Repair Template Research Articles

Employing template-directed CRISPR-based editing of the OsALS gene to create herbicide tolerance in Basmati rice.

Rice (Oryza sativa) is one of the primary food crops which contributes major portion of daily calorie intake. It is used as model crop for various genome editing studies. Basmati rice was also explored for establishing non-homologous end joining-based genome editing. But it was not clear whether homology-directed repair (HDR)-based genome editing can be done in Basmati rice. The current study was designed to establish HDR-based genome editing in Basmati rice to develop herbicide tolerance. There is severe weed spread when rice is grown via direct planted rice method in various countries to save labour and water resources. Therefore, the use of herbicides is necessary to control weeds. These herbicides can also affect cultivated rice which creates the need to develop herbicide-tolerant rice. In current study, we introduced a point mutation in Acetolactate Synthase gene to convert tryptophan to leucine at position 548. For this purpose, different constructs for HDR were tested with different RNA scaffold and orientation of repair templates. Out of four different architectures, the one having repair template identical to the target DNA strand precisely edited the target site. We successfully established template-directed CRISPR-Cas9 system in Super Basmati rice by detecting desired substitutions at the target site in Acetolactate Synthase locus. Moreover, this editing of Acetolactate Synthase gene resulted in the production of herbicide tolerance in Super Basmati rice. This study suggests that such type of HDR system can be used to precisely edit other genes for crop improvement.

MDM2 antagonists promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells

CRISPR-Cas9-mediated genome editing in sheep is of great use in both agricultural and biomedical applications. While targeted gene knockout by CRISPR-Cas9 through non-homologous end joining (NHEJ) has worked efficiently, the knockin efficiency via homology-directed repair (HDR) remains lower, which severely hampers the application of precise genome editing in sheep. Here, in sheep fetal fibroblasts (SFFs), we optimized several key parameters that affect HDR, including homology arm (HA) length and the amount of double-stranded DNA (dsDNA) repair template; we also observed synchronization of SFFs in G2/M phase could increase HDR efficiency. Besides, we identified three potent small molecules, RITA, Nutlin3, and CTX1, inhibitors of p53-MDM2 interaction, that caused activation of the p53 pathway, resulting in distinct G2/M cell-cycle arrest in response to DNA damage and improved CRISPR-Cas9-mediated HDR efficiency by 1.43- to 4.28-fold in SFFs. Furthermore, we demonstrated that genetic knockout of p53 could inhibit HDR in SFFs by suppressing the expression of several key factors involved in the HDR pathway, such as BRCA1 and RAD51. Overall, this study offersan optimized strategy for the usage of dsDNA repair template, more importantly, the application of MDM2 antagonists provides a simple and efficient strategy to promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells.

Fluorescent PCR-based Screening Methods for Precise Knock-in of Small DNA Fragments and Point Mutations in Zebrafish.

Generation of zebrafish (Danio rerio) models with targeted insertion of epitope tags and point mutations is highly desirable for functional genomics and disease modeling studies. Currently, CRISPR/Cas9-mediated knock-in is the method of choice for insertion of exogeneous sequences by providing a repair template for homology-directed repair (HDR). A major hurdle in generating knock-in models is the labor and cost involved in screening of injected fish to identify the precise knock-in events due to low efficiency of the HDR pathway in zebrafish. Thus, we developed fluorescent PCR-based high-throughput screening methods for precise knock-in of epitope tags and point mutations in zebrafish. Here, we provide a step-by-step guide that describes selection of an active sgRNA near the intended knock-in site, design of single-stranded oligonucleotide (ssODN) templates for HDR, quick validation of somatic knock-in using injected embryos, and screening for germline transmission of precise knock-in events to establish stable lines. Our screening method relies on the size-based separation of all fragments in an amplicon by fluorescent PCR and capillary electrophoresis, thus providing a robust and cost-effective strategy. Although we present the use of this protocol for insertion of epitope tags and point mutations, it can be used for insertion of any small DNA fragments (e.g., LoxP sites, in-frame codons). Furthermore, the screening strategy described here can be used to screen for precise knock-in of small DNA sequences in any model system, as PCR amplification of the target region is its only requirement. Key features This protocol expands the use of fluorescent PCR and CRISPR-STAT for screening of precise knock-in of small insertions and point mutations in zebrafish. Allows validation of selected sgRNA and HDR template within two weeks by somatic knock-in screening. Allows robust screening of point mutations by combining restriction digest with CRISPR-STAT. Graphical overview Overview of the three-phase knock-in pipeline in zebrafish (created with BioRender.com).

Gene Editing in Mouse Zygotes Using the CRISPR/Cas9 System.

Engineering of the mouse germline is a key technology in biomedical research for studying the function of genes in health and disease. Since the first knockout mouse was described in 1989, gene targeting was based on recombination of vector encoded sequences in mouse embryonic stem cell lines and their introduction into preimplantation embryos to obtain germline chimeric mice. This approach has been replaced in 2013 by the application of the RNA-guided CRISPR/Cas9 nuclease system, which is introduced into zygotes and directly creates targeted modifications in the mouse genome. Upon the introduction of Cas9 nuclease and guide RNAs into one-cell embryos, sequence-specific double-strand breaks are created that are highly recombinogenic and processed by DNA repair enzymes. Gene editing commonly refers to the diversity of DSB repair products that include imprecise deletions or precise sequence modifications copied from repair template molecules. Since gene editing can now be easily applied directly in mouse zygotes, it has rapidly become the standard procedure for generating genetically engineered mice. This article covers the design of guide RNAs, knockout and knockin alleles, options for donor delivery, preparation of reagents, microinjection or electroporation of zygotes, and the genotyping of pups derived from gene editing projects.

PCR cloning Intermediated Gibson assembly (PIG) for Constructing DNA Repair Templates in CRISPR-Cas9 Based Gene Editing.

The CRISPR-Cas9 gene-editing system has revolutionized genome engineering, allowing precise modifications to be made in a wide range of organisms. One significant challenge associated with CRISPR-Cas9 mediated gene editing is the construction of DNA repair templates containing homology arms, a screenable marker and a tag sequence of interest. Here, we present an efficient, two-step strategy to generate DNA repair templates in approximately one week, facilitating rapid and precise genome engineering applications.

Base editing therapy forges ahead

CRISPR/Cas-based gene editing is an innovative biotechnology that has revolutionized genetic engineering in recent years. The process involves induction of a double-strand break (DSB) at the desired DNA site and subsequent cellular repair. Two primary mechanisms drive DSB repair: non-homologous end joining and homologous recombination-mediated repair. Non-homologous end joining, the primary mode of DSB repair, is a simple high-efficiency process that is susceptible to errors, and unpredictable nucleotide insertion or deletion mutations. In contrast, point mutations account for more than 50% of human genetic disorders and are the most frequent type of genetic variation in nature. Base editing is a precise gene editing approach in which a single DNA base is substituted without introduction of DSBs or use of a repair template. This technique has promising therapeutic potential in gene therapy, owing to its high efficiency and controllable editing results. Since the invention of the first base editing tools, the technique has rapidly developed and undergone clinical trials. This review summarizes progress in gene therapy through base editing, including DNA and RNA base editing, with particular emphasis on recent clinical trial and preclinical research advancements, current limitations and remaining challenges, and prospects for further research and applications.

Production of light-coloured, low heat-absorbing Holstein Friesian cattle by precise embryo-mediated genome editing

Context Genome editing enables the introduction of beneficial sequence variants into the genomes of animals with high genetic merit in a single generation. This can be achieved by introducing variants into primary cells followed by producing a live animal from these cells by somatic cell nuclear transfer cloning. The latter step is associated with low efficiencies and developmental problems due to incorrect reprogramming of the donor cells, causing animal welfare concerns. Direct editing of fertilised one-cell embryos could circumvent this issue and might better integrate with genetic improvement strategies implemented by the industry. Methods In vitro fertilised zygotes were injected with TALEN editors and repair template to introduce a known coat colour dilution mutation in the PMEL gene. Embryo biopsies of injected embryos were screened by polymerase chain reaction and sequencing for intended biallelic edits before transferring verified embryos into recipients for development to term. Calves were genotyped and their coats scanned with visible and hyperspectral cameras to assess thermal energy absorption. Key results Multiple non-mosaic calves with precision edited genotypes were produced, including calves from high genetic merit parents. Compared to controls, the edited calves showed a strong coat colour dilution which was associated with lower thermal energy absorbance. Conclusions Although biopsy screening was not absolutely accurate, non-mosaic, precisely edited calves can be readily produced by embryo-mediated editing. The lighter coat colouring caused by the PMEL mutation can lower radiative heat gain which might help to reduce heat stress. Implications The study validates putative causative sequence variants to rapidly adapt grazing cattle to changing environmental conditions.

A robust pipeline for efficient knock-in of point mutations and epitope tags in zebrafish using fluorescent PCR based screening

BackgroundGenome editing using CRISPR/Cas9 has become a powerful tool in zebrafish to generate targeted gene knockouts models. However, its use for targeted knock-in remains challenging due to inefficient homology directed repair (HDR) pathway in zebrafish, highlighting the need for efficient and cost-effective screening methods. ResultsHere, we present our fluorescent PCR and capillary electrophoresis based screening approach for knock-in using a single-stranded oligodeoxynucleotide donor (ssODN) as a repair template for the targeted insertion of epitope tags, or single nucleotide changes to recapitulate pathogenic human alleles. For the insertion of epitope tags, we took advantage of the expected change in size of the PCR product. For point mutations, we combined fluorescent PCR with restriction fragment length polymorphism (RFLP) analysis to distinguish the fish with the knock-in allele. As a proof-of-principle, we present our data on the generation of fish lines with insertion of a FLAG tag at the tcnba locus, an HA tag at the gata2b locus, and a point mutation observed in Gaucher disease patients in the gba gene. Despite the low number of germline transmitting founders (1–5%), combining our screening methods with prioritization of founder fish by fin biopsies allowed us to establish stable knock-in lines by screening 12 or less fish per gene.ConclusionsWe have established a robust pipeline for the generation of zebrafish models with precise integration of small DNA sequences and point mutations at the desired sites in the genome. Our screening method is very efficient and easy to implement as it is PCR-based and only requires access to a capillary sequencer.

Stabilization of DNA fork junctions by Smc5/6 complexes revealed by single-molecule imaging.

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.

CD25 As Feedback Regulator of Oncogenic TCR Signaling in T-Cell Lymphoma

Introduction: CD25, also known as interleukin-2 receptor α chain (IL-2Rα), forms a heterotrimer with IL-2Rβ and common γ chain, and mediates IL-2 signaling. IL-2 signaling is essential for T- and NK-cell survival and proliferation. Additionally, CD25 is required for development of thymic regulatory T cells (Treg) and their suppressive function. Interestingly, CD25 is upregulated in a variety of lymphoid and myeloid malignancies, which do not depend on IL-2 signaling, suggesting IL-2-independent roles of CD25. Here, we seek to investigate whether CD25 has previously unrecognized functions in T cells and T-cell lymphomas. Results: Our previous study in B cells has discovered the cytoplasmic tail of CD25 as an essential structural element for site-specific recruitment of inhibitory phosphatases. CD25-mediated shuttling of inhibitory phosphatases balanced strength of oncogenic B-cell receptor (BCR)-signaling and was essential for cell-survival in B-cell malignancies. Interestingly, we found that CD25 expressed on B cells was monomeric and showed dynamic recruitment to BCR or its oncogenic mimics within lipid rafts. Studying gene expression data across different hematopoietic lineages, we found CD25 was strongly induced in T cell receptor (TCR)-activated T cells, as well as in T-cell malignancies that harbor oncogenic TCR-mimics, such as PTCL. Our mechanistic studies discovered that the serine residue 268 (S268) in the cytoplasmic tail of CD25 was a principal substrate of protein kinase C (PKC), and that S268 phosphorylation by PKC was critical for cell surface expression of CD25. The proximity-based labeling assays (Bio-ID) revealed that CD25-S268 but not its phospho-dead mutant recruits PKC and its adapter RACK1, which scaffold the inhibitory phosphatases SHIP1 and SHP1 for site-specific activation in lipid rafts. CRISPR-mediated knock-in of CD25-S268A that abrogates the formation of inhibitory phosphatase complexes in primary human T cells enabled us to specifically interrogate the functional relevance of CD25-S268 in the regulation of TCR signaling. By using single stranded homology-directed repair templates (ssHDRT) that contain a Cas9 shuttling sequence, we were able to achieve 70% knock-in efficiency in primary human T cells (Figure 1). Signaling studies with these cells revealed that S268A mutation significantly augmented TCR signaling strength. Additionally, S268A mutation resulted in acute inactivation of inhibitory phosphatases including SHP1. Consistent with these results, CD25 shuttling to TCR complex was promptly induced upon TCR stimulation and was mitigated by S268 mutation. Furthermore, immunophenotyping showed that loss of S268 phosphorylation resulted in increased expression of T-cell activation markers such as CD44 and CD69. Based on the ex vivo results, we generated a transgenic mouse model with germline knock-in of CD25-S268A, which would allow us to study S268-specific roles in CD25-mediated feedback regulation of TCR and oncogenic signaling in vivo. Conclusions: The role of CD25 in mediating IL-2 signaling has been extensively studied. However, increasing evidence suggests that CD25 plays a more sophisticated role in TCR signaling and T-lymphomagenesis. Here we discovered a previously unrecognized function of CD25 as a feedback regulator of TCR signaling. TCR activation or oncogenic TCR-mimics induce CD25-mediated recruitment of inhibitory phosphatases in lipid rafts to mitigate TCR signaling or curb excessive oncogenic signaling. Figure 1. CRISPR-mediated knock-out of CD25 alone, or followed by knock-in of CD25-S268-GFP or CD25-A268-GFP, in primary human T cells. The proportion of CD25-expressing GFP+ cells are analyzed by flow cytometry on day 4 and day 30 post CRISPR. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Virus-derived gene transfer agents benefit host cells by providing templates for DNA repair.

The potential benefits of producing gene transfer agents (GTAs) have long been speculated. A new study in PLOS Biology shows that DNA transfer by these phage-like elements allows cells with DNA damage to perform DNA repair and survive.

Prophage-like gene transfer agents promote Caulobacter crescentus survival and DNA repair during stationary phase.

Gene transfer agents (GTAs) are prophage-like entities found in many bacterial genomes that cannot propagate themselves and instead package approximately 5 to 15 kbp fragments of the host genome that can then be transferred to related recipient cells. Although suggested to facilitate horizontal gene transfer (HGT) in the wild, no clear physiological role for GTAs has been elucidated. Here, we demonstrate that the α-proteobacterium Caulobacter crescentus produces bona fide GTAs. The production of Caulobacter GTAs is tightly regulated by a newly identified transcription factor, RogA, that represses gafYZ, the direct activators of GTA synthesis. Cells lacking rogA or expressing gafYZ produce GTAs harboring approximately 8.3 kbp fragment of the genome that can, after cell lysis, be transferred into recipient cells. Notably, we find that GTAs promote the survival of Caulobacter in stationary phase and following DNA damage by providing recipient cells a template for homologous recombination-based repair. This function may be broadly conserved in other GTA-producing organisms and explain the prevalence of this unusual HGT mechanism.

High-efficiency retron-mediated single-stranded DNA production in plants.

Retrons are a class of retroelements that produce multicopy single-stranded DNA (ssDNA) and participate in anti-phage defenses in bacteria. Retrons have been harnessed for the overproduction of ssDNA, genome engineering and directed evolution in bacteria, yeast and mammalian cells. Retron-mediated ssDNA production in plants could unlock their potential applications in plant biotechnology. For example, ssDNA can be used as a template for homology-directed repair (HDR) in several organisms. However, current gene editing technologies rely on the physical delivery of synthetic ssDNA, which limits their applications. Here, we demonstrated retron-mediated overproduction of ssDNA in Nicotiana benthamiana. Additionally, we tested different retron architectures for improved ssDNA production and identified a new retron architecture that resulted in greater ssDNA abundance. Furthermore, co-expression of the gene encoding the ssDNA-protecting protein VirE2 from Agrobacterium tumefaciens with the retron systems resulted in a 10.7-fold increase in ssDNA production in vivo. We also demonstrated clustered regularly interspaced short palindromic repeats-retron-coupled ssDNA overproduction and targeted HDR in N. benthamiana. Overall, we present an efficient approach for in vivo ssDNA production in plants, which can be harnessed for biotechnological applications. Graphical Abstract.

Highly Efficient CRISPR/Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells.

Human pluripotent stem cells hold tremendous potential for both basic biology and cell-based therapies for a wide variety of diseases. The ability to manipulate the genome of these cells using the CRISPR/Cas9 technology has expanded this potential by providing a valuable tool to engineer or correct disease-associated mutations. Because of the high efficiency with which CRISPR/Cas9 creates targeted double-strand breaks, a major challenge has been the introduction of precise genetic modifications on one allele without indel formation on the non-targeted allele. To overcome this obstacle, we describe use of two oligonucleotide repair templates: one expressing the sequence change and the other maintaining the normal sequence. In addition, we have streamlined both the transfection and screening methodologies to make the protocols efficient, with small numbers of cells and a limited amount of labor-intensive clone passaging. This article provides a technically simple approach for generating valuable tools to model human disease in stem cells. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Application and optimization of CRISPR-based genome editing in human pluripotent stem cells Basic Protocol 2: Genetic modification of human pluripotent stem cells using a double-oligonucleotide CRISPR/Cas9 recombination system.

A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9.

Until recently, the favored method for making directed modifications to the budding yeast genome involved the introduction of a DNA template carrying the desired genetic changes along with a selectable marker, flanked by homology arms. This approach both limited the ability to make changes within genes due to disruption by the introduced selectable marker and prevented the use of that selectable marker for subsequent genomic manipulations. Following the discovery of CRISPR-Cas9-mediated genome editing, protocols were developed for modifying any DNA region of interest in a similar single transformation step without the need for a permanent selectable marker. This approach involves the generation of a DNA double-strand break (DSB) at the desired genomic location by the Cas9 nuclease, expressed on a plasmid which also expresses the guide RNA (gRNA) sequence directing the location of the DSB. The DSB is subsequently repaired via homologous recombination using a PCR-derived DNA repair template. Here, we describe in detail an improved method for incorporation of the gRNA-encoding DNA sequences into the Cas9 expression plasmid. Using Golden Gate cloning, annealed oligonucleotides bearing unique single-strand DNA overhangs are ligated into directional restriction enzyme sites. We describe the use of this CRISPR-Cas9 genome editing protocol to introduce multiple types of directed genetic changes into the yeast genome.

ALL-IN-ONE VIRUS-FREE MANUFACTURING PROCESS OF ALLOGENEIC CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS USING CRISPR/CAS9

Chimeric antigen receptor (CAR) T cell therapy has emerged as a promising therapeutic opportunity against blood cancers, leading to the six FDA-approved CAR T cell products. All commercially available products are produced following an autologous strategy, which represents a bottleneck for the widespread adoption of this technology due to the poor quality and quantity of patients’ T cells. Allogeneic T cells from healthy donors are an excellent alternative over the autologous ones, allowing the production of a ready-to-use therapy besides additional benefits, such as standardization of the final product, cost reduction through manufacturing scale-up, and the possibility of multiple genetic modifications. Safety concerns related to the use of cells from third-party donors can be easily eliminated through T cell receptor (TCR) knock-out with the demonstrated prevention of graft-versus-host disease development. Here we describe CRISPR/Cas9 editing for TRAC ( T Cell Receptor Alpha Constant )-targeted genomic integration of the CAR transgene and the consequent TCR knock-out in a fast and efficient virus-free manufacturing process. For the CRISPR/Cas9-mediated knock-in, we designed a plasmid containing the second-generation anti-CD19 CAR flanked by arms homologous to the desired cut site. This sequence was PCR amplified and purified, resulting in a highly concentrated double-stranded DNA (dsDNA) template for the homology-directed repair (HDR). Human T cells isolated from healthy donors were electroporated with the dsDNA-HDR templates (dsDNA-HDRT) and Cas9 ribonucleoproteins (RNPs) targeting the human TRAC locus. Cells were assayed on days 5 and 11 post-electroporation for CAR expression and killing efficiency, respectively. A mCherry transgene flanked by the same homology arms was also included as a control. Preliminary studies using the mCherry dsDNA-HDRT indicated a 1.2-fold improvement in the editing efficiency by adding the positively charged polymer poly-L-glutamic acid (40 μg) that binds to RNPs and reduces aggregates. Additionally, by culturing the electroporated cells with varying concentrations of an NHEJ inhibitor, we observed a 1.4 to 2-fold increase in mCherry knock-in, suggesting a favorable condition for the HDR. Based on the established optimized conditions, we electroporated T cells with the CAR dsDNA-HDRT (3.1 kb length, 6 μg) and got 88% of CAR+ cells five days after electroporation. The viability was 27% on day 3 and reached about 60% on day 7. On-target CAR gene integration was confirmed through an “in-out” PCR assay on the genomic DNA using primers specific to the TRAC locus and CAR transmembrane domain. We then analyzed the in vitro cytotoxicity of CAR T cells by coculturing them with GFP-expressing tumor cell lines. Complete growth inhibition of the RS4-11 cells (CD19 + ) was achieved at the 5:1, 10:1, and 50:1 effector-to-target ratios. For the K562 cells (CD19-), conversely, the killing activity was related to the T cells themselves since CAR expression did not further decrease the tumor cell number. These results demonstrate robust cytotoxic function and target specificity of the CAR T cell products. Combined, the data provide a proof-of-concept for the virus-free manufacturing process of allogeneic anti-CD19 CAR T cells, enabling CAR knock-in and TCR knock-out in an all-in-one strategy.

Natural biopolymer scaffold for meniscus tissue engineering.

Meniscal injuries caused by trauma, degeneration, osteoarthritis, or other diseases always result in severe joint pain and motor dysfunction. Due to the unique anatomy of the human meniscus, the damaged meniscus lacks the ability to repair itself. Moreover, current clinical treatments for meniscal injuries, including meniscal suturing or resection, have significant limitations and drawbacks. With developments in tissue engineering, biopolymer scaffolds have shown promise in meniscal injury repair. They act as templates for tissue repair and regeneration, interacting with surrounding cells and providing structural support for newly formed meniscal tissue. Biomaterials offer tremendous advantages in terms of biocompatibility, bioactivity, and modifiable mechanical and degradation kinetics. In this study, the preparation and composition of meniscal biopolymer scaffolds, as well as their properties, are summarized. The current status of research and future research prospects for meniscal biopolymer scaffolds are reviewed in terms of collagen, silk, hyaluronic acid, chitosan, and extracellular matrix (ECM) materials. Overall, such a comprehensive summary provides constructive suggestions for the development of meniscal biopolymer scaffolds in tissue engineering.

Insights into the molecular mechanisms of CRISPR/Cas9-mediated gene targeting at multiple loci in Arabidopsis.

Homologous recombination-mediated gene targeting (GT) enables precise sequence knockin or sequence replacement, and thus is a powerful tool for heritable precision genome engineering. We recently established a clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9)-mediated approach for heritable GT in Arabidopsis (Arabidopsis thaliana), but its broad utility was not tested, and the underlying molecular mechanism was unclear. Here, we achieved precise GT at 14 out of 27 tested endogenous target loci using the sequential transformation approach and obtained vector-free GT plants by backcrossing. Thus, the sequential transformation GT method provides a broadly applicable technology for precise genome manipulation. We show that our approach generates heritable GT in the egg cell or early embryo of T1 Arabidopsis plants. Analysis of imprecise GT events suggested that single-stranded transfer DNA (T-DNA)/VirD2 complexes produced during the Agrobacterium (Agrobacterium tumefaciens) transformation process may serve as the donor templates for homologous recombination-mediated repair in the GT process. This study provides new insights into the molecular mechanisms of CRISPR/Cas9-mediated GT in Arabidopsis.

Highly efficient generation of isogenic pluripotent stem cell models using prime editing.

The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson's disease in hPSCs.

Correcting a PLCζ Mutation in the Human Germ Line to Overcome Hereditary Infertility

Human germline gene correction by CRISPR/Cas9 holds great promise to eliminate transmission of genetic mutations. Recent studies have however made troublesome observations of mosaicism (the occurrence of multiple genetic events in one embryo) and loss-of-heterozygosity (LOH, loss of the allele of one of the parents), the latter being either caused by inter-homologue homologous recombination (IH-HR) or chromosome loss events. As a proof-of-concept we targeted a heterozygous base pair substitution mutation in PLCZ1, leading to subfertility. After CRISPR/Cas9 editing, we detected in 26% (7/27) of the targeted embryos that originated from mutant sperm only, wild-type alleles upon analysis, which were not obtained by exogenous repair template use. In these seven embryos, we observed a significant amount of LOH events (14%) that spanned regions beyond the PLCZ1 gene, but did not stretch the complete analysed region, pointing to IH-HR, next to a large proportion of samples (57%) showing potential but non-detectable (i.e. below the resolution of our assay) LOH. In addition, a minority of embryos (29%) showed LOH across the whole analysed region. Remarkably, also 10% of the embryos originating from wild-type sperm showed LOH events, underscoring the subpar specificity of the CRIPSR/Cas9 system. Single-cell analysis demonstrated mosaicism in 80% (4/5) of the targeted embryos, warranting caution for trophectoderm biopsy interpretation in future human embryo editing attempts. Lastly, a correction rate of 75% was observed in our single-cell data which illustrates that the correction rate was possibly underestimated when looking at the whole-embryo level.