulation of amyloid beta (Aβ) and glioblastoma phagocytosis in microglia. We showed that CRISPR genome editing could enhance the phagocytic activities of HMC3. We performed CRISPR gene knockout (KO) in HMC3 by the electroporation of pre-assembled Cas9 RNP. Co-introduction of DNA repair templates allowed site-specific knock-in (KI) of an epitope tag, a synthetic promoter and a fluorescent reporter gene. The editing efficiencies were determined genotypically by DNA sequencing and phenotypically by immunofluorescent staining and flow cytometry. The gene-edited HMC3 cells were examined in vitro by fluorescent Aβ and glioblastoma phagocytosis assays. Our platform enabled robust single (>90%) and double (>70%) KO without detectable off-target editing by high throughput DNA sequencing. We also inserted a synthetic SFFV promoter to efficiently upregulate the expression of endogenous CD14 and TREM2 genes associated with microglial phagocytosis. The CRISPR-edited HMC3 showed stable phenotypes and enhanced phagocytosis of fluorescence-labeled Aβ1-42 peptides. Confocal microscopy further confirmed the localization of Aβ1-42 aggregates in the acidified lysosomes. HMC3 mutants also changed the phagocytic characteristic toward apoptotic glioblastoma cells. CRISPR genome editing by Cas9 RNP electroporation is a robust approach to genetically modify HMC3 for functional studies such as the interrogation of Aβ and tumor phagocytosis, and is readily adoptable to investigate other aspects of microglial biology.

Phosphorothioated DNA Engineered Liposomes as a General Platform for Stimuli-Responsive Cell-Specific Intracellular Delivery and Genome Editing.

Intracellular protein delivery is highly desirable for protein drug-based cell therapy. Established technologies suffer from poor cell-specific cytosolic protein delivery, which hampers the targeting therapy of specific cell populations. A fusogenic liposome system enables cytosolic delivery, but its ability of cell-specific and controllable delivery is quite limited. Inspired by the kinetics of viral fusion, we designed a phosphorothioated DNA coatings-modified fusogenic liposome to mimic the function of viral hemagglutinin. The macromolecular fusion machine docks cargo-loaded liposomes at the membrane of target cells, triggers membrane fusion upon pH or UV light stimuli, and facilitates cytosolic protein delivery. Our results showed efficient cell-targeted delivery of proteins of various sizes and charges, indicating the phosphorothioated DNA plug-in unit on liposomes could be a general strategy for spatial-temporally controllable protein delivery both in vitro and in vivo.

Large DNA fragment knock-in and sequential gene editing in Plasmodium falciparum: a preliminary study using suicide-rescue-based CRISPR/Cas9 system.

CRISPR/Cas9 technology applied to Plasmodium falciparum offers the potential to greatly improve gene editing, but such expectations including large DNA fragment knock-ins and sequential gene editing have remained unfulfilled. Here, we achieved a major advance in addressing this challenge, especially for creating large DNA fragment knock-ins and sequential editing, by modifying our suicide-rescue-based system that has already been demonstrated to be highly efficient for conventional gene editing. This improved approach was confirmed to mediate efficient knock-ins of DNA fragments up to 6.3kb, to produce "marker-free" genetically engineered parasites and to show potential for sequential gene editing. This represents an important advancement in establishing platforms for large-scale genome editing, which might gain a better understanding of gene function for the most lethal cause of malaria and contribute to adjusting synthetic biology strategies to live parasite malaria vaccine development. Site-directed knock-in of large DNA fragments is highly efficient using suicide-rescue-based CRISPR/Cas9 system, and sequential gene insertion is feasible but further confirmation is still needed.