Gene Editing Efficiency Research Articles

Establishing a one-step marker-free CRISPR/Cas9 system for industrial Aspergillus niger using counter-selectable marker Ang-ace2.

To develop a one-step, marker-free CRISPR/Cas9 system for highly efficient genome editing in industrial Aspergillus niger, with a short genetic operation cycle. Firstly, evaluation of different promoters for sgRNA expression revealed tRNAGly15 as the most efficient, achieving a remarkable 100% gene editing efficiency. Furthermore, a counter-selectable marker, Ang-ace2, was identified for A. niger. Subsequently, a CRISPR/Cas9 plasmid was developed, utilizing a truncated AMA1 element and the Ang-ace2 conditional expression cassette driven by a Tet-on promoter. In the presence of doxycycline, the plasmid demonstrated a 33% loss efficiency in the progeny of A. niger spores after a single generation, resulting in a shortened genetic operation cycle of 16days for CRISPR/Cas9. The one-step marker-free CRISPR/Cas9 system was successfully developed in industrial A. niger, allowing for efficient gene editing while simultaneously reducing the editing time.

Trichostatin A for Efficient CRISPR-Cas9 Gene Editing of Human Pluripotent Stem Cells.

Genome-edited human-induced pluripotent stem cells (iPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. Despite the development of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system, the gene editing process is inefficient and can take several weeks to months to generate edited iPSC clones. We developed a strategy to improve the efficiency of the iPSC gene editing process via application of a small-molecule, trichostatin A (TSA), a Class I and II histone deacetylase inhibitor. We observed that TSA decreased global chromatin condensation and further resulted in increased gene-editing efficiency of iPSCs by twofold to fourfold while concurrently ensuring no increased off-target effects. The edited iPSCs could be clonally expanded while maintaining genomic integrity and pluripotency. The rapid generation of therapeutically relevant gene-edited iPSCs could be enabled by these findings.

Cascade dynamic assembly/disassembly of DNA nanoframework enabling the controlled delivery of CRISPR-Cas9 system.

CRISPR-Cas9 has been explored as a therapeutic agent for down-regulating target genes; the controlled delivery of Cas9 ribonucleoprotein (RNP) is essential for therapeutic efficacy and remains a challenge. Here, we report cascade dynamic assembly/disassembly of DNA nanoframework (NF) that enables the controlled delivery of Cas9 RNP. NF was prepared with acrylamide-modified DNA that initiated cascade hybridization chain reaction (HCR). Through an HCR, single-guide RNA was incorporated to NF; simultaneously, the internal space of NF was expanded, facilitating the loading of Cas9 protein. NF was designed with hydrophilic acylamino and hydrophobic isopropyl, allowing dynamic swelling and aggregation. The responsive release of Cas9 RNP was realized by introducing disulfide bond-containing N,N-bis(acryloyl)cystamine that was specifically in response to glutathione of cancer cells, triggering the complete disassembly of NF. In vitro and in vivo investigations demonstrated the high gene editing efficiency in cancer cells, the hypotoxicity in normal cells, and notable antitumor efficacy in a breast cancer mouse model.

Advances in CRISPR/Cas9-based research related to soybean [Glycine max (Linn.) Merr] molecular breeding.

Soybean [Glycine max (Linn.) Merr] is a source of plant-based proteins and an essential oilseed crop and industrial raw material. The increase in the demand for soybeans due to societal changes has coincided with the increase in the breeding of soybean varieties with enhanced traits. Earlier gene editing technologies involved zinc finger nucleases and transcription activator-like effector nucleases, but the third-generation gene editing technology uses clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The rapid development of CRISPR/Cas9 technology has made it one of the most effective, straightforward, affordable, and user-friendly technologies for targeted gene editing. This review summarizes the application of CRISPR/Cas9 technology in soybean molecular breeding. More specifically, it provides an overview of the genes that have been targeted, the type of editing that occurs, the mechanism of action, and the efficiency of gene editing. Furthermore, suggestions for enhancing and accelerating the molecular breeding of novel soybean varieties with ideal traits (e.g., high yield, high quality, and durable disease resistance) are included.

Engineered Extracellular Vesicle-Delivered CRISPR/Cas9 for Radiotherapy Sensitization of Glioblastoma.

Radiotherapy is a mainstay of glioblastoma (GBM) treatment; however, the development of therapeutic resistance has hampered the efficacy of radiotherapy, suggesting that additional treatment strategies are needed. Here, an in vivo loss-of-function genome-wide CRISPR screen was carried out in orthotopic tumors in mice subjected to radiation treatment to identify synthetic lethal genes associated with radiotherapy. Using functional screening and transcriptome analyses, glutathione synthetase (GSS) was found to be a potential regulator of radioresistance through ferroptosis. High GSS levels were closely related to poor prognosis and relapse in patients with glioma. Mechanistic studies demonstrated that GSS was associated with the suppression of radiotherapy-induced ferroptosis in glioma cells. The depletion of GSS resulted in the disruption of glutathione (GSH) synthesis, thereby causing the inactivation of GPX4 and iron accumulation, thus enhancing the induction of ferroptosis upon radiotherapy treatment. Moreover, to overcome the obstacles to broad therapeutic translation of CRISPR editing, we report a previously unidentified genome editing delivery system, in which Cas9 protein/sgRNA complex was loaded into Angiopep-2 (Ang) and the trans-activator of the transcription (TAT) peptide dual-modified extracellular vesicle (EV), which not only targeted the blood-brain barrier (BBB) and GBM but also permeated the BBB and penetrated the tumor. Our encapsulating EVs showed encouraging signs of GBM tissue targeting, which resulted in high GSS gene editing efficiency in GBM (up to 67.2%) with negligible off-target gene editing. These results demonstrate that a combination of unbiased genetic screens, and CRISPR-Cas9-based gene therapy is feasible for identifying potential synthetic lethal genes and, by extension, therapeutic targets.

A Digital PCR Method Based on Highly Specific Taq for Detecting Gene Editing and Mutations.

Digital PCR (dPCR) has great potential for assessing gene editing or gene mutation due to its ability to independently inspect each DNA template in parallel. However, current dPCR methods use a fluorescence-labeled probe to detect gene variation events, and their ability to distinguish variated sequences from the wild-type sequence is limited by the probe's tolerance to mismatch. To address this, we have developed a novel dPCR method that uses a primer instead of a probe to sense gene variation. The enhanced Taq DNA polymerase in the PCR system has a high mismatch sensitivity, which enables our dPCR method to distinguish gene mutations from wild-type sequences. Compared to current dPCR methods, our method shows superior precision in assessing gene editing efficiency and single-base DNA mutation. This presents a promising opportunity to advance gene editing research and rare gene mutation detection.

Effects of sgRNAs, Promoters, and Explants on the Gene Editing Efficiency of the CRISPR/Cas9 System in Chinese Kale.

The CRISPR/Cas9 system is extensively used for plant gene editing. This study developed an efficient CRISPR/Cas9 system for Chinese kale using multiple sgRNAs and two promoters to create various CRISPR/Cas9 vectors. These vectors targeted BoaZDS and BoaCRTISO in Chinese kale protoplasts and cotyledons. Transient transformation of Chinese kale protoplasts was assessed for editing efficiency at three BoaZDS sites. Notably, sgRNA: Z2 achieved the highest efficiency (90%). Efficiency reached 100% when two sgRNAs targeted BoaZDS with a deletion of a large fragment (576 bp) between them. However, simultaneous targeting of BoaZDS and BoaCRTISO yielded lower efficiency. Transformation of cotyledons led to Chinese kale mutants with albino phenotypes for boazds mutants and orange-mottled phenotypes for boacrtiso mutants. The mutation efficiency of 35S-CRISPR/Cas9 (92.59%) exceeded YAO-CRISPR/Cas9 (70.97%) in protoplasts, and YAO-CRISPR/Cas9 (96.49%) surpassed 35S-CRISPR/Cas9 (58%) in cotyledons. These findings introduce a strategy for enhancing CRISPR/Cas9 editing efficiency in Chinese kale.

Biotechnological Advances in Gene Therapy of Hematopoietic Stem Cells: Systematic Review and Meta-Analysis.

Gene therapy (GT) has emerged as a promising treatment option for disorders in the hematopoietic system, particularly primary immunodeficiencies (PID). Hematopoietic stem cells (HSCs) have gained attention due to their ability to support long-term hematopoiesis. In this study, we present a summary of research evaluating the most effective method of gene editing in HSCs for translational medicine. We conducted a systematic literature search in various databases, including Cochrane, LILACs, SciELO, and PubMed (MEDLINE), covering the period from January 1989 to June 10, 2023. The aim of this study was to identify articles that assessed the efficiency of gene editing in HSCs and clinical trials focusing on PID. Our research protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO; registration number CRD42022349850). Of the 470 studies identified in our search, 77 met the inclusion criteria. Among these, 61 studies were included in strategy 1 (gene therapy using HSC [GT-HSC]) of the systematic review (SR). We performed a meta-analysis on 17 of these studies. In addition, 16 studies were categorized under strategy 2 (clinical trials for PID). While clinical trials have demonstrated the potential benefits of GT-HSC, the safety and efficacy of gene editing still pose significant challenges. Various viral and nonviral approaches for gene delivery have been explored in basic and clinical research, with viral vectors being the most commonly used method in HSC therapeutics. Although promising, recent technologies such as CRISPR/Cas are not yet ready for efficient long-term restoration of the immune system as a whole.

Mini-PE, a prime editor with compact Cas9 and truncated reverse transcriptase

Prime editor (PE) is a versatile genome editing tool that does not need extra DNA donors or inducing double-strand breaks. However, invivo implementation of PE remains a challenge because of its oversized composition. In this study, we screened out the smallest truncated Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) with the F155Y mutation to keep gene editing efficiency. We discovered the most efficient gene editing variants of MMLV RT with the smallest size. After optimization of the pegRNAs and incorporation with nick sgRNAs, the mini-PE delivered up to 10% precise editing at target sites in human and mouse cells. It also edited the mouse Hsf1 gene in the mouse retina precisely after delivery with adeno-associated viruses (AAVs), although the editing efficiency was lower than 1%. We will focus on improving the editing efficiency of mini-PE and exploiting its therapeutic potential against human genetic diseases.

CRISPR-Cas9 as a tool for treating cystic fibrosis through gene editing

The most common cause of mortality is Cystic Fibrosis, a fatal genetic disease. However, in recent years, there have been a growing number of papers concentrating on CRISPR-Cas9, a gene-editing tool that is being used to permanently cure this genetic disease, named by a biopharmaceutical company EditasMedicine, invested in by Bill Gates. However, before the breadth of search and study of this technology continuously expands, challenges and remaining issues should be addressed. This paper reviews the mechanisms of cystic fibrosis and discusses its technical challenges, such as efficiency, safety and delivery of gene editing, potential side effects, and ethical issues, this paper also talks about the future applications of CRISPR-Cas9 in other diseases, so as to provide an alternative treatment method for the diseases with gene editing better results can be obtained.

Genome Editing in Ferret Airway Epithelia Mediated by CRISPR/Nucleases Delivered with Amphiphilic Shuttle Peptides.

Gene editing strategies are attractive for treating genetic pulmonary diseases such as cystic fibrosis (CF). However, challenges have included the development of safe and effective vector systems for gene editing of airway epithelia and model systems to report their efficiency and durability. The domestic ferret (Mustela putorius furo) has a high degree of conservation in lung cellular anatomy with humans, and has served as an excellent model for many types of lung diseases, including CF. In this study, we evaluated the efficiency of amphiphilic shuttle peptide S10 for protein delivery and gene editing using SpCas9, and AsCas12a (Cpf1) ribonucleoproteins (RNPs). These approaches were evaluated in proliferating ferret airway basal cells, polarized airway epithelia in vitro, and lungs in vivo, by accessing the editing efficiency using reporter ferrets and measuring indels at the ferret CFTR locus. Our results demonstrate that shuttle peptides efficiently enable delivery of reporter proteins/peptides and gene editing SpCas9 or Cpf1 RNP complexes to ferret airway epithelial cells in vitro and in vivo. We measured S10 delivery efficiency of green fluorescent protein (GFP)-nuclear localization signal (NLS) protein or SpCas9 RNP into ferret airway basal cells and fully differentiated ciliated and nonciliated epithelial cells in vitro. In vitro and in vivo gene editing efficiencies were determined by Cas/LoxP-gRNA RNP-mediated conversion of a ROSA-TG Cre recombinase reporter using transgenic primary cells and ferrets. S10/Cas9 RNP was more effective, relative to S10/Cpf1 RNP at gene editing of the ROSA-TG locus. Intratracheal lung delivery of the S10 shuttle combined with GFP-NLS protein or D-Retro-Inverso (DRI)-NLS peptide demonstrated efficiencies of protein delivery that were ∼3-fold or 14-fold greater, respectively, than the efficiency of gene editing at the ROSA-TG locus using S10/Cas9/LoxP-gRNA. Cpf1 RNPs was less effective than SpCas9 at gene editing of LoxP locus. These data demonstrate the feasibility of shuttle peptide delivery of Cas RNPs to the ferret airways and the potential utility for developing ex vivo stem cell-based and in vivo gene editing therapies for genetic pulmonary diseases such as CF.

Codon Usage Bias Analysis in Macronuclear Genomes of Ciliated Protozoa.

Ciliated protozoa (ciliates) are unicellular eukaryotes, several of which are important model organisms for molecular biology research. Analyses of codon usage bias (CUB) of the macronuclear (MAC) genome of ciliates can promote a better understanding of the genetic mode and evolutionary history of these organisms and help optimize codons to improve gene editing efficiency in model ciliates. In this study, the following indices were calculated: the guanine-cytosine (GC) content, the frequency of the nucleotides at the third position of codons (T3, C3, A3, G3), the effective number of codons (ENc), GC content at the 3rd position of synonymous codons (GC3s), and the relative synonymous codon usage (RSCU). Parity rule 2 plot analysis, Neutrality plot analysis, ENc plot analysis, and correlation analysis were employed to explore the main influencing factors of CUB. The results showed that the GC content in the MAC genomes of each of 21 ciliate species, the genomes of which were relatively complete, was lower than 50%, and the base compositions of GC and GC3s were markedly distinct. Synonymous codon analysis revealed that the codons in most of the 21 ciliates ended with A or T and four codons were the general putative optimal codons. Collectively, our results indicated that most of the ciliates investigated preferred using the codons with anof AT-ending and that codon usage bias was affected by gene mutation and natural selection.

Application of CRISPR in Filamentous Fungi and Macrofungi: From Component Function to Development Potentiality.

Fungi, particularly filamentous fungi and macrofungi, have a very powerful ability to produce secondary metabolites and can serve as excellent chassis cells for the production of enzymes or natural products of great value in synthetic biology. Thus, it is imperative to establish simple, reliable, and efficient techniques for their genetic modification. However, the heterokaryosis of some fungi and the dominance of nonhomologous end-joining (NHEJ) repair mechanisms in vivo have been greatly affecting the efficiency of fungal gene editing. In recent years, the CRISPR/Cas9 system has been applied as a widely used gene editing technology in life science research and has also played an important role in the genetic modification of filamentous and macrofungi. The various functional components (cas9, sgRNA, promoter, and screening marker) of the CRISPR/Cas9 system and its development, as well as the difficulties and potential of the CRISPR/Cas9 system in filamentous fungus and macrofungi, are the main topics of this paper.

P-802 TFAP2C is required for proper segregation of developmental markers regulating trophectoderm commitment during mouse preimplantation development

Abstract Study question What is the effect of CRISPR/Cas9-mediated knock-out of trophectoderm markers TFAP2A/C on polarization and lineage commitment in mouse? Summary answer CRISPR/Cas9-mediated KO revealed discrepancies in TFAP2A/C function, wherein TFAP2C is dispensable for blastocyst formation but controls the segregation of transcription factors in the mouse embryo. What is known already TFAP2C is an important transcription factor that is expressed solely in trophectoderm (TE) in mouse, and both TE and epiblast in human. Recently, genetic ablation of both TFAP2C and TEAD4 (player of the HIPPO-pathway) completely abolished polarization in mouse, which indicates that TFAP2C might already act before the first lineage segregation takes place. In addition, TFAP2C RNA depletion experiments in mouse embryos revealed a compensatory upregulation of TFAP2A, which indicates functional redundancy. Nonetheless, the question remains how exactly TFAP2C regulates polarization on a molecular level in mouse and whether there is also a functional implication of its isoform TFAP2A. Study design, size, duration Guide RNAs were designed targeting exon 5 of Tfap2c and exon 2 of the Tfap2a gene. CRISPR/Cas9 ribonucleoprotein complexes were delivered into mouse zygotes via electroporation. Additionally, appropriate non-targeted and scramble (inactive crRNA) control groups were included. Morphological analysis, immunofluorescence and next-generation sequencing (NGS) were applied to check for gene editing efficiency and the impact of KO on embryonic development and lineage segregation. Participants/materials, setting, methods Targeted mouse embryos and controls were cultured for a maximum of 4.5 days in vitro. They were stained for different developmental markers, including CDX2 (TE), SOX2 (early ICM), NANOG (epibast, EPI) and SOX17 (hypoblast, PrE) at different stages of development, such as polarization (E2.5), first (E3.5) and second (E4.5) lineage segregation. Immunostaining was used to determine cell number, TE/ICM fraction, marker localization and fluorescence intensity. Embryos were subjected to genetic analysis to determine on-target efficiency. Main results and the role of chance CRISPR/Cas9-mediated electroporation of mouse zygotes generated efficiently complete knock-out embryos. Of the 39 mouse zygotes targeted for TFAP2C, 38 (97%) of them were edited. From the 38 embryos edited for TFAP2C, 24 (62%) embryos displayed 100% frameshift mutations, and were considered KO. TFAP2C KO mouse embryos cultured until E4.5 were still able to form blastocysts (13/15, 87%), but were associated with inferior blastocyst quality compared to the control groups. Furthermore, TFAP2c-null blastocysts could hatch but herniated at multiple places, in contrast to wild-type blastocysts, which typically herniate at one place. In full frameshift TFAP2C KO mouse embryos, we observed delayed CDX2 expression at E2.5 (n = 7). At E3.5 (n = 5), however, CDX2 expression could still be pertained in KO embryos, whereas nuclear localization of SOX2 was disturbed. At E4.5 (n = 6), some primitive endoderm cells were observed in the presumed ICM (SOX17-positive), whereas no NANOG-positive (EPI) cells could be detected. However, some cells of presumed TE of E4.5 TFAP2C-KO embryos displayed erroneous SOX17 or NANOG expression, indicating that they adopted another cell fate irrespective of cell localization. Secondly, we generated TFAP2A KO mouse embryos (3/5, 60%) which exhibited complete morula arrest (100%; n = 3) at E4.5. Limitations, reasons for caution CRISPR/Cas9 is limited by the occurrence of mosaicism (more than one genotype present in an embryo) and potential off-target editing, which we will assess at in silico predicted off-target sites via NGS in mouse embryonic stem cells. The observations of the study will be consolidated by increasing the sample size. Wider implications of the findings Gene editing studies enable us to unravel the molecular interactions that are required for human preimplantation development. Obtaining novel insights into the molecular networks of the TFAP2-transcription factor family could improve our fundamental understanding on the spatial segregation of key transcription factors, which is crucial for successful implantation. Trial registration number Not applicable

Recipient Somatic Embryogenesis System Promotes CRISPR/Cas Genome Editing

Somatic embryogenesis (SE) is an important type of plant regeneration, which can be used as a recipient system of genome editing. It is vital to optimize the SE system to improve the mutated somatic cells regenerated into fertile plants after transformation. 2,4-D was used as inducer of somatic embryogenesis from mature seeds. Arabidopsis wild type Col-0 and ABA biosynthesis double mutant <i>nced6 / 9 </i>were grown in 15°C and 25°C respectively, to investigate the effect of sampled plants growth conditions on the SE rate of their seeds and to optimize SE protocol. On the other hand, construct Pbi121-U6-26-Cas9-sgRNA-ELF6-FastR was used for genome editing. The positive seeds after floral dip were separated into two parts: some for sowing and the rest seeds went though SE induction. The efficiency of gene editing was compared by sequencing. Our results show that the SE rate of <i>nced6 / 9</i> seeds from 25°C was significantly lower than that from 15°C. When transgenic positive seeds were sown directly, the target sequence was not found to be edited; However, when seeds went though somatic embryogenesis protocol, gene editing were found around PAM sequence, suggesting that SE system could improve gene editing after transformation. The sampled plants grown at low temperature (15°C) may affect SE ability of their seeds by ABA level. The mature seed somatic embryogenesis system can improve the efficiency of gene editing.

CRISPR-Cas9 in hiPSCs: A new era in personalized treatment for Stargardt disease

CRISPR-Cas9 in hiPSCs: A new era in personalized treatment for Stargardt disease

CRISPR-Cas9 Mediated Gene Correction of CFTR Mutations in Cystic Fibrosis: Evaluating Efficacy, Safety, and Long-Term Outcomes in Patient-Derived Lung Organoids

Cystic fibrosis (CF) is a genetic disorder caused by mutations in the *CFTR* gene, leading to severe respiratory and digestive problems. Current therapies including AAV gene therapy, small molecule modifiers, and RNA-based therapies only partially restore *CFTR* function, leaving a need for more effective therapies The aim of this review is to address CRISPR-1. Cas9-mediated genetic correction in patient derived lung organoids Efficacy, safety and long-term outcomes are evaluated as a novel strategy for CF therapy. Our goal was to achieve high gene editing efficiency, restore *CFTR* function to near normal levels, and evaluate the safety of this approach by monitoring off-target effects CRISPR-Cas9 achieved 75% average gene editing efficiency, with recovery of *CFTR* function up to 85%, AAV gene therapy (35% efficiency, 50). % Functional restoration), small molecule modulators (60% functional restoration), and... RNA-based therapy (57% functional restoration). Off-target effects were small, observed at 0.5%, confirming the accuracy of CRISPR-Cas9 in correcting *CFTR* mutations. The use of patient-derived lung organoids proved important for modeling the ailment and trying out healing interventions, providing excessive personalization ability and the cap potential to reveal long-time period stability. The look at concludes that CRISPR-Cas9, while carried out to patient-derived lung organoids, represents a promising development withinside the remedy of Cystic Fibrosis, providing advanced efficacy and personalization as compared to present methods. However, the excessive fee and moral concerns related to CRISPR-Cas9 necessitate similarly studies to optimize its utility and make sure its protection in medical settings.

Lentiviral Transduction-based CRISPR/Cas9 Editing of Schistosoma mansoni Acetylcholinesterase.

Recent studies on CRISPR/Cas9-mediated gene editing in Schistosoma mansoni have shed new light on the study and control of this parasitic helminth. However, the gene editing efficiency in this parasite is modest. To improve the efficiency of CRISPR/Cas9 genome editing in schistosomes, we used lentivirus, which has been effectively used for gene editing in mammalian cells, to deliver plasmid DNA encoding Cas9 nuclease, a sgRNA targeting acetylcholinesterase (SmAChE) and a mCherry fluorescence marker into schistosomes. MCherry fluorescence was observed in transduced eggs, schistosomula, and adult worms, indicating that the CRISPR components had been delivered into these parasite stages by lentivirus. In addition, clearly changed phenotypes were observed in SmAChE-edited parasites, including decreased SmAChE activity, reduced hatching ability of edited eggs, and altered behavior of miracidia hatched from edited eggs. Next-generation sequencing analysis demonstrated that the lentiviral transduction-based CRISPR/Cas9 gene modifications in SmAChE-edited schistosomes were homology-directed repair predominant but with much lower efficiency than that obtained using electroporation (data previously published by our laboratory) for the delivery of CRISPR components. Taken together, electroporation is more efficient than lentiviral transduction in the delivery of CRISPR/Cas9 into schistosomes for programmed genome editing. The exploration of tactics for enhancing CRISPR/Cas9 gene editing provides the basis for the future improvement of programmed genome editing in S. mansoni.

Rapidly generating homozygous mutate zebrafish in F0 generation by technical integration of CRISPR/Cas9 and gynogenesis

Gene editing technique has been widely applied for gene function characterization. However, such an approach is often time-consuming to obtain homozygous mutant. In this article, through timing the earlier injection of embryos, the gene editing efficiency is significantly improved, and together with the technique of the UV inactivated sperm and heat shock treatment for blocking the first cleavage, the gene-edited homozygous zebrafish are successfully obtained in F0 generation, rather than in the third or more generations as reported in the existing results. It is concluded that genetic purification of artificial gynogenesis can play critical role in more efficiently preparing the gene-edited homozygous individuals in fish.

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

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.