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Development of deaminase-free T-to-S base editor and C-to-G base editor by engineered human uracil DNA glycosylase.
DNA base editors enable direct editing of adenine (A), cytosine (C), or guanine (G), but there is no base editor for direct thymine (T) editing currently. Here we develop two deaminase-free glycosylase-based base editors for direct T editing (gTBE) and C editing (gCBE) by fusing Cas9 nickase (nCas9) with engineered human uracil DNA glycosylase (UNG) variants. By several rounds of structure-informed rational mutagenesis on UNG in cultured human cells, we obtain gTBE and gCBE with high activity of T-to-S (i.e., T-to-C or T-to-G) and C-to-G conversions, respectively. Furthermore, we conduct parallel comparison of gTBE/gCBE with those recently developed using other protein engineering strategies, and find gTBE/gCBE show the outperformance. Thus, we provide several base editors, gTBEs and gCBEs, with corresponding engineered UNG variants, broadening the targeting scope of base editors.
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Application of Base Editor-Mediated Genome Editing in Mouse Retina.
Base editor is a newly developed genome editing technology that enables conversion of single nucleotides without DNA double-strand breaks (DSB) and maintains a low rate of insertion-deletion (INDEL) errors. With these flexibility and safety, base editor has been widely used in many fields, including inherited retinal disease. The majority of retinal genome editing requires intravitreal and subretinal injection delivery of the therapeutic vector in order to transduce the target cells. Here, we provide an application guide of base editor as performed in the mouse retina.
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β Cell Replacement after Gene Editing of a Neonatal Diabetes-Causing Mutation at the Insulin Locus.
Permanent neonatal diabetes mellitus (PNDM) can be caused by insulin mutations. We generated induced pluripotent stem cells from fibroblasts of a patient with PNDM and undetectable insulin at birth due to a homozygous mutation in the translation start site of the insulin gene. Differentiation of mutant cells resulted in insulin-negative endocrine stem cells expressing MAFA, NKX6.1, and chromogranin A. Correction of the mutation in stem cells and differentiation to pancreatic endocrine cells restored insulin production and insulin secretion to levels comparable to those of wild-type cells. Grafting of corrected cells into mice, followed by ablating mouse β cells using streptozotocin, resulted in normal glucose homeostasis, including at night, and the stem cell-derived grafts adapted insulin secretion to metabolic changes. Our study provides proof of principle for the generation of genetically corrected cells autologous to a patient with non-autoimmune insulin-dependent diabetes. These cases should be readily amenable to autologous cell therapy.
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Efficient Method for Generating Point Mutations in the Vaccinia Virus Genome Using CRISPR/Cas9.
The vaccinia virus (VACV) was previously used as a vaccine for smallpox eradication. Nowadays, recombinant VACVs are developed as vaccine platforms for infectious disease prevention and cancer treatment. The conventional method for genome editing of the VACV is based on homologous recombination, which is poorly efficient. Recently, the use of CRISPR/Cas9 technology was shown to greatly improve the speed and efficiency of the production of recombinant VACV expressing a heterologous gene. However, the ability to rapidly recover viruses bearing single nucleotide substitutions is still challenging. Notwithstanding, ongoing studies on the VACV and its interaction with the host cell could benefit from viral gene targeted mutagenesis. Here, we present a modified version of the CRISPR/Cas9 system for the rapid selection of mutant VACV carrying point mutations. For this purpose, we introduced a silent mutation into the donor gene (which will replace the wildtype gene) that serves a double function: it is located in the PAM (NGG) sequence, which is essential for Cas9 cleavage, and it alters a restriction site. This silent mutation, once introduced into the VACV genome, allows for rapid selection and screening of mutant viruses carrying a mutation of interest in the targeted gene. As a proof of concept, we produced several recombinant VACVs, with mutations in the E9L gene, upon which, phenotypic analysis was performed.
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Versatile and efficient in vivo genome editing with compact Streptococcus pasteurianus Cas9.
Compact CRISPR-Cas9 systems that can be packaged into an adeno-associated virus (AAV) show promise for gene therapy. However, the requirement of protospacer adjacent motifs (PAMs) restricts the target scope. To expand this repertoire, we revisited and optimized a small Cas9 ortholog derived from Streptococcus pasteurianus (SpaCas9) for efficient genome editing in vivo. We found that SpaCas9 enables potent targeting of 5'-NNGYRA-3' PAMs, which are distinct from those recognized by currently used small Cas9s; the Spa-cytosine base editor (CBE) and Spa-adenine base editor (ABE) systems efficiently generated robust C-to-T and A-to-G conversions both in vitro and in vivo. In addition, by exploiting natural variation in the PAM-interacting domain, we engineered three SpaCas9 variants to further expand the targeting scope of compact Cas9 systems. Moreover, mutant mice with efficient disruption of the Tyr gene were successfully generated by microinjection of SpaCas9 mRNA and the corresponding single guide RNA (sgRNA) into zygotes. Notably, all-in-one AAV delivery of SpaCas9 targeting the Pcsk9 gene in adult mouse liver produced efficient genome-editing events and reduced its serum cholesterol. Thus, with distinct PAMs and a small size, SpaCas9 will broaden the CRISPR-Cas9 toolsets for efficient gene modifications and therapeutic applications.
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High-precision cytosine base editors by evolving nucleic-acid-recognition hotspots in deaminase.
Base editors (BEs), covalent fusions of a cytosine or adenine deaminase with a nuclease-impaired CRISPR protein, mediate site-specific conversion of C:G to T:A (CBEs) or A:T to G:C (ABEs) in the genome. Existing BEs modify all cytosines or adenines within the editing window, which limits their precision. Here we engineer nucleotide and context specificity of the Escherichia coli transfer RNA-specific adenosine deaminase (TadA) to pinpoint cytosine editing. Strategically sampling multiple nucleic-acid-recognition hotspots through directed evolution, we develop 16 TadA-derived NCN-specific deaminases that cover every possible -1 and +1 context for a target cytosine, providing on-demand deaminase choices for editor customization. We apply these variants to (1) correct disease-associated T:A-to-C:G transitions documented by ClinVar, achieving greater accuracy than conventional CBEs in 81.5% of cases, and (2) model two cancer-driver mutations-KRASG12D (ACC) and TP53R248Q (CCG)-in vitro. Our approach offers a general strategy to access precise base editors for potential clinical applications.
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Establishment of CRISPR/Cas9 mediated targeted mutagenesis in hop (Humulus lupulus).
The CRISPR/Cas9-based targeted genome editing has emerged as a versatile technique, widely employed in plant genome engineering, both to decipher gene function and as an alternative to classical breeding technique for traits improvement in plants. However, to date, no such platform has been developed for hop (Humulus lupulus L.), which is an economically important crop producing valuable secondary metabolites utilized in the brewing and pharmaceutical industries. Here, we present the first report on the successful establishment of efficient CRISPR/Cas9-based genome editing using the visible endogenous marker gene phytoene desaturase (PDS) involved in carotenoid biosynthesis to demonstrate successful genome editing in hop. Agrobacterium tumefaciens-mediated transformation of in vitro generated internodal explants was used for the stable integration of constructs expressing plant codon-optimized Cas9 and a pair of co-expressed guide RNAs to target the distinct genomic sites of the PDS gene of hop. Analysis of RNA-guided genome-editing events, including mutant lines screening and homozygosity assessment using the T7 endonuclease assay showed that 33.3% of transformed plants were successfully edited at the target site, displaying albino and mosaic regenerants. Intriguingly, the detected mutations were ranges of deletions (16 bp to 39 bp) which led to disruption of the exon-intron boundary, few base substitutions, and a 1 bp insertion at 3 bp upstream of the PAM region of the target site. The decrease in chlorophyll a/b, and carotenoid content in the mutant lines further confirmed the functional disruption of the HlPDS gene. Taken together, our results demonstrate that the CRISPR/Cas9 system can precisely edit the targeted genome sequences, which may revolutionize our way to overcome some of the obstacles that have plagued the traits improvement in hop.
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In vivo analysis of renal epithelial cells in zebrafish.
The zebrafish kidney has been used effectively for studying kidney development, repair and disease. New gene editing capability makes it a more versatile in vivo vertebrate model system to investigate renal epithelial cells in their native environment. In this chapter we focus on dissecting gene function in basic cellular biology of renal epithelial cells, including lumen formation and cell polarity, in intact zebrafish embryos.
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Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application.
In the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system, protoplasts are not only useful for rapidly validating the mutagenesis efficiency of various RNA-guided endonucleases, promoters, sgRNA designs, or Cas proteins, but can also be a platform for DNA-free gene editing. To date, the latter approach has been applied to numerous crops, particularly those with complex genomes, a long juvenile period, a tendency for heterosis, and/or self-incompatibility. Protoplast regeneration is thus a key step in DNA-free gene editing. In this report, we review the history and some future prospects for protoplast technology, including protoplast transfection, transformation, fusion, regeneration, and current protoplast applications in CRISPR/Cas-based breeding.
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Computational Tools and Resources Supporting CRISPR-Cas Experiments.
The CRISPR-Cas system has become a cutting-edge technology that revolutionized genome engineering. The use of Cas9 nuclease is currently the method of choice in most tasks requiring a specific DNA modification. The rapid development in the field of CRISPR-Cas is reflected by the constantly expanding ecosystem of computational tools aimed at facilitating experimental design and result analysis. The first group of CRISPR-Cas-related tools that we review is dedicated to aid in guide RNA design by prediction of their efficiency and specificity. The second, relatively new group of tools exploits the observed biases in repair outcomes to predict the results of CRISPR-Cas edits. The third class of tools is developed to assist in the evaluation of the editing outcomes by analysis of the sequencing data. These utilities are accompanied by relevant repositories and databases. Here we present a comprehensive and updated overview of the currently available CRISPR-Cas-related tools, from the perspective of a user who needs a convenient and reliable means to facilitate genome editing experiments at every step, from the guide RNA design to analysis of editing outcomes. Moreover, we discuss the current limitations and challenges that the field must overcome for further improvement in the CRISPR-Cas endeavor.
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