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A history of genome editing in Saccharomyces cerevisiae.
Genome editing is a form of highly precise genetic engineering which produces alterations to an organism's genome as small as a single base pair with no incidental or auxiliary modifications; this technique is crucial to the field of synthetic biology, which requires such precision in the installation of novel genetic circuits into host genomes. While a new methodology for most organisms, genome editing capabilities have been used in the budding yeast Saccharomyces cerevisiae for decades. In this review, I will present a brief history of genome editing in S. cerevisiae, discuss the current gold standard method of Cas9-mediated genome editing, and speculate on future directions of the field.
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ScCas9 recognizes NNG protospacer adjacent motif in genome editing of rice.
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Prime editing: a search and replace tool with versatile base changes.
Prime editing is a newly developed CRISPR/Cas system-based genome editing technique. The effector of prime editor (PE) is termed PE2, which is generated by fusing a reverse transcriptase (RT) with a Cas9 H840A nickase. The guide RNA of PE is termed prime editing guide RNA (pegRNA), which consists of a single guide RNA (sgRNA) with a 3' extension containing the RT template (RTT) and primer binding site (PBS). PE can install all 12 types of point mutations, small insertions and deletions and combinations thereof. Since its emergence in 2019, with the high versatility and specificity, PE has been applied to many living organisms, including animals, plants and bacteria. This led to many explorations of PE on gene therapy and genetic improvement in agriculture. In this review, we systematically describe the development, characteristics, optimizations, applications and security of PE. In addition, we discuss the future applications of PE. We expect that this review will help researchers to grasp and better use PE.
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Enhancing glycosylase base-editor activity by fusion to transactivation modules.
Base editors (BEs) are a group of genetic tools with potential in both scientific and medical research. Recently, a glycosylase BE (GBE), which converts C to G, has been constructed. However, the editing efficiency and targeting scope remains to be further exploited. Here, we renovate the GBE by first fusing it to various transactivation modules including Vp64, leading to a higher conversion of C to G relative to GBE in HEK293T cells. Further, higher editing efficiency, enhanced editing purity, and an enlarged editing window are acquired by the combination of SunTag system, GBE, and VP64. Finally, a SpRY-Cas9 variant is used to expand the targeting scope for Vp64-GBE. Vp64-SpRY-GBE and SpRY-GBE target genomic sites with non-NGG PAM, and Vp64-SpRY-GBE demonstrates better performance compared with SpRY-GBE. The construction of GBE variants with superior performance and versatile editing scope broadens the toolbox of BEs and may contribute to genetic therapies with C-to-G mutation.
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A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9.
BACKGROUND: Adenine base editors (ABEs) are promising therapeutic gene editing tools that can efficiently convert targeted A•T to G•C base pairs in the genome. However, the large size of commonly used ABEs based on SpCas9 hinders its delivery in vivo using certain vectors such as adeno-associated virus (AAV) during preclinical applications. Despite a number of approaches having previously been attempted to overcome that challenge, including split Cas9-derived and numerous domain-deleted versions of editors, whether base editor (BE) and prime editor (PE) systems can also allow deletion of those domains remains to be proven. In this study, we present a new small ABE (sABE) with significantly reduced size. RESULTS: We discovered that ABE8e can tolerate large single deletions in the REC2 (Δ174-296) and HNH (Δ786-855) domains of SpCas9, and these deletions can be stacked together to create a new sABE. The sABE showed higher precision than the original ABE8e, with proximally shifted protospacer adjacent motif (PAM) editing windows (A3- A15), and comparable editing efficiencies to 8e-SaCas9-KKH. The sABE system efficiently generated A-G mutations at disease-relevant loci (T1214C in GAA and A494G in MFN2) in HEK293T cells and several canonical Pcsk9 splice sites in N2a cells. Moreover, the sABE enabled in vivo delivery in a single adeno-associated virus (AAV) vector with slight efficiency. Furthermore, we also successfully edited the genome of mouse embryos by microinjecting mRNA and sgRNA of sABE system into zygotes. CONCLUSIONS: We have developed a substantially smaller sABE system that expands the targeting scope and offers higher precision of genome editing. Our findings suggest that the sABE system holds great therapeutic potential in preclinical applications.
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A Cloning-Free Method for CRISPR/Cas9-Mediated Genome Editing in Fission Yeast.
The CRISPR/Cas9 system, which relies on RNA-guided DNA cleavage to induce site-specific DNA double-strand breaks, is a powerful tool for genome editing. This system has been successfully adapted for the fission yeast Schizosaccharomyces pombe by expressing Cas9 and the single-guide RNA (sgRNA) from a plasmid. In the procedures published to date, the cloning step that introduces a specific sgRNA target sequence into the plasmid is the most tedious and time-consuming. To increase the efficiency of applying the CRISPR/Cas9 system in fission yeast, we here developed a cloning-free procedure that uses gap repair in fission yeast cells to assemble two linear DNA fragments, a gapped Cas9-encoding plasmid and a PCR-amplified sgRNA insert, into a circular plasmid. Both fragments contain only a portion of the ura4 or bsdMX marker so that only the correctly assembled plasmid can confer uracil prototrophy or blasticidin resistance. We show that this gap-repair-based and cloning-free CRISPR/Cas9 procedure permits rapid and efficient point mutation knock-in, endogenous N-terminal tagging, and genomic sequence deletion in fission yeast.
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Guide RNA Design for CRISPR/Cas9-Mediated Potato Genome Editing.
The activity of the pool of sgRNA molecules designed for different regions of potato coilin and phytoene desaturase genes was compared in vitro. Due to the presence of nucleotides unpaired with DNA, sgRNA is able not only to inhibit but also to stimulate the activity of the Cas9-sgRNA complex in vitro. Although the first six nucleotides located in the DNA substrate proximally to the PAM site at the 3' end are the binding sites for cas9, they had no significant effect on the activity of the Cas9-sgRNA complex.
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Coloring target and off-target effects of genetically modified nucleases by blue & white colony assays.
More sensitive evaluation of the off-target effects of gene editing nucleases is crucial for human gene therapy. Here we report chromogenic assays designed for sensitive evaluation of gene editing activities using CRISPR/Cas9 test system. Based on beta-galactosidase alpha complementation, qualitative and quantitative evaluations of the target and off-target effects of CRISPR/Cas9 were well established through the color alteration of the E.coli colonies. In addition to target effect analysis, these new assays provide extremely sensitive and efficient tool to profile the off-target effects with one or more bases mismatched between the targets and the gRNAs. Moreover, these assays allow the identification of gene editing effects for off-targets with one base mismatched PAM sites.
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Enhanced Genome Editing Activity with Novel Chimeric ScCas9 Variants in Rice.
The Streptococcus canis Cas9 protein (ScCas9) recognizes the NNG protospacer adjacent motif (PAM), offering a wider range of targets than that offered by the commonly used S. pyogenes Cas9 protein (SpCas9). However, both ScCas9 and its evolved Sc++ variant still exhibit low genome editing efficiency in plants, particularly at the less preferred NTG and NCG PAM targets. In this study, a chimeric SpcRN++ variant is engineered by grafting the recognition (REC) domain of SpCas9 into the Sc++ variant, incorporating the R221K/N394K mutations, and retaining the positively charged loop of S. anginosus Cas9. The SpcRN++ variant exhibits a higher genome editing capacity and wider target range than the Sc++ variant in rice protoplasts and stable transgenic plants. Further evidence indicates that nSpcRN++-based A3A/Y130F and TadA8e exhibit enhanced cytosine and adenine editing efficiency in plants. Finally, herbicide-resistant rice germplasms are produced by targeting the OsACC gene using nSpcRN++-based adenine base editors. These results demonstrate that SpcRN++ is a powerful tool for genome editing in plants, and this integrative protein engineering strategy holds promise for engineering other Cas9 proteins.
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Structure-optimized sgRNA selection with PlatinumCRISPr for efficient Cas9 generation of knockouts.
A single guide RNA (sgRNA) directs Cas9 nuclease for gene-specific scission of double-stranded DNA. High Cas9 activity is essential for efficient gene editing to generate gene deletions and gene replacements by homologous recombination. However, cleavage efficiency is below 50% for more than half of randomly selected sgRNA sequences in human cell culture screens or model organisms. We used in vitro assays to determine intrinsic molecular parameters for maximal sgRNA activity including correct folding of sgRNAs and Cas9 structural information. From the comparison of over 10 data sets, we find major constraints in sgRNA design originating from defective secondary structure of the sgRNA, sequence context of the seed region, GC context, and detrimental motifs, but we also find considerable variation among different prediction tools when applied to different data sets. To aid selection of efficient sgRNAs, we developed web-based PlatinumCRISPr, an sgRNA design tool to evaluate base-pairing and sequence composition parameters for optimal design of highly efficient sgRNAs for Cas9 genome editing. We applied this tool to select sgRNAs to efficiently generate gene deletions in Drosophila Ythdc1 and Ythdf, that bind to N 6 methylated adenosines (m6A) in mRNA. However, we discovered that generating small deletions with sgRNAs and Cas9 leads to ectopic reinsertion of the deleted DNA fragment elsewhere in the genome. These insertions can be removed by standard genetic recombination and chromosome exchange. These new insights into sgRNA design and the mechanisms of CRISPR-Cas9 genome editing advance the efficient use of this technique for safer applications in humans.
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