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High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus.
The revolution in CRISPR-mediated genome editing has enabled the mutation and insertion of virtually any DNA sequence, particularly in cell culture where selection can be used to recover relatively rare homologous recombination events. The efficient use of this technology in animal models still presents a number of challenges, including the time to establish mutant lines, mosaic gene editing in founder animals, and low homologous recombination rates. Here we report a method for CRISPR-mediated genome editing in Xenopus oocytes with homology-directed repair (HDR) that provides efficient non-mosaic targeted insertion of small DNA fragments (40-50 nucleotides) in 4.4-25.7% of F0 tadpoles, with germline transmission. For both CRISPR/Cas9-mediated HDR gene editing and indel mutation, the gene-edited F0 embryos are uniformly heterozygous, consistent with a mutation in only the maternal genome. In addition to efficient tagging of proteins in vivo, this HDR methodology will allow researchers to create patient-specific mutations for human disease modeling in Xenopus.
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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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Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems function as an adaptive immune system in bacteria and archaea for defense against invading viruses and plasmids (Barrangou and Marraffini, 2014). The effector nucleases from some class 2 CRISPR-Cas systems have been repurposed for heterologous targeting in eukaryotic cells ( Jinek et al., 2012 ; Cong et al., 2013 ; Mali et al., 2013 ; Zetsche et al., 2015 ). However, the genomic environments of eukaryotes are distinctively different from that of prokaryotes in which CRISPR-Cas systems have evolved. Mammalian heterochromatin was found to be a barrier to target DNA access by Streptococcus pyogenes Cas9 (SpCas9), and nucleosomes, the basic units of the chromatin, were also found to impede target DNA access and cleavage by SpCas9 in vitro ( Knight et al., 2015 ; Hinz et al., 2015 ; Horlbeck et al., 2016 ; Isaac et al., 2016 ). Moreover, many CRISPR-Cas systems characterized to date often exhibit inactivity in mammalian cells and are thus precluded from gene editing applications even though they are active in bacteria or on purified DNA substrates. Thus, there is a need to devise a means to alleviate chromatin inhibition to increase gene editing efficiency, especially on difficult-to-access genomic sites, and to enable use of otherwise inactive CRISPR-Cas nucleases for gene editing need. Here we describe a proxy-CRISPR protocol for restoring nuclease activity of various class 2 CRISPR-Cas nucleases on otherwise inaccessible genomic sites in human cells via proximal targeting of a catalytically dead Cas9 ( Chen et al., 2017 ). This protocol is exemplified here by using Campylobacter jejuni Cas9 (CjCas9) as nuclease and catalytically dead SpCas9 (SpdCas9) as proximal DNA binding protein to enable CjCas9 to cleave the target for gene editing using single stranded DNA oligo templates.
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Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing.
Genetic and molecular modifications of the large dsDNA chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like green algae. However, to date, only a few of these algal species and virtually none of their viruses have been genetically manipulated due to lack of practical methods for genetic transformation and genome editing. Attempts at using Agrobacterium-mediated transfection of chlorovirus host Chlorella variabilis NC64A with a specially-designed binary vector resulted in successful transgenic cell selection based on expression of a hygromycin-resistance gene, initial expression of a green fluorescence gene and demonstration of integration of Agrobacterium T-DNA. However, expression of the integrated genes was soon lost. To develop gene editing tools for modifying specific chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we tested multiple methods for delivery of Cas9/sgRNA RNP complexes into infected cells including cell wall-degrading enzymes, electroporation, silicon carbide (SiC) whiskers, and cell-penetrating peptides (CPPs). In one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting virus CA-4B-encoded gene 034r, which encodes a glycosyltransferase. Analysis of DNA sequences from the two mutant viruses showed highly targeted nucleotide sequence modifications in the 034r gene of each virus that were fully consistent with Cas9/RNP-directed gene editing. However, in ten subsequent experiments, we were unable to duplicate these results and therefore unable to achieve a reliable system to genetically edit chloroviruses. Nonetheless, these observations provide strong initial suggestions that Cas9/RNPs may function to promote editing of the chlorovirus genome, and that further experimentation is warranted and worthwhile.
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IL2RG deficient X-SCID pigs generated via electroporation-mediated gene editing using CRISPR/Cas9.
Interleukin 2 receptor gamma (IL2RG) knockout (KO) is frequently used to generate X-linked severe combined immunodeficiency (X-SCID) models. Humans and pigs have anatomical and physiological similarities; hence, X-SCID pigs are being actively developed. IL2RG KO X-SCID pigs are typically achieved through somatic cell nuclear transfer using genetically modified somatic cells as nuclear donors. Therefore, we investigated the possibility of generating IL2RG KO pigs through electroporation-mediated zygotic gene editing using CRISPR/Cas9. First, we designed and selected an efficient guide RNA targeting the porcine IL2RG gene. Subsequently, we obtained one mosaic mutant male piglet carrying a frame-shift mutation (-19 bp) in IL2RG without off-target events, after the transfer of gene-edited zygotes. We also confirmed that F1 female pigs successfully inherited the -19bp mutation (XKOX). Subsequently, we obtained 30 offspring, including 10 IL2RG-KO male pigs (XKOY), from three F1 XKOX litters. The XKOY pigs showed thymic tissue hypoplasia, and flow cytometry analysis revealed absence of cytotoxic T lymphocytes, helper T, and natural killer cells. Typically, X-linked disorders predominantly affect males. Therefore, female carriers pigs are usually maintained and mated with WT males, generating animal models of the disease. In this study, we successfully generated IL2RG-modified F0 male hemizygous pigs rescued by mosaic genotype via electroporation-mediated zygotic gene editing and subsequently generated IL2RG KO pigs by mating F0 pigs with WT females. Our results demonstrated the feasibility of using zygote electroporation to generate IL2RG-deficient pigs. Furthermore, while obtaining a fertile IL2RG mosaic male pig capable of transmitting the mutation was serendipitous, it presents a potential alternative approach for generating X-linked immunodeficiency models.
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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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Methods for Applying Prime Editing and Inverse Prime Editing in Mammalian Cell Culture.
Prime editing (PE) is a powerful method for introducing point mutations into the genomes of living organisms. PE utilizes a Cas9 nickase fused to an engineered Moloney Murine Leukemia Virus reverse transcriptase (MLV-RT), paired with an extended guide RNA known as pegRNA, which contains a primer binding site (PBS) and a reverse transcriptase template (RTT) complementary to the non-target strand DNA. Recently described inverse prime editing (iPE) also employs reverse transcriptase and pegRNAs; however, it utilizes an RNA template complementary to the target strand, resulting in the polymerization of target-strand DNA in the opposite direction. In this work, we provide a practical protocol for using either PE or iPE to introduce point mutation(s) or small-to-medium sized insertions and deletions in cell culture, and demonstrate how to assess editing efficiency via flow cytometry and next-generation sequencing. We include recommendations for prime editor selection and straightforward guidelines for pegRNA design, intended for researchers unfamiliar with genome editing technologies.
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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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Unlocking crops' genetic potential: Advances in genome and epigenome editing of regulatory regions.
Genome editing tools could precisely and efficiently target plant genomes leading to the development of improved crops. Besides editing the coding regions, researchers can employ editing technologies to target specific gene regulatory elements or modify epigenetic marks associated with distal regulatory regions, thereby regulating gene expression in crops. This review outlines several prominent genome editing technologies, including CRISPR-Cas9, TALENs, and ZFNs and recent advancements. The applications for genome and epigenome editing especially of regulatory regions in crop plants is also discussed, including efforts to enhance abiotic stress tolerance, yield, disease resistance and plant phenotype. Additionally, the review addresses the potential of epigenetic modifications, such as DNA methylation and histone modifications, to alter gene expression for crop improvement. Finally, the limitations and future scope of utilizing various genome editing tools to manipulate regulatory elements for gene regulation to unlock the full potential of these tools in plant breeding has been discussed.
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Inhibiting the growth of melanoma cells via hTERT gene editing using CRISPR-dCas9-dnmt3a system.
CRISPR-Cas9 gene-editing technology has pushed the boundaries of genetic modification. The principle of this method is based on the purposeful defense system of DNA degradation and will be one of the most powerful instruments for gene editing shortly. The purpose of this study was to evaluate the capability of this approach to manage melanoma cells. The present study used EF1a-hsaCas9-U6-gRNA as a hybrid vector of sgRNA and Cas9 for the transfection of A-375 melanoma cells. Transfection efficiency was enhanced by examining the two concentrations of 4 and 8 µg/mL of hexadimethrine bromide (trade name Polybrene). The existence of Cas9 in transfected cells was detected by flow cytometry. The expression level of the metabisulfite-modified hTERT gene was measured by real-time PCR technique. The presence of telomerase in cells was determined by flow cytometry and western blotting analysis. The hTERT gene promoter methylation was also evaluated by HRM assay. Finally, the induction of apoptosis in transfected A375 cells was assessed using flow cytometry. The results showed that the presence of gRNA significantly increased the transfection efficiency (up to about 7.75 times higher). The hTERT expression levels in A-375 cells were significantly decreased at different concentrations of Polybrene (in a dose-dependent manner) and various amounts of transfection (P < 0.05). The expression of hTERT in basal cells was not significantly different from the group transfected without gRNA (P˃0.05) but was significantly higher than the group transfected with gRNA (P < 0.05). The results of flow cytometry and western blotting analysis showed a decrease in hTERT level compared to cells transfected without gRNA as well as basal cells. The methylation of hTERT gene promoter in the cells transfected with gRNA at a concentration of 80 μg/mL in the presence of both 4 μg/mL and 8 μg/mL of Polybrene was significantly increased compared to those transfected without sRNA (P < 0.05). The flow cytometry results indicated no significant difference in the induction of apoptosis in the transfected cells compared to the basal cells (P < 0.05). Evidence suggests that the designed CRISPR/Cas9 system reduces the expression of the hTERT gene and telomerase presence, thereby inhibiting the growth of melanoma cells.
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