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Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors.
Targeted saturation mutagenesis of crop genes could be applied to produce genetic variants with improved agronomic performance. However, tools for directed evolution of plant genes, such as error-prone PCR or DNA shuffling, are limited1. We engineered five saturated targeted endogenous mutagenesis editors (STEMEs) that can generate de novo mutations and facilitate directed evolution of plant genes. In rice protoplasts, STEME-1 edited cytosine and adenine at the same target site with C > T efficiency up to 61.61% and simultaneous C > T and A > G efficiency up to 15.10%. STEME-NG, which incorporates the nickase Cas9-NG protospacer-adjacent motif variant, was used with 20 individual single guide RNAs in rice protoplasts to produce near-saturated mutagenesis (73.21%) for a 56-amino-acid portion of the rice acetyl-coenzyme A carboxylase (OsACC). We also applied STEME-1 and STEME-NG for directed evolution of the OsACC gene in rice and obtained herbicide resistance mutations. This set of two STEMEs will accelerate trait development and should work in any plants amenable to CRISPR-based editing.
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Genome editing in K562 cells suggests a functional role for the XmnI Gg polymorphism: a widely used genetic marker in β-thalassemia and sickle cell disease patients.
The XmnI Gg -158 C/T polymorphism has been widely associated with fetal hemoglobin (HbF) levels, the severity of disease, and the response to the drug hydroxyurea (HU) in both β-thalassemia (β-thal) and sickle cell disease (SCD) patients. However, the functional significance of this single nucleotide polymorphism (SNP) remains unclear. To gain insight, green fluorescence protein (GFP) cassettes harboring the XmnI C or T alleles in their left homology arms (i.e. Gg promoters) were knocked into the Gg gene(s) of K562 cells via CRISPR/Cas9. Subsequently, the GFP fluorescence levels were compared in the ensuing cell populations and isolated clones. In both instances, median fluorescence intensities (MFI) of the knockin cells having the inserted XmnI T allele were higher than those having the XmnI C allele. Our results suggest that the XmnI T allele can increase Gg expression in K562 cells. The possible functional significance of the XmnI Gg -158 C/T polymorphism provides a rationale for the aforementioned associations. Furthermore, the XmnI polymorphism as a functional SNP substantiates its importance as a prognostic marker.
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Structure basis for single-strand nucleic acid targeting by IscB and variants.
Transposon-encoded IscB was defined as the evolutionary ancestor of CRISPR-Cas9. This compact RNA-guided endonuclease has since been engineered for genome-editing applications. We previously repurposed IscB and related Cas9s as efficient RNA editors by removing their double-stranded DNA (dsDNA) recognition module, the target-adjacent motif (TAM)/protospacer adjacent motif-interacting domain. Here, we report four cryo-electron microscopy structures of IscB, with or without TAM-interaction domain (TID), in complex with single-stranded nucleic acid (ssNA) targets. Structures reveal that, regardless of TID presence, IscB engages ssNA using the same mechanism. IscB initially facilitates formation of a 10-nt seed duplex with ssNA; further base-pairing is blocked by an alternatively positioned HNH nuclease that acts as a roadblock. In this intermediate state, neither HNH nor RuvC is competent for target cleavage. Only upon full duplex formation is the HNH roadblock dislodged by the duplex extension between guide RNA and ssNA. HNH and RuvC nuclease active sites become exposed as the result. A similar set of conformational rearrangements likely governs IscB activity during dsDNA target interrogation. Guided by the structural and mechanistic insights, we introduced mutations to either improve ssNA binding or ease HNH dislodging. Both approaches improved the RNA-targeting efficiency of IscB in vitro and in human cells.
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Development of novel quality control material based on CRISPR/Cas9 editing and xenografts for MLH1 protein deficiency testing.
BACKGROUND: Mismatch repair deficiency (dMMR) status induced by MLH1 protein deficiency plays a pivotal role in therapeutic decision-making for cancer patients. Appropriate quality control (QC) materials are necessary for monitoring the accuracy of MLH1 protein deficiency assays used in clinical laboratories. METHODS: CRISPR/Cas9 technology was used to edit the MLH1 gene of GM12878Cas9 cells to establish MLH1 protein-deficient cell lines. The positive cell lines were screened and validated by Sanger sequencing, Western blot (WB), and next-generation sequencing (NGS) and were then used to prepare formalin-fixed, paraffin-embedded (FFPE) samples through xenografting. These FFPE samples were tested by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for suitability as novel QC materials for MLH1 protein deficiency testing. RESULTS: We successfully cultured 358 monoclonal cells, with a survival rate of 37.3% (358/960) of the sorted monoclonal cells. Through Sanger sequencing, cell lines with MLH1 gene mutation were identified. Subsequently, two cell lines with MLH1 protein deficiency were identified by WB and named as GM12878Cas9_6 and GM12878Cas9_10. The NGS results further confirmed that the MLH1 gene mutation in these two cell lines would cause the formation of stop codons and terminate the expression of the MLH1 protein. The H&E staining and IHC results also verified the deficiency of the MLH1 protein, and FFPE samples from xenografts proved their similarity and consistency with clinical samples. CONCLUSIONS: We successfully established MLH1 protein-deficient cell lines. Followed by xenografting, we developed novel FFPE QC materials with homogenous, sustainable, and typical histological structures advantages that are suitable for the standardization of clinical IHC methods.
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Increasing the performance of pooled CRISPR-Cas9 drop-out screening.
Components of the type II CRISPR-Cas complex in bacteria have been used successfully in eukaryotic cells to facilitate rapid and accurate cell line engineering, animal model generation and functional genomic screens. Such developments are providing new opportunities for drug target identification and validation, particularly with the application of pooled genetic screening. As CRISPR-Cas is a relatively new genetic screening tool, it is important to assess its functionality in a number of different cell lines and to analyse potential improvements that might increase the sensitivity of a given screen. To examine critical aspects of screening quality, we constructed ultra-complex libraries containing sgRNA sequences targeting a collection of essential genes. We examined the performance of screening in both haploid and hypotriploid cell lines, using two alternative guide design algorithms and two tracrRNA variants in a time-resolved analysis. Our data indicate that a simple adaptation of the tracrRNA substantially improves the robustness of guide loss during a screen. This modification minimises the requirement for high numbers of sgRNAs targeting each gene, increasing hit scoring and creating a powerful new platform for successful screening.
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Development of a novel gene editing lexicon for hemophilia: methodology and results.
BACKGROUND: Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9)-based targeted gene editing platforms are being developed to treat genetic diseases like hemophilia. Such novel therapy involves complex concepts and terminology that require aligned language to engage key stakeholders in the hemophilia community. Thus, a globally aligned gene editing lexicon - a consistent language to communicate the fundamentals of gene editing in hemophilia, designed to be credible and accessible for people with hemophilia and caregivers while avoiding unnecessary complexity - is required to address this need. OBJECTIVES: To establish an aligned language and communications framework that facilitates informed consent and shared decision-making regarding gene editing and treatment considerations in hemophilia. METHODS: Through an innovative partnership with global experts in hemophilia, gene editing, and biotechnology, initial insights were gathered via interviews, workshops, and analysis of existing language within the hemophilia community. Qualitative research involving lived experience experts (people with hemophilia and caregivers; n = 43) and hematologists (n = 24) informed the lexicon development, which was further validated by a steering committee of global experts in the hemophilia and gene editing fields. Finally, optimized language recommendations were developed for a clear, consistent gene editing lexicon. RESULTS: Key themes included insights into audience mindsets, guiding language principles, and optimized terminology for key topics like gene editing concepts and posttreatment considerations. Audience mindsets revealed cautious optimism around gene therapy, with more skepticism around gene editing. Guiding language principles indicated a preference for plainspoken over technical language, definitions that link to patient benefits, and explanations that highlight the precise nature of gene editing. CONCLUSION: This collaborative approach ensures broad adoption of the lexicon within the hemophilia community and readiness for beta testing.
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Genome Editing Using the CRISPR-Cas9 System to Generate a Solid-Red Germline of Nile Tilapia (Oreochromis niloticus).
In recent years, there has been increasing demand for red tilapia, which are commercial strains of hybrids of different tilapiine species or red variants of highly inbred Nile tilapia. However, red tilapia phenotypes are genetically unstable and affected by environmental factors, resulting in nonuniform coloration with black or dark-red color blotches that reduce their market value. Solute carrier family 45 member 2 (SLC45A2) is a membrane transporter that mediates melanin biosynthesis and is evolutionarily conserved from fish to humans. In the present study, we describe the generation of a stable and heritable red tilapia phenotype by inducing loss-of-function mutations in the slc45a2 gene. For this purpose, we identified the slc45a2 gene in Nile tilapia and designed highly specific guide RNAs (gRNA) for its genomic sequence. Multiplex microinjection of slc45a2-specific ribonucleoproteins to Nile tilapia zygotes induced up to 97-99% albinism, including loss of melanin in the eye. Next-generation sequencing of the injected zygotes demonstrated that all injected fish carried mutant alleles with variable mutagenesis efficiencies. Sanger sequencing of the genomic target region in the slc45a2 gene from fin clips, sperm, and F1 offspring of a highly mutant male identified various genomic indels and germline transmission of the sperm-identified indels. Overall, this work demonstrates the generation of somatic and germline slc45a2 mutant alleles, which leads to complete albinism in Nile tilapia.
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COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa.
Designer nucleases allow specific and precise genomic modifications and represent versatile molecular tools for the correction of disease-associated mutations. In this study, we have exploited an ex vivo CRISPR/Cas9-mediated homology-directed repair approach for the correction of a frequent inherited mutation in exon 80 of COL7A1, which impairs type VII collagen expression, causing the severe blistering skin disease recessive dystrophic epidermolysis bullosa. Upon CRISPR/Cas9 treatment of patient-derived keratinocytes, using either the wild-type Cas9 or D10A nickase, corrected single-cell clones expressed and secreted similar levels of type VII collagen as control keratinocytes. Transplantation of skin equivalents grown from corrected keratinocytes onto immunodeficient mice showed phenotypic reversion with normal localization of type VII collagen at the basement membrane zone, compared with uncorrected keratinocytes, as well as fully stratified and differentiated skin layers without indication of blister development. Next-generation sequencing revealed on-target efficiency of up to 30%, whereas nuclease-mediated off-target site modifications at predicted genomic loci were not detected. These data demonstrate the potential of the CRISPR/Cas9 technology as a possible ex vivo treatment option for genetic skin diseases in the future.
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Correction of IVS I-110(G>A) β-thalassemia by CRISPR/Cas-and TALEN-mediated disruption of aberrant regulatory elements in human hematopoietic stem and progenitor cells.
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A homologous recombination-proficient Yarrowia lipolytica chassis for multiplex genome manipulation.
Homologous recombination (HR) greatly facilitates precise genome editing. However, most organisms prefer error-prone non-homologous end joining (NHEJ) for DNA double-strand break (DSB) repair. Here, the NHEJ-proficient Yarrowia lipolytica was transformed into a HR-proficient strain by systematic engineering of recombination machinery, regulating the multiinvasion-induced rearrangement (MIR) process, and expressing cognate single-stranded DNA-annealing protein (SSAP)-single-stranded DNA-binding protein (SSB) pairs. These strategies improved HR efficiency by 38.9, 1.6, and 1.2-fold compared with the NHEJ-deficient strain for multifragment multisite integration, and multi- and single-fragment single-site integration, respectively. Moreover, HR efficiency remained high at 58% even with 50-base pair (bp) homology arms (HAs) and reached 11% for simultaneously integrating two mega-DNA fragments (18.0 kb and 13.5 kb) at two genome sites. This strain also enabled simultaneous editing, repression, and activation of multiple genes, while cellular robustness parameters showed marked increases over the NHEJ-deficient strain. Our work provides a HR-proficient Y. lipolytica chassis allowing efficient and precise genome editing of this increasingly important microbe.
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