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The Promise and Challenge of In Vivo Delivery for Genome Therapeutics.
CRISPR-based genome editing technologies are poised to enable countless new therapies to prevent, treat, or cure diseases with a genetic basis. However, the safe and effective delivery of genome editing enzymes represents a substantial challenge that must be tackled to enable the next generation of genetic therapies. In this Review, we summarize recent progress in developing enzymatic tools to combat genetic disease and examine current efforts to deliver these enzymes to the cells in need of correction. Viral vectors already in use for traditional gene therapy are being applied to enable in vivo CRISPR-based therapeutics, as are emerging technologies such as nanoparticle-based delivery of CRISPR components and direct delivery of preassembled RNA-protein complexes. Success in these areas will allow CRISPR-based genome editing therapeutics to reach their full potential.
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Protocol for establishing knockout cell clones by deletion of a large gene fragment using CRISPR-Cas9 with multiple guide RNAs.
Genome editing is a powerful tool for establishing gene knockout or mutant cell lines. Here, we present a protocol for establishing knockout cell clones by deletion of large gene fragments using CRISPR-Cas9 with multiple guide RNAs. We describe steps for designing guide RNAs, cloning them into CRISPR-Cas9 vectors, cell seeding, transfection into cultured cells, clonal selection, and screening assays. This protocol can delete gene regions over 100 kbp, including GC-rich domains, and is applicable to various cell lines. For complete details on the use and execution of this protocol, please refer to Saito et al.,1 Saito and Endo et al.,2 and Higashi et al.3.
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Cas9 Protein Outperforms mRNA in Lipid Nanoparticle-Mediated CFTR Repair.
Lipid nanoparticles (LNPs) are currently one of the most clinically advanced delivery systems for nucleic acid cargo and hold great potential for clinical applications in gene editing and the treatment of genetic diseases. LNP-mediated delivery of Cas9 with single guide RNA (sgRNA) and homology-directed repair DNA template (ssDNA) enables efficient and precise editing in vitro and in vivo. Comparative analysis of LNP delivery of Cas9 as protein or mRNA for relevant clinical targets, such as cystic fibrosis (CF), which is caused by mutations in the CFTR gene, is imperative in the design of corrective therapeutics for genetic diseases. Here, we show that delivery of Cas9 protein LNPs outperforms Cas9 mRNA LNPs when evaluated for in vivo lung editing as well as corrective CRISPR/Cas9 editing and functional recovery of the CFTR protein. These results demonstrate the ability to optimize the use of CRISPR/Cas9 LNPs for cystic fibrosis applications.
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Protocol for Allele-Specific Epigenome Editing Using CRISPR/dCas9.
The discovery and adaptation of CRISPR/Cas systema for epigenome editing has allowed for a straightforward design of targeting modules that can direct epigenome editors to virtually any genomic site. This advancement in DNA-targeting technology brings allele-specific epigenome editing into reach, a "super-specific" variation of epigenome editing whose goal is an alteration of chromatin marks at only one selected allele of the genomic target locus. This technology could be useful for the treatment of diseases caused by a mutant allele with a dominant effect, because allele-specific epigenome editing allows the specific silencing of the mutated allele leaving the healthy counterpart expressed. Moreover, it may allow the direct correction of aberrant imprints in imprinting disorders where editing of DNA methylation is required exclusively in a single allele. Here, we describe a basic protocol for the design and application of allele-specific epigenome editing systems using allele-specific DNA methylation at the NARF gene in HEK293 cells as an example. An sgRNA/dCas9 unit is used for allele-specific binding to the target locus containing a SNP in the seed region of the sgRNA or the PAM region. The dCas9 protein is connected to a SunTag allowing to recruit up to 10 DNMT3A/3L units fused to a single-chain Fv fragment, which specifically binds to the SunTag peptide sequence. The plasmids expressing dCas9-10x SunTag, scFv-DNMT3A/3L, and sgRNA, each of them co-expressing a fluorophore, are introduced into cells by co-transfection. Cells containing all three plasmids are enriched by FACS, cultivated, and later the genomic DNA and RNA can be retrieved for DNA methylation and gene expression analysis.
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Introduction of a point mutation in the KRAS gene of in vitro fertilized porcine zygotes via electroporation of the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides.
This study aimed to investigate the efficiency of KRAS gene editing via CRISPR/Cas9 delivery by electroporation and analyzed the effects of the non-homologous end-joining pathway inhibitor Scr7 and single-stranded oligodeoxynucleotide (ssODN) homology arm length on introducing a point mutation in KRAS. Various concentrations (0-2 µM) of Scr7 were evaluated; all concentrations of Scr7 including 0 µM resulted in the generation of blastocysts with a point mutation and the wild-type sequence or indels. No significant differences in the blastocyst formation rates of electroporated zygotes were observed among ssODN homology arm lengths, irrespective of the gRNA (gRNA1 and gRNA2). The proportion of blastocysts carrying a point mutation with or without the wild-type sequence and indels was significantly higher in the ssODN20 group (i.e., the group with a ssODN homology arm of 20 bp) than in the ssODN60 group (gRNA1: 25.7% vs. 5.4% and gRNA2: 45.5% vs. 5.9%, p < .05). In conclusion, the CRISPR/Cas9 delivery with ssODN via electroporation is feasible for the generation of point mutations in porcine embryos. Further studies are required to improve the efficiency and accuracy of the homology-directed repair.
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Narrow path charted for editing genes of human embryos.
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Increasing Knockin Efficiency in Mouse Zygotes by Transient Hypothermia.
Integration of a point mutation to correct or edit a gene requires the repair of the CRISPR-Cas9-induced double-strand break by homology-directed repair (HDR). This repair pathway is more active in late S and G2 phases of the cell cycle, whereas the competing pathway of nonhomologous end-joining (NHEJ) operates throughout the cell cycle. Accordingly, modulation of the cell cycle by chemical perturbation or simply by the timing of gene editing to shift the editing toward the S/G2 phase has been shown to increase HDR rates. Using a traffic light reporter in mouse embryonic stem cells and a fluorescence conversion reporter in human-induced pluripotent stem cells, we confirm that a transient cold shock leads to an increase in the rate of HDR, with a corresponding decrease in the rate of NHEJ repair. We then investigated whether a similar cold shock could lead to an increase in the rate of HDR in the mouse embryo. By analyzing the efficiency of gene editing using single nucleotide polymorphism changes and loxP insertion at three different genetic loci, we found that a transient reduction in temperature after zygote electroporation of CRISPR-Cas9 ribonucleoprotein with a single-stranded oligodeoxynucleotide repair template did indeed increase knockin efficiency, without affecting embryonic development. The efficiency of gene editing with and without the cold shock was first assessed by genotyping blastocysts. As a proof of concept, we then confirmed that the modified embryo culture conditions were compatible with live births by targeting the coat color gene tyrosinase and observing the repair of the albino mutation. Taken together, our data suggest that a transient cold shock could offer a simple and robust way to improve knockin outcomes in both stem cells and zygotes.
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On recent advances in human engineering.
Advances in embryology, genetics, and regenerative medicine regularly attract attention from scientists, scholars, journalists, and policymakers, yet implications of these advances may be broader than commonly supposed. Laboratories culturing human embryos, editing human genes, and creating human-animal chimeras have been working along lines that are now becoming intertwined. Embryogenic methods are weaving traditional in vivo and in vitro distinctions into a new “in vivitro” (in life in glass) fabric. These and other methods known to be in use or thought to be in development promise soon to bring society to startling choices and discomfiting predicaments, all in a global effort to supply reliably rejuvenating stem cells, to grow immunologically non-provocative replacement organs, and to prevent, treat, cure, or even someday eradicate diseases having genetic or epigenetic mechanisms. With humanity's human-engineering era now begun, procedural prohibitions, funding restrictions, institutional controls, and transparency rules are proving ineffective, and business incentives are migrating into the most basic life-sciences inquiries, wherein lie huge biomedical potentials and bioethical risks. Rights, health, and heritage are coming into play with bioethical presumptions and formal protections urgently needing reassessment.
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AcrPred: A hybrid optimization with enumerated machine learning algorithm to predict Anti-CRISPR proteins.
CRISPR-Cas, as a tool for gene editing, has received extensive attention in recent years. Anti-CRISPR (Acr) proteins can inactivate the CRISPR-Cas defense system during interference phase, and can be used as a potential tool for the regulation of gene editing. In-depth study of Anti-CRISPR proteins is of great significance for the implementation of gene editing. In this study, we developed a high-accuracy prediction model based on two-step model fusion strategy, called AcrPred, which could produce an AUC of 0.952 with independent dataset validation. To further validate the proposed model, we compared with published tools and correctly identified 9 of 10 new Acr proteins, indicating the strong generalization ability of our model. Finally, for the convenience of related wet-experimental researchers, a user-friendly web-server AcrPred (Anti-CRISPR proteins Prediction) was established at http://lin-group.cn/server/AcrPred, by which users can easily identify potential Anti-CRISPR proteins.
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CRISPR/Cas9-germline editing of Biomphalaria glabrata: A breakthrough in genetic modification of snails that transmit schistosomiasis.
Human schistosomiasis remains one of the most devastating parasitic diseases worldwide, and the development of genetically modified vector snails has long been a goal in the field. Here, we report the successful creation of genetically modified Biomphalaria glabrata, an important intermediate host, using CRISPR/Cas9 gene editing. We targeted the fibrinogen-related protein 3.1 (FREP3.1) gene, confirmed stable germline transmission of the mutated gene, and established two different homozygous FREP3.1-edited lines. Disruption of the FREP 3.1 gene did not alter snail susceptibility to Schistosoma mansoni infection, possibly due to a limited role of FREP3.1 in resistance or to functional redundancy and/or compensatory expression within the highly diverse FREP gene family. Our study demonstrates successful germline editing, effective ex ovo culture of decapsulated embryos, and the generation of viable, genetically modified B. glabrata snails, thereby establishing a foundation for future genetic strategies to control schistosomiasis.
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