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Cost-effective generation of A-to-G mutant mice by zygote electroporation of adenine base editor ribonucleoproteins.
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Enhanced Bacterial Immunity and Mammalian Genome Editing via RNA-Polymerase-Mediated Dislodging of Cas9 from Double-Strand DNA Breaks.
The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double-stranded break (DSB), Cas9 remains stably bound to DNA. Here, we show persistent Cas9 binding blocks access to the DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches from one direction toward the Cas9-DSB complex. By exploiting these RNA-polymerase/Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of protospacer adjacent motif (PAM) sequences and a simple method of improving selection of highly active sgRNAs for genome editing.
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Efficient Gene Silencing by Adenine Base Editor-Mediated Start Codon Mutation.
Traditional CRISPR/Cas9-based gene knockouts are generated by introducing DNA double-strand breaks (DSBs), but this may cause excessive DNA damage or cell death. CRISPR-based cytosine base editors (CBEs) and adenine base editors (ABEs) can facilitate C-to-T or A-to-G exchanges, respectively, without DSBs. CBEs have been employed in a gene knockout strategy: CRISPR-STOP or i-STOP changes single nucleotides to induce in-frame stop codons. However, this strategy is not applicable for some genes, and the unwanted mutations in CBE systems have recently been reported to be surprisingly significant. As a variant, the ABE systems mediate precise editing and have only rare unwanted mutations. In this study, we implemented a new strategy to induce gene silencing (i-Silence) with an ABE-mediated start codon mutation from ATG to GTG or ACG. Using both in vitro and in vivo model systems, we showed that the i-Silence approach is efficient and precise for producing a gene knockout. In addition, the i-Silence strategy can be employed to analyze ~17,804 human genes and can be used to mimic 147 kinds of pathogenic diseases caused by start codon mutations. Altogether, compared to other methods, the ABE-based i-Silence method is a safer gene knockout strategy, and it has promising application potential.
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ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions.
Maize ARGOS8 is a negative regulator of ethylene responses. A previous study has shown that transgenic plants constitutively overexpressing ARGOS8 have reduced ethylene sensitivity and improved grain yield under drought stress conditions. To explore the targeted use of ARGOS8 native expression variation in drought-tolerant breeding, a diverse set of over 400 maize inbreds was examined for ARGOS8 mRNA expression, but the expression levels in all lines were less than that created in the original ARGOS8 transgenic events. We then employed a CRISPR-Cas-enabled advanced breeding technology to generate novel variants of ARGOS8. The native maize GOS2 promoter, which confers a moderate level of constitutive expression, was inserted into the 5'-untranslated region of the native ARGOS8 gene or was used to replace the native promoter of ARGOS8. Precise genomic DNA modification at the ARGOS8 locus was verified by PCR and sequencing. The ARGOS8 variants had elevated levels of ARGOS8 transcripts relative to the native allele and these transcripts were detectable in all the tissues tested, which was the expected results using the GOS2 promoter. A field study showed that compared to the WT, the ARGOS8 variants increased grain yield by five bushels per acre under flowering stress conditions and had no yield loss under well-watered conditions. These results demonstrate the utility of the CRISPR-Cas9 system in generating novel allelic variation for breeding drought-tolerant crops.
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CRISPR/Cas9-mediated gene knockout for DNA methyltransferase Dnmt3a in CHO cells displays enhanced transgenic expression and long-term stability.
CHO cells are the preferred host for the production of complex pharmaceutical proteins in the biopharmaceutical industry, and genome engineering of CHO cells would benefit product yield and stability. Here, we demonstrated the efficacy of a Dnmt3a-deficient CHO cell line created by CRISPR/Cas9 genome editing technology through gene disruptions in Dnmt3a, which encode the proteins involved in DNA methyltransferases. The transgenes, which were driven by the 2 commonly used CMV and EF1α promoters, were evaluated for their expression level and stability. The methylation levels of CpG sites in the promoter regions and the global DNA were compared in the transfected cells. The Dnmt3a-deficent CHO cell line based on Dnmt3a KO displayed an enhanced long-term stability of transgene expression under the control of the CMV promoter in transfected cells in over 60 passages. Under the CMV promoter, the Dnmt3a-deficent cell line with a high transgene expression displayed a low methylation rate in the promoter region and global DNA. Under the EF1α promoter, the Dnmt3a-deficient and normal cell lines with low transgene expression exhibited high DNA methylation rates. These findings provide insight into cell line modification and design for improved recombinant protein production in CHO and other mammalian cells.
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Engineering Broad-Spectrum Bacterial Blight Resistance by Simultaneously Disrupting Variable TALE-Binding Elements of Multiple Susceptibility Genes in Rice.
Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice, employs the transcription activator-like effectors (TALEs) to induce the expression of the OsSWEET family of putative sugar transporter genes, which function in conferring disease susceptibility (S) in rice plants. To engineer broad-spectrum bacterial blight resistance, we used CRISPR/Cas9-mediated gene editing to disrupt the TALE-binding elements (EBEs) of two S genes, OsSWEET11 and OsSWEET14, in rice cv. Kitaake, which harbors the recessive resistance allele of Xa25/OsSWEET13. The engineered rice line MS14K exhibited broad-spectrum resistance to most Xoo strains with a few exceptions, suggesting that the compatible strains may contain new TALEs. We identified two PthXo2-like TALEs, Tal5LN18 and Tal7PXO61, as major virulence factors in the compatible Xoo strains LN18 and PXO61, respectively, and found that Xoo encodes at least five types of PthXo2-like effectors. Given that PthXo2/PthXo2.1 target OsSWEET13 for transcriptional activation, the genomes of 3000 rice varieties were analyzed for EBE variationsin the OsSWEET13 promoter, and 10 Xa25-like haplotypes were identified. We found that Tal5LN18 and Tal7PXO61 bind slightly different EBE sequences in the OsSWEET13 promoter to activate its expression. CRISPR/Cas9 technology was then used to generate InDels in the EBE of the OsSWEET13 promoter in MS14K to creat a new germplasm with three edited OsSWEET EBEs and broad-spectrum resistance against all Xoo strains tested. Collectively, our findings illustrate how to disarm TALE-S co-evolved loci to generate broad-spectrum resistance through the loss of effector-triggered susceptibility in plants.
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Structural basis for Cas9 off-target activity.
The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here, we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of noncanonical base-pairing interactions within the guide:off-target heteroduplex. Off-target substrates containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping or multiple noncanonical base pairs rather than RNA bulge formation. Finally, PAM-distal mismatches result in duplex unpairing and induce a conformational change in the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.
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CRISPR/Cas9-mediated somatic and germline gene correction to restore hemostasis in hemophilia B mice.
Hemophilia B (HB) is an X-linked disorder caused by defects of F9 encoded coagulation factor IX, which is an ideal model for gene therapy. Most existing HB gene therapies are based on viral mediated gene supplementation, which could increase immunoreaction. In this study, CRISPR/Cas9 system was used for gene correction in an F9 mutant HB mouse model in both adult mice (in vivo) and in germline cells (ex vivo). In vivo, naked Cas9-sgRNA plasmid and donor DNA were delivered to HB mice livers to recover the mutation via hydrodynamic tail vein (HTV) injection. 62.5% of the HTV-treated mice showed a detectable gene correction (>1%) in the F9 alleles of hepatocytes, which was sufficient to remit the coagulation deficiency. Ex vivo, three different forms of Cas9 were microinjected into germline cells of HB mice to investigate their efficiency and safety in gene correction. Cas9 protein showed higher gene recovery rates, less embryo toxicity, and lower mosaic repair percentage, making it more suitable for germline gene therapy. Our study strongly supports that CRISPR/Cas9-mediated genome editing is feasible in gene therapy of genetic disorders.
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Human Globozoospermia-Related Gene Spata16 Is Required for Sperm Formation Revealed by CRISPR/Cas9-Mediated Mouse Models.
A recent genetic analysis of infertile globozoospermic patients identified causative mutations in three genes: a protein interacting with C kinase 1 (PICK1), dpy 19-like 2 (DPY19L2), and spermatogenesis associated 16 (SPATA16). Although mouse models have clarified the physiological functions of Pick1 and Dpy19l2 during spermatogenesis, Spata16 remains to be determined. Globozoospermic patients carried a homozygous point mutation in SPATA16 at 848G→A/R283Q. We generated CRISPR/Cas9-mediated mutant mice with the same amino acid substitution in the fourth exon of Spata16 to analyze the mutation site at R284Q, which corresponded with R283Q of mutated human SPATA16. We found that the point mutation in Spata16 was not essential for male fertility; however, deletion of the fourth exon of Spata16 resulted in infertile male mice due to spermiogenic arrest but not globozoospermia. This study demonstrates that Spata16 is indispensable for male fertility in mice, as well as in humans, as revealed by CRISPR/Cas9-mediated mouse models.
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Somatic genome editing with CRISPR/Cas9 generates and corrects a metabolic disease.
Germline manipulation using CRISPR/Cas9 genome editing has dramatically accelerated the generation of new mouse models. Nonetheless, many metabolic disease models still depend upon laborious germline targeting, and are further complicated by the need to avoid developmental phenotypes. We sought to address these experimental limitations by generating somatic mutations in the adult liver using CRISPR/Cas9, as a new strategy to model metabolic disorders. As proof-of-principle, we targeted the low-density lipoprotein receptor (Ldlr), which when deleted, leads to severe hypercholesterolemia and atherosclerosis. Here we show that hepatic disruption of Ldlr with AAV-CRISPR results in severe hypercholesterolemia and atherosclerosis. We further demonstrate that co-disruption of Apob, whose germline loss is embryonically lethal, completely prevented disease through compensatory inhibition of hepatic LDL production. This new concept of metabolic disease modeling by somatic genome editing could be applied to many other systemic as well as liver-restricted disorders which are difficult to study by germline manipulation.
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