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An engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity.
Compact CRISPR-Cas systems offer versatile treatment options for genetic disorders, but their application is often limited by modest gene-editing activity. Here we present enAsCas12f, an engineered RNA-guided DNA endonuclease up to 11.3-fold more potent than its parent protein, AsCas12f, and one-third of the size of SpCas9. enAsCas12f shows higher DNA cleavage activity than wild-type AsCas12f in vitro and functions broadly in human cells, delivering up to 69.8% insertions and deletions at user-specified genomic loci. Minimal off-target editing is observed with enAsCas12f, suggesting that boosted on-target activity does not impair genome-wide specificity. We determine the cryo-electron microscopy (cryo-EM) structure of the AsCas12f-sgRNA-DNA complex at a resolution of 2.9 Å, which reveals dimerization-mediated substrate recognition and cleavage. Structure-guided single guide RNA (sgRNA) engineering leads to sgRNA-v2, which is 33% shorter than the full-length sgRNA, but with on par activity. Together, the engineered hypercompact AsCas12f system enables robust and faithful gene editing in mammalian cells.
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ZNF410 represses fetal globin by singular control of CHD4.
Known fetal hemoglobin (HbF) silencers have potential on-target liabilities for rational β-hemoglobinopathy therapeutic inhibition. Here, through transcription factor (TF) CRISPR screening, we identify zinc-finger protein (ZNF) 410 as an HbF repressor. ZNF410 does not bind directly to the genes encoding γ-globins, but rather its chromatin occupancy is concentrated solely at CHD4, encoding the NuRD nucleosome remodeler, which is itself required for HbF repression. CHD4 has two ZNF410-bound regulatory elements with 27 combined ZNF410 binding motifs constituting unparalleled genomic clusters. These elements completely account for the effects of ZNF410 on fetal globin repression. Knockout of ZNF410 or its mouse homolog Zfp410 reduces CHD4 levels by 60%, enough to substantially de-repress HbF while eluding cellular or organismal toxicity. These studies suggest a potential target for HbF induction for β-hemoglobin disorders with a wide therapeutic index. More broadly, ZNF410 represents a special class of gene regulator, a conserved TF with singular devotion to regulation of a chromatin subcomplex.
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CRISPR/Cas9-Assisted Genome Editing in Murine Embryonic Stem Cells.
The study of gene function in normal human physiology and pathophysiology is complicated by countless factors such as genetic diversity (~98 million SNPs identified in the human genome as of June 2015), environmental exposure, epigenetic imprinting, maternal/in utero exposure, diet, exercise, age, sex, socioeconomic factors, and many other variables. Inbred mouse lines have allowed researchers to control for many of the variables that define human diversity but complicate the study of the human genome, gene/protein function, cellular and molecular pathways, and countless other genetic diseases. Furthermore, genetically modified mouse models enable us to generate and study mice whose genomes differ by as little as a single point mutation while controlling for non-genomic variables. This allows researchers to elucidate the quintessential function of a gene, which will further not only our scientific understanding, but provide key insight into human health and disease. Recent advances in CRISPR/Cas9 genome editing have revolutionized scientific protocols for introducing mutations into the mammalian genome. The ensuing chapter provides an overview of CRISPR/Cas9 genome editing in murine embryonic stem cells for the generation of genetically modified mouse models.
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CRISPR/Cas9 in plant biotechnology: applications and challenges.
The application of plant biotechnology to enhance beneficial traits in crops is now indispensable because of food insecurity due to increasing global population and climate change. The recent biotechnological development of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated system 9 (Cas9) allows for a more simple and precise method of gene editing, which is now preferred compared to Zinc Finger Nucleases (ZFNs) and Transcription Activator-like Effector Nucleases (TALENs). In this review, recent progress in utilizing CRISPR/Cas9-mediated gene editing in plants to enhance certain traits in beneficial crops, including rice, soybean, and oilseed rape, is discussed. In addition, novel methods of applying the CRISPR/Cas9 system in live cell imaging are also extensively reviewed. Despite all the applications, the existing delivery methods of CRISPR/Cas9 fail to provide consistent results and are inefficient for in planta transformation. Hence, research should be focused on improving current delivery methods or developing novel ones to facilitate CRISPR/Cas9-based gene editing studies. Strict regulations on the sale and commercial growth of gene-edited crops have restricted more efforts in applying CRISPR/Cas9 technology in plant species. Therefore, a shift in public viewpoint toward gene editing would help to propel scientific progress rapidly.
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Targeting Plant ssDNA Viruses with Engineered Miniature CRISPR-Cas14a.
CRISPR-Cas14a is a highly compact protein with great potential to be used as a guided genome editing tool for single-stranded (ss) DNA cleavage. Recently isolated from noncultured archaea, its unrestricted and sequence-independent cleavage makes it an ideal tool for engineering resistance against economically important plant ssDNA viruses.
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Investigating the Efficacy of the CRISPR/Cas9 Gene-Editing System for Targeting the HBB FSC 36-37 (-T) Mutation Locus in Hematopoietic Stem Cells.
The emergence of genome editing using the CRISPR/Cas9 system has opened up new possibilities and significantly improved the potential for long-term gene therapy of beta-thalassemia. In Iran, FSC 36/37 (-T) is one of the most common mutations among affected individuals, with the highest frequency in the West region (20.8%) and the South-West region (14%). In the context of a proof-of-concept investigation, we present a comprehensive design and assess the efficacy of a CRISPR construct specifically engineered to employ homology-directed repair for targeting the FSC 36/37 (-T) mutation in the HBB gene. The selected sgRNAs were designed and cloned into an optimized CRISPR plasmid. The guide RNAs were transferred to HEK293 cells. T7EI analysis and a non-denaturing PAGE system were employed to assess the effectiveness of the guide RNAs. Hematopoietic stem cells were isolated using the MACS system, and transfection was performed with the Lonza Nucleofector device. The edited cells were monitored using TaqMan-qPCR and RFLP-PCR techniques. Furthermore, the Cas9 enzyme's off-target cleavage sites were meticulously examined to ensure the specificity of the editing process. This research revealed successful genome editing in 30% of the clones analyzed in HEK293 cells and 23.91% of the examined clones in HSCs. Our findings demonstrate the potential of CRISPR/Cas9-mediated genome editing as a promising strategy for addressing genetic mutations associated with thalassemia and other monogenic diseases.
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Exploration of the pneumocandin biosynthetic gene cluster based on efficient CRISPR/Cas9 gene editing strategy in Glarea lozoyensis.
Pneumocandin B0 (PB0) is a lipopeptide produced by the fungus Glarea lozoyensis. The existing challenges with the low-yield and the extended-fermentation cycle emphasize necessity for strain improvement. In this study, we optimized conditions to obtain high-quality protoplasts and screened effective selection markers, leading to the construction of three CRISPR/Cas9 gene editing systems. Utilizing a constitutive Cas9 expression recipient strain, combined with dual sgRNAs targeting, we achieved highly efficient editing of target genes. We successfully knocked out 10 genes within the pneumocandin putative biosynthetic gene cluster and analyzed their roles in PB0 production. Our findings reveal that 4 of 10 genes are directly involved in PB0 production. Specially, the deletion of gltrt or gl10050 resulted in reduced PB0 production, while the absence of glhyp or glhtyC led to the complete loss of PB0 biosynthesis. Notably, the deletion of glhyp caused the silencing of nearly all cluster genes, whereas overexpression of glhyp led to a 2.38-fold increase in PB0 production. Therefore, this study provides the first comprehensive exploration of the functions of 10 genes within the pneumocandin putative biosynthetic gene cluster. Our findings provide valuable technical strategies for constructing bioengineering strains with purposefully enhanced PB0 production.
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Regulating genome editing technologies: a comparison of expert recommendations in the U.K. and in the U.S.A.
The present paper aims to inform the bioethical debate on the regulation of human genome editing technologies with a specific focus on the role of scientific experts and their interactions with the general public in the formulation of policy. It reviews and compares two of the major contributions to this debate in the U.K. and in the U.S.A., comparing expert approaches towards regulation on genome editing technologies. The results of this analysis offer important lessons that should be appreciated in building an international regulatory framework. On the basis of these results, I conclude that the experts should embrace a socially responsible approach and encourage active public engagement.
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Guide RNA Engineering Enables Dual Purpose CRISPR-Cpf1 for Simultaneous Gene Editing and Gene Regulation in Yarrowia lipolytica.
Yarrowia lipolytica has fast become a biotechnologically significant yeast for its ability to accumulate lipids to high levels. While there exists a suite of synthetic biology tools for genetic engineering in this yeast, there is a need for multipurposed tools for rapid strain generation. Here, we describe a dual purpose CRISPR-Cpf1 system that is capable of simultaneous gene disruption and gene regulation. Truncating guide RNA spacer length to 16 nt inhibited nuclease activity but not binding to the target loci, enabling gene activation and repression with Cpf1-fused transcriptional regulators. Gene repression was demonstrated using a Cpf1-Mxi1 fusion achieving a 7-fold reduction in mRNA, while CRISPR-activation with Cpf1-VPR increased hrGFP expression by 10-fold. High efficiency disruptions were achieved with gRNAs 23-25 bp in length, and efficiency and repression levels were maintained with multiplexed expression of truncated and full-length gRNAs. The developed CRISPR-Cpf1 system should prove useful in metabolic engineering, genome wide screening, and functional genomics studies.
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Protocol for the saturation and multiplexing of genetic variants using CRISPR-Cas9.
Here, we present a multiplexed assay for variant effect protocol to assess the functional impact of all possible genetic variations within a particular genomic region. We describe steps for saturation genome editing by designing and cloning of single-guide RNA (sgRNA). We then detail steps for nucleofection of sgRNA, testing drug response on variants, and amplification of genomic DNA for next-generation sequencing. For complete details on the use and execution of this protocol, please refer to Sahu et al.1.
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