Editing Efficiency Research Articles

CRISPR/Cas9-Based Genome Editing for Protein Expression and Secretion in Kluyveromyces lactis.

The budding yeast Kluyveromyces lactis has emerged as a promising microbial chassis in industrial biotechnology. However, a lack of efficient molecular genetic manipulation tools and strategies has hindered the development of K. lactis as a biomanufacturing platform. In this study, we developed and applied a CRISPR/Cas9-based genome editing method to K. lactis. Single-gene editing efficiency was increased to 80% by disrupting the nonhomologous end-joining-related gene KU80 and performing a series of process optimizations. Subsequently, the CRISPR/Cas9 system was explored based on different sgRNA delivery modes for simultaneous multigene editing. With the aid of the color indicator, the editing efficiencies of two and three genes reached 73.3 and 36%, respectively, in the KlΔKU80 strain. Furthermore, the CRISPR/Cas9 system was used for multisite integration to enhance lactase production and combinatorial knockout of TMED10 and HSP90 to characterize the extracellular secretion of lactase in K. lactis. Generally, genome editing is a powerful tool for constructing K. lactis cell factories for protein and chemical production.

Miniature CRISPR-Cas12 Systems: Mechanisms, Engineering, and Genome Editing Applications.

The therapeutic application of CRISPR-based gene editing technology is hindered by the delivery challenges of large Cas nucleases. The emergence of miniature editing tools derived from type V CRISPR systems and their ancestor TnpB nucleases presents promising solutions to counter these obstacles. Notably, the type V CRISPR-Cas12f and -Cas12n systems exhibit not only a concise gene size but also remarkable precision in targeted editing, thereby underscoring their potential as supreme gene editing tools. Although both systems are considered as intermediates in the evolution of TnpB to mature Cas12 effectors, they exhibit distinct biochemical and structural characteristics, demonstrating the diversity and complexity of TnpB's evolutionary outcomes. The diverse evolutionary branches indicate the existence of numerous unexplored compact CRISPR systems in nature, the mining and development of which could potentially revolutionize gene manipulation techniques and pave the way for innovative applications in gene therapy. In this Account, we summarize the recent advances from our group with the research and development of Cas12f and Cas12n genome editing systems, including the identification, characterization, and engineering for improving the editing efficiency. Additionally, we discuss the evolutionary process of the ancestral nuclease TnpB growing into various type V CRISPR systems, giving insight into the discovery of novel compact gene editing systems.

IgRNA Prediction and Selection AI Models (igRNA-PS) for Bystander-less ABE Base Editing

CRISPR derived base editing techniques tend to edit multiple bases in the targeted region, which impedes precise reversion of disease-associated single nucleotide variations (SNVs). We designed an imperfect gRNA (igRNA) editing strategy to achieve bystander-less single-base editing. To predict the performance and provide ready-to-use igRNAs, we employed a high-throughput method to edit 5000 loci, each with approximate 19 systematically designed ABE igRNAs. Through deep learning of the relationship of editing efficiency, original gRNA sequence and igRNA sequence, AI models were constructed and tested, designated igRNA Prediction and Selection AI models (igRNA-PS). The models have three functions, First, they can identify the major editing site from the bystanders on a gRNA protospacer with a near 90% accuracy. second, a modified single-base editing efficiency (SBE), considering both single-base editing efficiency and product purity, can be predicted for any given igRNAs. Third, for an editing locus, a set of 64 igRNAs derived from a gRNA can be generated, evaluated through igRNA-PS to select for the best performer, and provided to the user. In this work, we overcome one of the most significant obstacles of base editors, and provide a convenient and efficient approach for single-base bystander-less ABE base editing.

Synergistic combination of RAD51-SCR7 improves CRISPR-Cas9 genome editing efficiency by preventing R-loop accumulation

CRISPR-Cas9 has emerged as a powerful tool for genome editing. However, Cas9 genome editing faces challenges, including low efficiency and off-target effects. Here, we report that combined treatment with RAD51, a key factor in homologous recombination, and SCR7, a DNA ligase IV small-molecule inhibitor, enhances CRISPR-Cas9-mediated genome-editing efficiency in human embryonic kidney 293T and human induced pluripotent stem cells, as confirmed by cyro- transmission electron microscopy and functional analyses. First, our findings reveal the crucial role of RAD51 in homologous recombination (HR)-mediated DNA repair process. Elevated levels of exogenous RAD51 promote a post-replication step via single-strandDNA gap repair process, ensuring the completion of DNA replication. Second, using the all-in-one CRISPR-Cas9-RAD51 system, highly expressed RAD51 improved the multiple endogenous gene knockin/knockout efficiency and insertion/deletion (InDel) mutation by activating the HR-based repair pathway in concert with SCR7. Sanger sequencing shows distinct outcomes for RAD51-SCR7 in the ratio of InDel mutations in multiple genome sites. Third, RAD51-SCR7 combination can induce efficient R-loop resolution and DNA repair by enhanced HR process, which leads to DNA replication stalling and thus is advantageous to CRISPR-Cas9-based stable genome editing. Our study suggests promising applications in genome editing by enhancing CRISPR-Cas9 efficiency through RAD51 and SCR7, offering potential advancements in biotechnology and therapeutics.

Increasing the efficiency of CRISPR/Cas9-mediated genome editing in the citrus postharvest pathogen Penicillium digitatum

BackgroundPenicillium digitatum is a fungal plant pathogen that causes the green mold disease in harvested citrus fruits. Due to its economical relevance, many efforts have focused on the development of genetic engineering tools for this fungus. Adaptation of the CRISPR/Cas9 technology was previously accomplished with self-replicative AMA1-based plasmids for marker-free gene editing, but the resulting efficiency (10%) limited its practical implementation. In this study, we aimed to enhance the efficiency of the CRISPR/Cas9-mediated gene editing in P. digitatum to facilitate its practical use.ResultsIncreasing the culture time by performing additional culture streaks under selection conditions in a medium that promotes slower growth rates significantly improved the gene editing efficiency in P. digitatum up to 54–83%. To prove this, we disrupted five candidate genes that were chosen based on our previous high-throughput gene expression studies aimed at elucidating the transcriptomic response of P. digitatum to the antifungal protein PdAfpB. Two of these genes lead to visual phenotypic changes (PDIG_53730/pksP, and PDIG_54100/arp2) and allowed to start the protocol optimization. The other three candidates (PDIG_56860, PDIG_33760/rodA and PDIG_68680/dfg5) had no visually associated phenotype and were targeted to confirm the high efficiency of the protocol.ConclusionGenome editing efficiency of P. digitatum was significantly increased from 10% to up to 83% through the modification of the selection methodology, which demonstrates the feasibility of the CRISPR/Cas9 system for gene disruption in this phytopathogenic fungus. Moreover, the approach described in this study might help increase CRISPR/Cas9 gene editing efficiencies in other economically relevant fungal species for which editing efficiency via CRISPR/Cas9 is still low.

Precise in vivo RNA base editing with a wobble-enhanced circular CLUSTER guide RNA.

Recruiting the endogenous editing enzyme adenosine deaminase acting on RNA (ADAR) with tailored guide RNAs for adenosine-to-inosine (A-to-I) RNA base editing is promising for safely manipulating genetic information at the RNA level. However, the precision and efficiency of editing are often compromised by bystander off-target editing. Here, we find that in 5'-UAN triplets, which dominate bystander editing, G•U wobble base pairs effectively mitigate off-target events while maintaining high on-target efficiency. This strategy is universally applicable to existing A-to-I RNA base-editing systems and complements other suppression methods such as G•A mismatches and uridine (U) depletion. Combining wobble base pairing with a circularized format of the CLUSTER approach achieves highly precise and efficient editing (up to 87%) of a disease-relevant mutation in the Mecp2 transcript in cell culture. Virus-mediated delivery of the guide RNA alone realizes functional MeCP2 protein restoration in the central nervous system of a murine Rett syndrome model with editing yields of up to 19% and excellent bystander control in vivo.

CRISPR Base Editing to Create Potential Charcot-Marie-Tooth Disease Models with High Editing Efficiency: Human Induced Pluripotent Stem Cell Harboring SH3TC2 Variants.

Human induced pluripotent stem cells (hiPSCs) represent a powerful tool to investigate neuropathological disorders in which the cells of interest are inaccessible, such as in the Charcot-Marie-Tooth disease (CMT), the most common inherited peripheral neuropathy. Developing appropriate cellular models becomes crucial in order to both study the disease's pathophysiology and test new therapeutic approaches. The generation of hiPS cellular models for disorders caused by a single nucleotide variation has been significantly improved following the development of CRISPR-based editing tools. In this study, we efficiently and quickly generated, by CRISPR editing, the two first hiPSCs cellular models carrying alterations involved in CMT4C, also called AR-CMTde-SH3TC2. This subtype of CMT is associated with alterations in the SH3TC2 gene and represents the most prevalent form of autosomal recessive demyelinating CMT. We aimed to develop models for two different SH3TC2 nonsense variants, c.211C>T, p.Gln71* and the most common AR-CMTde-SH3TC2 alteration, c.2860C>T, p.Arg954*. First, in order to determine the best CRISPR strategy to adopt on hiPSCs, we first tested a variety of sgRNAs combined with a selection of recent base editors using the conveniently cultivable and transfectable HEK-293T cell line. The chosen CRISPR base-editing strategy was then applied to hiPSCs derived from healthy individuals to generate isogenic CMT disease models with up to 93% editing efficiency. For point mutation generation, we first recommend to test your strategies on alternative cell line such as HEK-293T before hiPSCs to evaluate a variety of sgRNA-BE combinations, thus boosting the chance of achieving edited cellular clones with the hard-to-culture and to transfect hiPSCs.

Conformational dynamics of CasX (Cas12e) in mediating DNA cleavage revealed by single-molecule FRET.

CasX (also known as Cas12e), a Class 2 CRISPR-Cas system, shows promise in genome editing due to its smaller size compared to the widely used Cas9 and Cas12a. Although the structures of CasX-sgRNA-DNA ternary complexes have been resolved and uncover a distinctive NTSB domain, the dynamic behaviors of CasX are not well characterized. In this study, we employed single-molecule and biochemical assays to investigate the conformational dynamics of two CasX homologs, DpbCasX and PlmCasX, from DNA binding to target cleavage and fragment release. Our results indicate that CasX cleaves the non-target strand and the target strand sequentially with relative irreversible dynamics. The two CasX homologs exhibited different cleavage patterns and specificities. The dynamic characterization of CasX also reveals a PAM-proximal seed region, providing guidance for CasX-based effector design. Further studies elucidate the mechanistic basis for why modification of sgRNA and the NTSB domain can affect its activity. Interestingly, CasX has less effective target search efficiency than Cas9 and Cas12a, potentially accounting for its lower genome editing efficiency. This observation opens a new avenue for future protein engineering.

Development of an Agrobacterium-mediated CRISPR/Cas9 gene editing system in jute (Corchorus capsularis)

Jute (Corchorus capsularis L.) is the second most important natural plant fiber source after cotton. However, developing an efficient gene editing system for jute remains a challenge. In this study, the transgenic hairy root system mediated by Agrobacterium rhizogenes strain K599 was developed for Meifeng 4, an elite jute variety widely cultivated in China. The transgenic hairy root system for jute was verified by subcellular localization and bimolecular fluorescence complementation (BiFC) assays. The CHLOROPLASTOS ALTERADOS 1 (CcCLA1) gene, which is involved in the development of chloroplasts, was targeted for editing at two sites in Meifeng 4. Based on this hairy root transformation, the gRNA scaffold was placed under the control of cotton ubiquitin GhU6.7 and -GhU6.9 promoters, respectively, to assess the efficiency of gene editing. Results indicated the 50.0% (GhU6.7) and 38.5% (GhU6.9) editing events in the target 2 alleles (gRNA2), but no mutation was detected in the target 1 allele (gRNA1) in transgenic-positive hairy roots. CcCLA1 gene editing at gRNA2 under the control of GhU6.7 in Meifeng 4 was also carried out by Agrobacterium tumefaciens-mediated transformation. Two CcCLA1 mutants were albinic, with a gene editing efficiency of 5.3%. These findings confirm that the CRISPR/Cas9 system, incorporating promoter GhU6.7, can be used as a gene editing tool for jute.

In situ targeted base editing of bacteria in the mouse gut

Microbiome research is now demonstrating a growing number of bacterial strains and genes that affect our health1. Although CRISPR-derived tools have shown great success in editing disease-driving genes in human cells2, we currently lack the tools to achieve comparable success for bacterial targets in situ. Here we engineer a phage-derived particle to deliver a base editor and modify Escherichia coli colonizing the mouse gut. Editing of a β-lactamase gene in a model E. coli strain resulted in a median editing efficiency of 93% of the target bacterial population with a single dose. Edited bacteria were stably maintained in the mouse gut for at least 42 days following treatment. This was achieved using a non-replicative DNA vector, preventing maintenance and dissemination of the payload. We then leveraged this approach to edit several genes of therapeutic relevance in E. coli and Klebsiella pneumoniae strains in vitro and demonstrate in situ editing of a gene involved in the production of curli in a pathogenic E. coli strain. Our work demonstrates the feasibility of modifying bacteria directly in the gut, offering a new avenue to investigate the function of bacterial genes and opening the door to the design of new microbiome-targeted therapies.

Development of super-infective ternary vector systems for enhancing the Agrobacterium-mediated plant transformation and genome editing efficiency.

Agrobacterium-mediated transformation remains a cornerstone of plant biology, fueling advancements in molecular genetics, new genomic techniques (NGTs), and the biotech industry. However, recalcitrant crops and technical hurdles persist as bottlenecks. The goal was to develop super-infective ternary vector systems that integrate a novel salicylic acid-degrading enzyme, GABA, and ethylene-degrading enzymes, targeting the transformation of crops by neutralizing plant defense system on Agrobacterium. Firstly, both the effect and activity of introducing enzymes were validated in EHA105, an important Agrobacterium strain. Our study demonstrates that all ternary vector (Tv) system variants significantly enhance reporter expression in transient assays with Nicotiana benthamiana and Cannabis sativa. Specifically, incorporating a constitutive virG mutation with novel enzyme combinations increased GFP and RUBY expression in C. sativa by >5-fold and 13-fold, respectively. The Tv system, combined with a geminivirus replicon, markedly boosted GUS gene expression in tomato, enhancing genome editing efficiency. Notably, compared to controls, Tv-VS demonstrated up to 18-fold and 4.5-fold increases in genome editing efficiency in C. sativa and tomato, respectively. Additionally, stable transformation rates in tomato and Arabidopsis improved significantly, with Tv-VS showing a remarkable 2.5-fold increase in transformation efficiency compared to control strains. The research marks notable progress in Agrobacterium-mediated plant transformation. The innovative ternary vectors overcome plant defense mechanisms, enabling genetic manipulation in previously challenging plant species. This development is anticipated to broaden the applications of plant genetic engineering, contributing to advancements in crop genome editing.

PE6c greatly enhances prime editing in transgenic rice plants.

Prime editing is a versatile CRISPR/Cas-based precise genome-editing technique for crop breeding. Four new types of prime editors (PEs) named PE6a-d were recently generated using evolved and engineered reverse transcriptase (RT) variants from three different sources. In this study, we tested the editing efficiencies of four PE6 variants and two additional PE6 constructs with double-RT modules in transgenic rice (Oryza sativa) plants. PE6c, with an evolved and engineered RT variant from the yeast Tf1 retrotransposon, yielded the highest prime-editing efficiency. The average fold change in the editing efficiency of PE6c compared with PEmax exceeded 3.5 across 18 agronomically important target sites from 15 genes. We also demonstrated the feasibility of using two RT modules to improve prime-editing efficiency. Our results suggest that PE6c or its derivatives would be an excellent choice for prime editing in monocot plants. In addition, our findings have laid a foundation for prime-editing-based breeding of rice varieties with enhanced agronomically important traits.

The HPE1 RNA-binding protein modulates chloroplast RNA editing to promote photosynthesis under cold stress in Arabidopsis.

Cold stress has severe negative consequences for plant growth and crop yield. Here, we report that an Arabidopsis thaliana mutant that lacks the HPE1 gene, which encodes an RNA-binding protein, maintains higher photosynthetic activity under cold stress, together with higher accumulation of thylakoid proteins. We showed that HPE1 interacts with MORF2 and MORF9 and thereby mediates RNA editing in chloroplasts. Loss of HPE1 function increased the editing efficiency at four RNA editing sites, rpoC-488, ndhB-149, ndhB-746 and matK-706, under cold stress and altered the expression of nuclear photosynthesis-related genes and cold-responsive genes. We propose that HPE1-mediated RNA editing acts as a trigger for retrograde signaling that affects photosynthesis under cold stress.

Engineering miniature IscB nickase for robust base editing with broad targeting range.

IscB has a similar domain organization to Cas9, but the small size of IscB is better suited for delivery by adeno-associated virus. To improve the low editing efficiency of OgeuIscB (IscB from human gut metagenome) in mammalian cells, we developed high-efficiency miniature base editors by engineering OgeuIscB nickase and its cognate ωRNA, termed IminiBEs. We demonstrated the robust editing efficiency of IminiCBE (67% on average) or IminiABE (52% on average). Fusing non-specific DNA-binding protein Sso7d to IminiBEs increased the editing efficiency of low-efficiency sites by around two- to threefold, and we termed it SIminiBEs. In addition, IminiCBE and SIminiCBE recognize NNRR, NNRY and NNYR target-adjacent motifs, which broaden the canonical NWRRNA target-adjacent motif sites for the wild-type IscB nickase. Overall, IminiBEs and SIminiBEs are efficient miniature base editors for site-specific genomic mutations.

Utilizing codon degeneracy in the design of donor DNA for CRISPR/Cas9-mediated gene editing to streamline the screening process for single amino acid mutations.

Chlamydomonas reinhardtii, a unicellular green alga, has been widely used as a model organism for studies of algal, plant and ciliary biology. The generation of targeted amino acid mutations is often necessary, and this can be achieved using CRISPR/Cas9 induced homology-directed repair to install genomic modifications from exogenous donor DNA. Due to the low gene editing efficiency, the technical challenge lies in identifying the mutant cells. Direct sequencing is not practical, and pre-screening is required. Here, we report a strategy for generating and screening for amino acid point mutations using the CRISPR/Cas9 gene editing system. The strategy is based on designing donor DNA using codon degeneracy, which enables the design of specific primers to facilitate mutant screening by PCR. An in vitro assembled RNP complex, along with a dsDNA donor and an antibiotic resistance marker, was electroporated into wild-type cells, followed by PCR screening. To demonstrate this principle, we have generated the E102K mutation in centrin and the K40R mutation in α-tubulin. The editing efficiencies at the target sites for Centrin, TUA1, TUA2 were 4, 24 and 8% respectively, based on PCR screening. More than 80% of the mutants with the expected size of PCR products were precisely edited, as revealed by DNA sequencing. Subsequently, the precision-edited mutants were biochemically verified. The introduction of codon degeneracy did not affect the gene expression of centrin and α-tubulins. Thus, this approach can be used to facilitate the identification of point mutations, especially in genes with low editing rates.

ABE-ultramax for high-efficiency biallelic adenine base editing in zebrafish

Advancements in CRISPR technology, particularly the development of base editors, revolutionize genetic variant research. When combined with model organisms like zebrafish, base editors significantly accelerate and refine in vivo analysis of genetic variations. However, base editors are restricted by protospacer adjacent motif (PAM) sequences and specific editing windows, hindering their applicability to a broad spectrum of genetic variants. Additionally, base editors can introduce unintended mutations and often exhibit reduced efficiency in living organisms compared to cultured cell lines. Here, we engineer a suite of adenine base editors (ABEs) called ABE-Ultramax (Umax), demonstrating high editing efficiency and low rates of insertions and deletions (indels) in zebrafish. The ABE-Umax suite of editors includes ABEs with shifted, narrowed, or broadened editing windows, reduced bystander mutation frequency, and highly flexible PAM sequence requirements. These advancements have the potential to address previous challenges in disease modeling and advance gene therapy applications.

P-805 Sperm and embryo genome editing by CRISPR-Cas9 in a mouse model

Abstract Study question We wonder whether it is possible to edit a specific gene in individual mammalian spermatozoa as well as embryo by using CRISPR-Cas9? Summary answer We were able to successfully edit the coat pigment gene on individual spermatozoa as well as embryos by injecting CRISPR-Cas9 during Piezo-ICSI. What is known already Earlier investigations into embryo genome editing with CRISPR-Cas9 were conducted during the S-phase or zygote stage, encountering obstacles such as mosaicism and loss of heterozygosity. To overcome these challenges, performing genome editing at the gamete level is considered more favorable. While it may be achievable in oocytes, sperm genome editing can be ambitious due to the tightly supercoiled DNA around the protamine cores. In initial attempts, simple permeabilization of the sperm membrane allowed CRISPR-Cas9 penetration into the cell; however, the attempts at genomic editing proved to be unsuccessful. Study design, size, duration In the past several months, oocytes were divided into two groups: half of them were used to perform embryo genome editing, whilst the other half was used to edit exclusively the sperm genome through oocyte-mediated sperm decondensation (OMSD), where a single spermatozoon was injected into an enucleated oocyte and used the ooplasmic machinery for DNA decondensation and editing. In both models, CRISPR-Cas9 was used to knock out Tyr gene to create an albino phenotype. Participants/materials, setting, methods B6D2F1 mice were used to retrieve oocytes and spermatozoa. A cohort of oocytes used for the OSMD approach were enucleated, whilst the others were maintained with an intact nucleus. All oocytes, enucleated or not, were injected with a single spermatozoon with Tyr-sgRNA and Cas9 protein. Embryos were cultured until the 8-cell stage and processed for T7E1 cleavage analysis. Genome editing efficiency was calculated following the manufacturer protocol. Main results and the role of chance Of the 40 oocytes used for the study, 20 intact oocytes were used to perform embryo genome editing in the standard fashion, while 20 were enucleated for OMSD experiments. After undergoing Piezo-ICSI with CRISPR-Cas9 solution, 90.0% (18/20) of intact oocytes fertilized. After 48 hours in culture, diploid embryos reached the 8-cell stage at 88.9% (16/18). The 584-bp region of the target site of extracted DNA was amplified and gene modification of the edited diploid embryo cohort was confirmed in 87.5% (14/16). Compared to the OMSD cohort, enucleated oocytes fertilized at 85.0% (17/20) while cleavage rate was lower at 64.7% (11/17) and sperm genome modification at 81.8% (9/11). Both diploid embryos and haploid androgenetic embryos evidence an editing efficiency of 36.8%. Therefore, we verified that genome editing on embryos and sperm achieved the same editing yield. Limitations, reasons for caution While the experimental observations are still limited, it is important to note that these results in the embryo model may be underestimated by the T7E1 cleavage assay that only recognize heterozygous modification. The technique needs further refinement to optimize targeting and editing efficiency. Wider implications of the findings Sperm genome decondensation through an OMSD approach allows CRISPR-Cas9 to edit the sperm genome in order to correct eventual pathogenic mutations present in the gamete. Moreover, these would be the only method to absolutely ensure uniform generation of resulting embryos. Trial registration number not applicable

P-806 Heritable genome editing in haploid mouse embryonic stem cells

Abstract Study question Can we edit a specific gene by CRISPR-Cas9 in haploid mouse embryonic stem cells? Summary answer We successfully carried out heritable genome editing of a coat pigmentation gene in haploid mouse embryonic stem cells via electroporation. What is known already Embryonic stem cells have been widely used as a substrate to carry out genetic studies due to their availability, low cost and ease of handling. CRISPR-Cas9 has been successful in editing genes in the human model. Gene editing occurred by homology directed repair or non-homologous end joining. The utilization of a haploid genome would confirm the feasibility of non-homologous end joining repair mechanism. Study design, size, duration In the past 3 months, diploid as positive control and experimental haploid embryonic stem cells were independently cultured for genome editing experiments. Electroporation was used as a means to introduce CRISPR-Cas9 into cells. The negative control cohort consisted of embryonic stem cells electroporated without delivering any CRISPR solution. CRISPR-Cas9 was designed to knock out the Tyr gene to create an albino phenotype. Participants/materials, setting, methods Diploid embryonic stem cells were derived from C57BL/6 mice. The haploid counterpart instead was obtained from haploid androgenetic embryos using spermatozoa from Oct4-EGFP mice. CRISPR solution was prepared by combining Tyr gRNA and Cas9 protein. For each experiment, one million cells were suspended in CRISPR solution and an electroporation buffer. Then, electric pulses were applied for electroporation with 4 different parameters. Gene modification at the target site was confirmed using T7E1 cleavage assay. Main results and the role of chance A 584 base pairs region around the CRISPR target site was amplified in all the diploid and haploid mouse embryonic stem cells. After performing T7E1 cleavage assay, editing efficiency was calculated following the manufacturer protocol. Precisely, after testing out different electroporation conditions on diploid mESCs, 1350V/20ms/2 pulses together with the combination of three sgRNAs (exon 1 and exon 2) and Cas9 protein, provided the best genome editing efficiency at 36.8% with a viability of 80%. Interestingly, when delivering only one type of gRNA (exon 2) together with Cas9 protein, we saw an efficiency rising to 77.6%. We then used the same condition to edit the Tyr gene in the genome of haploid androgenetic stem cells and confirmed that also in these cells, we were able to obtain a comparable genome editing efficiency at 68.4%. Limitations, reasons for caution While these experimental observations are limited, it is encouraging that it is possible to target and edit a haploid genome with a single allele. Wider implications of the findings The utilization of a haploid cell culture model paves the way to utilize CRISPR-Cas9 for gamete genome editing. This will provide a consistent and reliable method to correct inheritable genetic conditions on gametes allowing uniform gene editing of all cell progeny. Trial registration number Not applicable

P-794 Unraveling the function of GATA2 and GATA3 in mouse blastocyst formation via CRISPR/Cas9 gene editing

Abstract Study question What is the effect of the CRISPR/Cas9 mediated knock-out (KO) of Gata2 and Gata3 on mouse preimplantation development/blastocyst formation? Summary answer Efficient generation of Gata3/Gata2 KO mouse embryos was achieved. Gata3 and Gata2 KO embryos could form blastocysts, suggesting a possible dispensability for blastocyst formation. What is known already GATA2 and GATA3 are important trophectoderm markers in mouse and human embryos. Prior studies, involving RNAi knockdown or conditional KO of Gata3, demonstrated high rates of morula arrest of Gata3-/- embryos, indicating its essential role in blastocyst formation. However, conflicting results showed embryonic lethality in Gata3-/- embryos only at 11-12 dpf. Besides, conditional KO of Gata2 suggested its dispensability in blastocyst formation, as Gata2-/- embryos could form blastocysts. It is of interest to validate these findings and potential functional redundancy between GATA2 and GATA3 in mouse embryos, by generating CRISPR/Cas9-mediated single and double KOs of Gata2 and Gata3. Study design, size, duration Upon assessment of the on-/ off-target effects of CRISPR/Cas9 components in mouse embryonic stem cells (mESC), CRISPR/Cas9 ribonucleoprotein (RNP) complexes were electroporated in mouse zygotes. Suitable control groups (non-targeted and scrambled targeted (inactive crRNA)) were incorporated. Embryos were genotyped by next generation sequencing (NGS), to assess the effect of gene KO on embryo morphology. Additionally, the expression pattern of Gata2 and Gata3 was examined in wild type and CRISPR/Cas9 targeted embryos, with immunofluorescent staining (IFS). Participants/materials, setting, methods Following CRISPR/Cas9 mediated targeting of Gata2 and Gata3, mouse embryos were cultured and morphologically monitored until day 4.5, alongside relevant control groups (scrambled, media control). Subsequently, DNA was extracted, and embryos were genotyped by PCR amplification of the region surrounding the target site, followed by NGS. IFS of mouse embryos at different developmental stages (E2.5, E3.0, E3.5 and E4.5) was performed to assess expression patterns and localization of both Gata2 and Gata3 throughout development. Main results and the role of chance Following the design of two gRNAs targeting either Gata2 or Gata3, on-target editing efficiency was tested in mESCs. The selected Gata2-targeting design resulted in a frameshift efficiency of 75%, whereas the Gata3-targeting design resulted in a frameshift efficiency of 78%. No off-target effects were observed in Gata2 and Gata3 targeted mESCs when examining in silico predicted off-target sites through PCR and NGS. Furthermore, 51% of Gata3 targeted and 41% of Gata2 targeted mouse embryos contained frameshift mutations in all cells. Notably, the majority of Gata3-/- (93%, n = 22) and Gata2-/- (94%, n = 16) embryos developed to blastocysts, similar to media control and scrambled groups. No statistically significant difference between Gata3-/- (n = 24, p = 0.326) and Gata2-/- (n = 17, p = 0.978) embryo morphology and scrambled (n = 96) embryo morphology was detected. IFS of control embryos (n = 16) showed that nuclear expression of GATA2 precedes nuclear expression of GATA3, indicating that GATA2 might control the expression of Gata3. Following IFS of Gata3 targeted embryos (n = 4), no nuclear signal for both GATA2 and GATA3 was observed, indicating that GATA3 could regulate the expression of Gata2. Furthermore, we created 100% double KO mouse embryos for Gata2 and Gata3 in two ways, enabling comprehensive exploration of functional redundancy in future studies. Limitations, reasons for caution Mosaicism (>1 genotype present in one embryo), off-target effects and chromosomal arrangements pose challenges regarding CRISPR/Cas9 germline genome editing. Zygote-stage delivery of RNP complexes is used to address mosaicism, and off-target effects are screened in mESCs via NGS. Future efforts aim to increase the sample sizes for stronger support. Wider implications of the findings Our goal is to identify the molecular pathways underlying unsuccessful implantation in mouse and human embryos. Utilizing CRISPR/Cas9 to generate KO embryos enhances our understanding of key molecular mechanisms, potentially shedding light on pregnancy failure due to failure of trophectoderm specification, formation or maintenance. Trial registration number not applicable

Chromatin context-dependent effects of epigenetic drugs on CRISPR-Cas9 editing.

The efficiency and outcome of CRISPR/Cas9 editing depends on the chromatin state at the cut site. It has been shown that changing the chromatin state can influence both the efficiency and repair outcome, and epigenetic drugs have been used to improve Cas9 editing. However, because the target proteins of these drugs are not homogeneously distributed across the genome, the efficacy of these drugs may be expected to vary from locus to locus. Here, we systematically analyzed this chromatin context-dependency for 160 epigenetic drugs. We used a human cell line with 19 stably integrated reporters to induce a double-stranded break in different chromatin environments. We then measured Cas9 editing efficiency and repair pathway usage by sequencing the mutational signatures. We identified 58 drugs that modulate Cas9 editing efficiency and/or repair outcome dependent on the local chromatin environment. For example, we find a subset of histone deacetylase inhibitors that improve Cas9 editing efficiency throughout all types of heterochromatin (e.g. PCI-24781), while others were only effective in euchromatin and H3K27me3-marked regions (e.g. apicidin). In summary, this study reveals that most epigenetic drugs alter CRISPR editing in a chromatin-dependent manner, and provides a resource to improve Cas9 editing more selectively at the desired location.