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CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1-10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement. We call this method efficient additions with ssDNA inserts-CRISPR (Easi-CRISPR) because it is a highly efficient technology (efficiency is typically 30-60% and reaches as high as 100% in some cases). The protocol takes â^¼2 months to generate the founder mice.

Although single-component Class 2 CRISPR systems, such as type II Cas9 or type V Cas12a (Cpf1), are widely used for genome editing in eukaryotic cells, the application of multi-component Class 1 CRISPR has been less developed. Here we demonstrate that type I-E CRISPR mediates distinct DNA cleavage activity in human cells. Notably, Cas3, which possesses helicase and nuclease activity, predominantly triggered several thousand base pair deletions upstream of the 5′-ARG protospacer adjacent motif (PAM), without prominent off-target activity. This Cas3-mediated directional and broad DNA degradation can be used to introduce functional gene knockouts and knock-ins. As an example of potential therapeutic applications, we show Cas3-mediated exon-skipping of the Duchenne muscular dystrophy ( DMD ) gene in patient-induced pluripotent stem cells (iPSCs). These findings broaden our understanding of the Class 1 CRISPR system, which may serve as a unique genome editing tool in eukaryotic cells distinct from the Class 2 CRISPR system. Class 1 CRISPR systems are not as developed for genome editing as Class 2 systems are. Here the authors show that Cas3 can be used to generate functional knockouts and knock-ins, as well as Cas3-mediated exon-skipping in DMD cells.

Enzyme turnover numbers (k cats) are essential for a quantitative understanding of cells. Because k cats are traditionally measured in low-throughput assays, they can be inconsistent, labor-intensive to obtain, and can miss in vivo effects. We use a data-driven approach to estimate in vivo k cats using metabolic specialist Escherichia coli strains that resulted from gene knockouts in central metabolism followed by metabolic optimization via laboratory evolution. By combining absolute proteomics with fluxomics data, we find that in vivo k cats are robust against genetic perturbations, suggesting that metabolic adaptation to gene loss is mostly achieved through other mechanisms, like gene-regulatory changes. Combining machine learning and genome-scale metabolic models, we show that the obtained in vivo k cats predict unseen proteomics data with much higher precision than in vitro k cats. The results demonstrate that in vivo k cats can solve the problem of inconsistent and low-coverage parameterizations of genome-scale cellular models.

Summary Plants offer fast, flexible and easily scalable alternative platforms for the production of pharmaceutical proteins, but differences between plant and mammalian N‐linked glycans, including the presence of β‐1,2‐xylose and core α‐1,3‐fucose residues in plants, can affect the activity, potency and immunogenicity of plant‐derived proteins. Nicotiana benthamiana is widely used for the transient expression of recombinant proteins so it is desirable to modify the endogenous N‐glycosylation machinery to allow the synthesis of complex N‐glycans lacking β‐1,2‐xylose and core α‐1,3‐fucose. Here, we used multiplex CRISPR/Cas9 genome editing to generate N. benthamiana production lines deficient in plant‐specific α‐1,3‐fucosyltransferase and β‐1,2‐xylosyltransferase activity, reflecting the mutation of six different genes. We confirmed the functional gene knockouts by Sanger sequencing and mass spectrometry‐based N‐glycan analysis of endogenous proteins and the recombinant monoclonal antibody 2G12. Furthermore, we compared the CD64‐binding affinity of 2G12 glycovariants produced in wild‐type N. benthamiana, the newly generated FX‐KO line, and Chinese hamster ovary (CHO) cells, confirming that the glyco‐engineered antibody performed as well as its CHO‐produced counterpart.

With the groundbreaking advancements in genome editing technologies, particularly CRISPR-Cas9, creating knockout mutants has become highly efficient. However, the CRISPR-Cas9 system introduces DNA double-strand breaks, increasing the risk of chromosomal rearrangements and posing a major obstacle to simultaneous multiple gene knockout. Base-editing systems, such as Target-AID, are safe alternatives for precise base modifications without requiring DNA double-strand breaks, serving as promising solutions for existing challenges. Nevertheless, the absence of adequate tools to support Target-AID-based gene knockout highlights the need for a comprehensive system to design guide RNAs (gRNAs) for the simultaneous knockout of multiple genes. Here, we aimed to develop KOnezumi-AID, a command-line tool for gRNA design for Target-AID-mediated genome editing. KOnezumi-AID facilitates gene knockout by inducing the premature termination codons or promoting exon skipping, thereby generating experiment-ready gRNA designs for mouse and human genomes. Additionally, KOnezumi-AID exhibits batch processing capacity, enabling rapid and precise gRNA design for large-scale genome editing, including CRISPR screening. In summary, KOnezumi-AID is an efficient and user-friendly tool for gRNA design, streamlining genome editing workflows and advancing gene knockout research.

G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes. However, arrestin signaling in the collective absence of functional G proteins has never been demonstrated. Here we achieve a state of “zero functional G” at the cellular level using HEK293 cells depleted by CRISPR/Cas9 technology of the Gs/q/12 families of Gα proteins, along with pertussis toxin-mediated inactivation of Gi/o. Together with HEK293 cells lacking β-arrestins (“zero arrestin”), we systematically dissect G protein- from arrestin-driven signaling outcomes for a broad set of GPCRs. We use biochemical, biophysical, label-free whole-cell biosensing and ERK phosphorylation to identify four salient features for all receptors at “zero functional G”: arrestin recruitment and internalization, but—unexpectedly—complete failure to activate ERK and whole-cell responses. These findings change our understanding of how GPCRs function and in particular of how they activate ERK1/2. Arrestins terminate signaling from GPCRs, but several lines of evidence suggest that they are also able to transduce signals independently of G proteins. Here, the authors systematically ablate G proteins in cell lines, and show that arrestins are unable to act as genuine signal initiators.

Due to an unexpected cell-penetrating property, zinc-finger nucleases (ZFNs) can be delivered to several mammalian cell types as proteins. Dose-dependent disruption of an endogenous gene was achieved with reduced activity at known off-target sites. Zinc-finger nucleases (ZFNs) are versatile reagents that have redefined genome engineering. Realizing the full potential of this technology requires the development of safe and effective methods for delivering ZFNs into cells. We demonstrate the intrinsic cell-penetrating capabilities of the standard ZFN architecture and show that direct delivery of ZFNs as proteins leads to efficient endogenous gene disruption in various mammalian cell types with minimal off-target effects.

The CRISPR/Cas9 system has recently been adapted for generating knockout mice to investigate physiological functions and pathological mechanisms. Here, we report a highly efficient procedure for brain-specific disruption of genes of interest in vivo . We constructed pX330 plasmids expressing humanized Cas9 and single-guide RNAs (sgRNAs) against the Satb2 gene, which encodes an AT-rich DNA-binding transcription factor and is responsible for callosal axon projections in the developing mouse brain. We first confirmed that these constructs efficiently induced double-strand breaks (DSBs) in target sites of exogenous plasmids both in vitro and in vivo . We then found that the introduction of pX330-Satb2 into the developing mouse brain using in utero electroporation led to a dramatic reduction of Satb2 expression in the transfected cerebral cortex, suggesting DSBs had occurred in the Satb2 gene with high efficiency. Furthermore, we found that Cas9-mediated targeting of the Satb2 gene induced abnormalities in axonal projection patterns, which is consistent with the phenotypes previously observed in Satb2 mutant mice. Introduction of pX330-NeuN using our procedure also resulted in the efficient disruption of the NeuN gene. Thus, our procedure combining the CRISPR/Cas9 system and in utero electroporation is an effective and rapid approach to achieve brain-specific gene knockout in vivo .

• Plant cellulose is synthesized by a large plasma membrane-localized cellulose synthase (CesA) complex. However, an overall functional determination of secondary cell wall (SCW) CesAs is still lacking in trees, especially one based on gene knockouts. • Here, the Cas9/gRNA-induced knockouts of PtrCesA4, 7A, 7B, 8A and 8B genes were produced in Populus trichocarpa. Based on anatomical, immunohistochemical and wood composition evidence, we gained a comprehensive understanding of five SCW PtrCesAs at the genetic level. • Complete loss of PtrCesA4, 7A/B or 8A/B led to similar morphological abnormalities, indicating similar and nonredundant genetic functions. The absence of the gelatinous (G) layer, one-layer-walled fibres and a 90% decrease in cellulose in these mutant woods revealed that the three classes of SCW PtrCesAs are essential for multilayered SCW structure and wood G-fibre. In addition, the mutant primary and secondary phloem fibres lost the n(G + L)- and G-layers and retained the thicker S-layers (L, lignified; S, secondary). Together with polysaccharide immunolocalization data, these findings suggest differences in the role of SCW PtrCesAs-synthesized cellulose in wood and phloem fibre wall structures. • Overall, this functional understanding of the SCW PtrCesAs provides further insights into the impact of lacking cellulose biosynthesis on growth, SCW, wood G-fibre and phloem fibre wall structures in the tree.

Repeated artificial selection of a complex trait facilitates the identification of genes underlying the trait, especially if multiple selected descendant lines are available. Here we developed a pedigree-based approach to identify genes underlying the Green Revolution (GR) phenotype. From a pedigree analysis, we selected 30 cultivars including the “miracle rice” IR8, a GR landmark, its ancestors and descendants, and also other related cultivars for identifying high-yield genes. Through sequencing of these genomes, we identified 28 ancestral chromosomal blocks that were maintained in all the high-yield cultivars under study. In these blocks, we identified six genes of known function, including the GR gene sd1, and 123 loci with genes of unknown function. We randomly selected 57 genes from the 123 loci for knockout or knockdown studies and found that a high proportion of these genes are essential or have phenotypic effects related to rice production. Notably, knockout lines have significant changes in plant height (P < 0.003), a key GR trait, compared with wild-type lines. Some gene knockouts or knockdowns were especially interesting. For example, knockout of Os10g0555100, a putative glucosyltransferase gene, showed both reduced growth and altered panicle architecture. In addition, we found that in some retained chromosome blocks several GR-related genes were clustered, although they have unrelated sequences, suggesting clustering of genes with similar functions. In conclusion, we have identified many high-yield genes in rice. Our method provides a powerful means to identify genes associated with a specific trait.