Precise Insertion Research Articles

Potential Contribution of Ultrasonography Assistance for the Safe and Steady Procedure of Endoscopic Intracerebral Hematoma Evacuation: A Retrospective Cohort Study.

Endoscopic hematoma evacuation is one of the most promising procedures for the treatment of intracerebral hemorrhage (ICH) to avoid severe outcomes, such as death or dependency. However, the effect of the procedure on the functional outcome remains controversial. Thus, standardization and sophistication are required to enhance the surgical results. This study aimed to evaluate the potential efficacy of ultrasonography (US) in endoscopic hematoma evacuation. This study included 39 consecutive patients with spontaneous supratentorial ICH who underwent endoscopic hematoma evacuation between April 2019 and July 2021. The patients were divided into two groups, namely, surgery with or without US assistance. Rebleeding and evacuation rate were set as the primary endpoints, and operation time, requirement for repeat puncture, and modified Rankin scale at discharge were set as the secondary endpoints. During surgery, the burr hole was placed, and the dura mater was widely opened. The US probe was applied on the brain surface via the burr hole to detect the depth and direction of the hematoma cavity. With US assistance, the hematoma cavity was punctured with a cannula, and the transparent port was introduced into the hematoma cavity along the tract. The hematoma was gently evacuated with the irrigation-suction instrument. Of the 39 cases, 9 underwent endoscopic hematoma evacuation with US assistance. Rebleeding was noted in 0 and 2 (6.7%) patients with and without US assistance, respectively (p = 0.43). The mean hematoma evacuation rates were 78.6 and 80.6% in patients with and without US assistance, respectively (p = 0.80). In all cases with US assistance, the cavity could be reached with a single tap. However, repeat puncture was required in 20 (66.7%) cases without US assistance (p = 0.04). In one case, an unexpected residual hematoma was detected using US, which was applied after hematoma evacuation and before wound closure. The operation time was not extended even if US was used during the surgery. US-assisted hematoma evacuation is an effective procedure that can assist in the precise insertion of the puncture cannula and exclusion of the residual hematoma. US might contribute toward improving the accuracy of each step of the procedure, thus leading to better clinical outcomes.

Occluded insertion loss from intracochlear pressure measurements during acoustic shock wave exposure

IntroductionInjuries to the peripheral auditory system are among the most common results of high intensity impulsive noise exposure. Hearing protection can mitigate this injury, but careful assessment of the insertion loss they provide is necessary. Insertion loss is typically measured using microphone-based acoustic manikins to measure the decrease in sound pressure level transmitted into the ear canal, which precisely measure the change in air conducted sound, but neglect alternate pathways to the inner ear such as bone conduction. In a previous study we reported intracochlear pressures in cadaveric human specimens to acoustic shock waves, which revealed a substantial bone conducted component (Greene, et al., 2018). Here we evaluate insertion loss to several hearing protection devices (HPDs) in those same specimens using intracochlear pressure measurements. MethodsHuman cadaver heads were exposed to impulsive acoustic pressure waves with peak overpressures of 7 and 28 kPa (171 & 183 dB SPL). Ear canal (EAC), middle ear, and intracochlear sound pressure levels were measured bilaterally with fiber-optic pressure sensors. Surface-mounted sensors measured SPL and skull strain near the opening of each EAC and at the forehead. Responses were measured with specimen ears unoccluded, as reported previously, as well as fitted with four types of HPDs. Impulse peak insertion loss (IPIL) and impulse spectrum insertion loss (ISIL) were calculated for each HPD. ResultsFor all HPDs, IPIL generally increases with exposure level, though ISIL tended to be more consistent, and the spectral characteristics across frequency appear to be highly dependent on exposure level. ISIL measured in the ear canal tended to overestimate insertion loss measured in the cochlea, particularly at frequencies > 1 kHz; however, low signal-to-noise in intracochlear pressures limited comparisons. As a proof of concept, 36 low-level unoccluded exposures, were averaged together, and the resulting signal-to-noise ratio was improved by up to 15 dB. ConclusionsInsertion loss measured in the cochlea was lower than in the ear canal, suggesting substantial contributions from transmission pathways in parallel with air conduction (e.g., bone conduction) were present, which will require novel strategies to mitigate. However, high variance was observed, and noise reduction strategies should be utilized in future studies to facilitate more precise insertion loss estimates.

Camera-based navigation in the hybrid operating room

Camera-based navigation in the hybrid operating room represents apossibility for precise, low-complication and efficient pedicle screw insertion in spinal surgery. In addition to increasing patient safety, the use of camera-based navigation as an orientation aid for the surgeon reduces radiation exposure. Camera-based navigation focuses on the surgeon's anatomical knowledge of landmarks, preoperative image acquisition, and subsequent information integration by the navigation software. The information provided from volume tomography (cone beam computed tomography, CBCT) and surface referencing through the video input from four optical cameras and the associated surface markers is collected, processed, optimized and customized by the software used. The result is the creation of atrajectory that allows the surgeon to analyze and evaluate complex anatomical structures more easily and facilitates the performance of the planned procedure. Minimally invasive insertion of pedicle screws using surface-reference navigation (augmented reality surgical navigation; ARSN) provides comparable accuracy to conventional fluoroscopic insertion of pedicle screws while reducing radiation by eliminating the need for postoperative computed tomographic imaging.

Precise and Scarless Insertion of Transposable Elements by Cas9-Mediated Genome Engineering.

Transposable element insertions can have broad effects on gene expression, ranging from new regulatory functions to pathogenic consequences by transplanting new cis-regulating elements or perturbing existing ones. Genetic manipulation of such DNA sequences can help decipher their mechanism of action. Here, we describe a CRISPR-Cas9-mediated two-step approach to preciselyinsert transposable elements into into the genome ofcultured human cells, without scar or reporter gene. First, a double-selection cassette is inserted into the desired target locus. Once a clone containing a single copy of this cassette has been isolated, a second editing step is performed to exchange the double-selection cassette with a markerless transposable element sequence. More generally, this method can be used for knocking in any large insert without genetic markers.

Precise Patellar Tendon Insertion Protection and Osteotomy Surface Advantage of Transtibial Tuberosity-High Tibial Osteotomy.

Medial opening wedge high tibial osteotomy (HTO) is successful in the treatment of knee osteoarthritis with medial compartment stenosis and tibial varus deformity, but patella infera is the main complication. This study aims to design a new medial tibial open osteotomy scheme, transtibial tuberosity-high tibial osteotomy (TT-HTO), which can fully protect the patellar tendon insertion. In addition, the area of the osteotomy surface and wedge volume were evaluated in TT-HTO, biplanar distal tibial tuberosity osteotomy (biplanar-DTO), and uniplanar-DTO to evaluate the potential advantages of this technology in bone healing. The tibial tubercle was divided into four equal sections from proximal to distal, which were defined as zones A, B, C, and D. From September to December 2020, the imaging examinations of 200 patients (95 males and 105 females) with a mean age of 40.6 years (range 19-60 years) were evaluated to observe the zonation of the tibial tubercle where the insertion of the patellar tendon is located. Then, 59 patients (23 males and 36 females) with a mean age 59.6 years (range 43-77 years), for a total of 69 knees (32 right and 37 left), who underwent routine knee surgery were observed and verified. According to the position of the patellar tendon insertion, TT-HTO was designed. Fifteen tibial sawbones were divided equally into three groups: TT-HTO; biplanar-DTO; and uniplanar-DTO. The total area of the osteotomy surface was compared using the graph paper method. The wedge volume at wedge heights of 10 mm was compared among osteotomy types using the plasticine Archimedes principle. One-way repeated-measures analysis of variance was used to compare the total area of the osteotomy surface and the wedge volume. The osteotomy line of TT-HTO passes through the boundary point of zones B and C of the tibial tubercle to fully protect the insertion point of the patellar tendon. The total area of the osteotomy surface in TT-HTO and biplanar-DTO was significantly larger than that in uniplanar-DTO (P < 0.05). The wedge volume in uniplanar-DTO was significantly smaller than that in TT-HTO and biplanar-DTO (P < 0.05). No significant differences in the osteotomy surface and the wedge volume were identified between TT-HTO and biplanar-DTO. TT-HTO can protect the patellar tendon insertion and avoid postoperative patella infera. The osteotomy surface is large and located in an area of cancellous bone, which ensures its good healing characteristics.

Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression

BackgroundChimeric antigen receptor (CAR) T cells have demonstrated high clinical response rates against hematological malignancies (e.g., CD19+ cancers) but have shown limited activity in patients with solid tumors. Recent work...

Feature-Based Compliance Control for Precise Peg-in-Hole Assembly

This article focuses on solving the problem of precise peg-in-hole assembly. The main contribution of this article is to propose a control method to improve the assembly precision. The method is capable to realize the precise insertion assembly tasks of cylindrical or prismatic objects with clearance or interference fit. In this article, a feature space and a response space are first constructed according to the relative pose and equivalent forces and moments. Then, the contact states are segmented in the feature space and the segmentation boundaries are mapped into the response space. Furthermore, a feature-based compliance control (FBCC) method is proposed based on boundary mapping. In FBCC method, a direction matrix is designed to execute accurate adjustment and an integral element is applied to eliminate residual responses. Finally, the simulations and experiments demonstrate the superiority and robustness of FBCC method.

Controlled oxygen incorporation into DC sputtered vanadium coatings for high-efficiency infrared shielding

The wide-band infrared thermochromic modulation of monoclinic vanadium dioxide (VO2(M)) has significant potential for energy-saving fenestration applications in the building industries. Adopting a precise oxygen insertion annealing treatment (POAT) step, the typical reactive sputtering fabrication of VO2(M) could be modified for superior control over the phase purity and surface morphology, leading to a superior thermochromic output. This work demonstrates the POAT fabrication of VO2 (M) coatings using the sputtered vanadium metal films. Ex-situ oxygen annealing provided excellent control over the fabrication process, optimized by systematic feedback obtained from the confocal Raman and X-ray spectroscopy studies. The coating exhibited superior homogeneity with ∼62% infrared (IR) modulation at 2500 nm, along with high solar (ΔTsol = 8.73%) and IR (ΔTIR = 22.30) regulation efficiencies. The optimized films exhibited a completely reversible cyclic stability for continuous operation. The morphological, optical, and electrical characterizations for the samples prepared in different oxygen flow environments were performed and linked to their thermochromic outputs.

Assessment of Nurses’ Practices toward Enteral Feeding Tube for Premature Neonates

Background: To ensure safe and successful enteral feeding, precise tube insertion in the stomach's body is critical. In fact, feeding tube malposition has been linked to gastric bleeding, aspiration, and gastro-oesophageal reflux problems. Mechanical complications such as tube obstruction, displacement, or dislodgement, as well as infection-related complications, occur infrequently as a result of formula handling, storage, and administration. Objective: This study aims to assess nurses’ practices toward enteral feeding tube for premature neonates. Methodology: This study is a complementary section of a larger study, where a quasi-experimental design using test-retest approach for study group and control group participants employed in Kirkuk City Hospitals being, evaluated in several periods, in this pare the focus is only upon the pre-test period. Data collection is done by self-administrated questionnaire form, and it was given for nurses to answer after taking their agreement. A non - probability purposive sample selected from nurses who were working at the neonatal intensive critical care units. The sample was forty nurses, (20) nurses enrolled as a control group and (20) nurses enrolled as a study group. The study group participants were exposed to an interventional program. The selection criteria included Only pediatric' nurses who have from (1-15) years of experience at critical care units. Results: The finding of this study revealed that nurses have low level of practice regarding the insertion and initiating continuous enteral feeding at the pretest. Conclusion: This study concluded that nurses at the NICU have middle to low level of practice concerning the standard procedures of insertion enteral feeding tube, and initiating enteral feeding.

A precise gene delivery approach for human induced pluripotent stem cells using Cas9 RNP complex and recombinant AAV6 donor vectors.

Genome editing in human induced pluripotent stem cells (hiPSCs) offers a potential tool for studying gene functions in disease models and correcting genetic mutations for cell-based therapy. Precise transgene insertion in hiPSCs represents a significant challenge. In the past decade, viral transduction has been widely used due to its high transduction efficiency; however, it can result in random transgene integration and variable transgene copy numbers. Non-viral-based strategies are generally safer but limited by their low transfection efficiency in hiPSCs. Recently, genome engineering using adeno-associated virus (AAV) vectors has emerged as a promising gene delivery approach due to AAVs’ low immunogenicity, toxicity, and ability to infect a broad range of cells. The following protocol describes the workflow for genome editing in hiPSCs using the CRISPR/Cas9 ribonucleoprotein (RNP) complex combined with the recombinant AAV serotype 6 (AAV6) donor vectors to introduce a gene of interest (GOI) fused with mCherry fluorescent reporter gene into the AAVS1 safe harbor site. This approach leads to efficient transgene insertion and is applicable to precise genome editing of hiPSCs or other types of stem cells for research purposes.

Review: Sustainable Clinical Development of CAR-T Cells - Switching From Viral Transduction Towards CRISPR-Cas Gene Editing.

T cells modified for expression of Chimeric Antigen Receptors (CARs) were the first gene-modified cell products approved for use in cancer immunotherapy. CAR-T cells engineered with gammaretroviral or lentiviral vectors (RVs/LVs) targeting B-cell lymphomas and leukemias have shown excellent clinical efficacy and no malignant transformation due to insertional mutagenesis to date. Large-scale production of RVs/LVs under good-manufacturing practices for CAR-T cell manufacturing has soared in recent years. However, manufacturing of RVs/LVs remains complex and costly, representing a logistical bottleneck for CAR-T cell production. Emerging gene-editing technologies are fostering a new paradigm in synthetic biology for the engineering and production of CAR-T cells. Firstly, the generation of the modular reagents utilized for gene editing with the CRISPR-Cas systems can be scaled-up with high precision under good manufacturing practices, are interchangeable and can be more sustainable in the long-run through the lower material costs. Secondly, gene editing exploits the precise insertion of CARs into defined genomic loci and allows combinatorial gene knock-ins and knock-outs with exciting and dynamic perspectives for T cell engineering to improve their therapeutic efficacy. Thirdly, allogeneic edited CAR-effector cells could eventually become available as “off-the-shelf” products. This review addresses important points to consider regarding the status quo, pending needs and perspectives for the forthright evolution from the viral towards gene editing developments for CAR-T cells.

Overexpression of Soybean GmWRI1a Stably Increases the Seed Oil Content in Soybean

WRINKLED1 (WRI1), an APETALA2/ethylene-responsive-element-binding protein (AP2/EREBP) subfamily transcription factor, plays a crucial role in the transcriptional regulation of plant fatty acid biosynthesis. In this study, GmWRI1a was overexpressed in the soybean cultivar ‘Dongnong 50’ using Agrobacterium-mediated transformation to generate three transgenic lines with high seed oil contents. PCR and Southern blotting analysis showed that the T-DNA was inserted into the genome at precise insertion sites and was stably inherited by the progeny. Expression analysis using qRT-PCR and Western blotting indicated that GmWRI1a and bar driven by the CaMV 35S promoter were significantly upregulated in the transgenic plants at different developmental stages. Transcriptome sequencing results showed there were obvious differences in gene expression between transgenic line and transgenic receptor during seed developmental stages. KEGG analysis found that the differentially expressed genes mainly annotated to metabolic pathways, such as carbohydrated metabolism and lipid metabolism. A 2-year single-location field trial revealed that three transgenic lines overexpressing GmWRI1a (GmWRI1a-OE) showed a stable increase in seed oil content of 4.97–10.35%. Importantly, no significant effect on protein content and yield was observed. Overexpression of GmWRI1a changed the fatty acid composition by increasing the linoleic acid (C18:2) content and decreasing the palmitic acid (C16:0) content in the seed. The three GmWRI1a-OE lines showed no significant changes in agronomic traits. The results demonstrated that the three GmWRI1a overexpression lines exhibited consistent increases in seed oil content compared with that of the wild type and did not significantly affect the seed yield and agronomic traits. The genetic engineering of GmWRI1a will be an effective strategy for the improvement of seed oil content and value in soybean.

A localization method for guide pin insertion points in the treatment of femoral neck fracture with closed reduction and percutaneous cannulated screw fixation.

BackgroundClosed reduction and percutaneous cannulated screw fixation is the preferred surgical method for treating femoral neck fractures in elderly patients under 70 years old. The key to the operation is the precise insertion of the guide pin after the successful reduction. However, there is no universally accepted standard for the localization of the guide pin insertion point. This study introduces our invention of a localization method for guide pin insertion points.MethodsWe used fluoroscopic frequency, operative time, union rates, and complications to retrospectively evaluate the treatment and outcome of femoral neck fractures using our localization method for guide pin insertion points. The study included ten patients treated at Affiliated Hospital of Shandong University of Traditional Chinese Medicine between 2010 and 2014 for femoral neck fracture using our localization method for guide pin insertion points.ResultsOur results showed that the mean number of fluoroscopies was 5 (range, 4–8), and the mean operative time was 32 minutes (range, 25–51 minutes). All 10 patients achieved union at an average of 3.4 months (range, 2.8–6.2 months) without complications after the operation. The average follow-up time was 13.2 months (range, 12.2–16 months). The treatment of femoral neck fracture with our localization method for guide pin insertion points resulted in satisfactory curative efficacy without complications.ConclusionsOur localization method offers distinct advantages, including the accuracy and simplicity of the operation, a low rate of complications, and adequate biomechanical stability. As such, it provides a feasible method for clinical treatment.

Ultraflexible and Stretchable Intrafascicular Peripheral Nerve Recording Device with Axon-Dimension, Cuff-Less Microneedle Electrode Array.

Peripheral nerve mapping tools with higher spatial resolution are needed to advance systems neuroscience, and potentially provide a closed-loop biomarker in neuromodulation applications. Two critical challenges of microscale neural interfaces are 1) how to apply them to small peripheral nerves, and 2) how to minimize chronic reactivity. A flexible microneedle nerve array (MINA) is developed, which is the first high-density penetrating electrode array made with axon-sized silicon microneedles embedded in low-modulus thin silicone. The design, fabrication, acute recording, and chronic reactivity to an implanted MINA, are presented. Distinctive units are identified in the rat peroneal nerve. The authors also demonstrate a long-term, cuff-free, and suture-free fixation manner using rose bengal as a light-activated adhesive for two time-points. The tissue response is investigated at 1-week and 6-week time-points, including two sham groups and two MINA-implanted groups. These conditions are quantified in the left vagus nerve of rats using histomorphometry. Micro computed tomography (micro-CT) is added to visualize and quantify tissue encapsulation around the implant. MINA demonstrates a reduction in encapsulation thickness over previously quantified interfascicular methods. Future challenges include techniques for precise insertion of the microneedle electrodes and demonstrating long-term recording.

Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase

The development of mouse models of human disease and synthetic biology research by targeted transgenesis of large DNA constructs represent a significant genetic engineering hurdle. We developed an efficient, precise, single-copy integration of large transgenes directly into zygotes using multiple mouse genetic backgrounds. We used in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) with a transgene “landing pad” composed of dual heterologous Bxb1 attachment (att) sites in cis, within the Gt(ROSA)26Sor safe harbor locus. RMCE of donor was achieved by microinjection of vector DNA carrying cognate attachment sites flanking the donor transgene with Bxb1-integrase mRNA. This approach achieves perfect vector-free integration of donor constructs at efficiencies > 40% with up to ~ 43 kb transgenes. Coupled with a nanopore-based Cas9-targeted sequencing (nCATS), complete verification of precise insertion sequence was achieved. As a proof-of-concept we describe the development of C57BL/6J and NSG Krt18-ACE2 models for SARS-CoV2 research with verified heterozygous N1 animals within ~ 4 months. Additionally, we created a series of mice with diverse backgrounds carrying a single att site including FVB/NJ, PWK/PhJ, NOD/ShiLtJ, CAST/EiJ and DBA/2J allowing for rapid transgene insertion. Combined, this system enables predictable, rapid development with simplified characterization of precisely targeted transgenic animals across multiple genetic backgrounds.

Efficient gene targeting in soybean using Ochrobactrum haywardense-mediated delivery of a marker-free donor template.

Gene targeting (GT) for precise gene insertion or swap into pre-defined genomic location has been a bottleneck for expedited soybean precision breeding. We report a robust selectable marker-free GT system in soybean, one of the most economically important crops. An efficient Oh H1-8 (Ochrobactrum haywardense H1-8)-mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants 6–8 weeks after transformation. This approach generated up to 3.4% targeted insertion of the donor sequence into the target locus in T0 plants, with ∼ 90% mutation rate observed at the genomic target site. The GT was demonstrated in two genomic sites using two different donor DNA templates without the need for a selectable marker within the template. High-resolution Southern-by-Sequencing analysis identified T1 plants with precise targeted insertion and without unintended plasmid DNA. Unlike previous low-frequency GT reports in soybean that involved particle bombardment–mediated delivery and extensive selection, the method described here is fast, efficient, reproducible, does not require a selectable marker within the donor DNA, and generates nonchimeric plants with heritable GT.

Minimally invasive repair of asymmetric pectus excavatum: An alternative technique to treating asymmetric morphology

Minimally invasive repair of pectus excavatum or the Nuss procedure has become the standard operation for pectus excavatum repair. Pectus excavatum can be broadly divided into two categories: symmetric or asymmetric morphology. To optimize surgical outcomes of asymmetric pectus excavatum repair, previous work has proposed morphology-tailored bar shaping technique; the bar to be inserted is shaped asymmetrically to counter-balance the outer contour of the chest prior to the passage of the introducer across the chest. We describe an alternate approach that emphasizes precise introducer chest insertion and extraction and that highlights the direction of the introducer passage is from the higher asymmetric side to the lower contralateral side. The shape of the bar is determined after the introducer has been placed into the chest. This technique allows simultaneous compression of the higher asymmetric chest and elevation of the contralateral depressed side by the metal bar achieving excellent symmetric chest appearance. Level of EvidenceLevel V, Operative Technique

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni.

The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.

Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9.

CRISPR-mediated genome editing has become a powerful tool for the genetic modification of biological traits. However, developing an efficient, site-specific, gene knock-in system based on homology-directed DNA repair (HDR) remains a significant challenge in plants, especially in woody species like poplar. Here, we show that simultaneous inhibition of non-homologous end joining (NHEJ) recombination cofactor XRCC4 and overexpression of HDR enhancer factors CtIP and MRE11 can improve HDR efficiency for gene knock-in. Using this approach, the BleoR gene was integrated onto the 3′ end of the MKK2 MAP kinase gene to generate a BleoR-MKK2 fusion protein. Based on fully edited nucleotides evaluated by TaqMan real-time PCR, the HDR-mediated knock-in efficiency was up to 48% when using XRCC4 silencing incorporated with a combination of CtIP and MRE11 overexpression compared with no HDR enhancement or NHEJ silencing. Furthermore, this combination of HDR enhancer overexpression and NHEJ repression also increased genome targeting efficiency and gave 7-fold fewer CRISPR-induced insertions and deletions (InDels), resulting in no functional effects on MKK2-based salt stress responses in poplar. Therefore, this approach may be useful not only in poplar and plants or crops but also in mammals for improving CRISPR-mediated gene knock-in efficiency.

Optimized CRISPR-Cas9-based Strategy for Complex Gene Targeting in Murine Embryonic Stem Cells for Germline Transmission.

Although CRISPR-Cas9 genome editing can be performed directly in single-cell mouse zygotes, the targeting efficiency for more complex modifications such as the insertion of two loxP sites, multiple mutations in cis, or the precise insertion or deletion of longer DNA sequences often remains low (Cohen, 2016). Thus, targeting and validation of correct genomic modification in murine embryonic stem cells (ESCs) with subsequent injection into early-stage mouse embryos may still be preferable, allowing for large-scale screening in vitro before transfer of thoroughly characterized and genetically defined ESC clones into the germline. This procedure can result in a reduction of animal numbers with cost effectiveness and compliance with the 3R principle of animal welfare regulations. Here, we demonstrate that after transfection of homology templates and PX458 CRISPR-Cas9 plasmids, EGFP-positive ESCs can be sorted with a flow cytometer for the enrichment of CRISPR-Cas9-expressing cells. Cell sorting obviates antibiotic selection and therefore allows for more gentle culture conditions and faster outgrowth of ESC clones, which are then screened by qPCR for correct genomic modifications. qPCR screening is more convenient and less time-consuming compared to analyzing PCR samples on agarose gels. Positive ESC clones are validated by PCR analysis and sequencing and can serve for injection into early-stage mouse embryos for the generation of chimeric mice with germline transmission. Therefore, we describe here a simple and straightforward protocol for CRISPR-Cas9-directed gene targeting in ESCs. Graphical abstract.