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Structural and functional insights into internal domain replacement in SpCas9 for protein engineering.
The CRISPR-Cas9 system has emerged as a powerful tool for precise genome editing, with ongoing research focused on enhancing its reliability and expanding its versatility. One effective strategy involves the integration of foreign functional domains into Cas9 to confer new capabilities. However, successful integration requires identification of insertion sites that preserve the protein's structural integrity and function. In this study, we identified a C-terminal region of Streptococcus pyogenes Cas9 (SpCas9), spanning residues 1242-1263, as a viable site for domain replacement. Structural and biochemical analyses of a SpCas9 variant lacking this region confirmed its dispensability for SpCas9 activity. As a proof of concept, we substituted this segment with the evolved E. coli tRNA adenosine deaminase (TadA), a key component of adenine base editors. Functional evaluation of this engineered SpCas9-TadA variant demonstrated deamination efficiency comparable to that of the ABE8e, with the potential to modulate the editing window through linker design. These results highlight the potential of targeted engineering of this region to develop more precise and versatile genome editing tools.
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Quantitative assessment of timing, efficiency, specificity and genetic mosaicism of CRISPR/Cas9-mediated gene editing of hemoglobin beta gene in rhesus monkey embryos.
Gene editing technologies offer new options for developing novel biomedical research models and for gene and stem cell based therapies. However, applications in many species demand high efficiencies, specificity, and a thorough understanding of likely editing outcomes. To date, overall efficiencies, rates of off-targeting and degree of genetic mosaicism have not been well-characterized for most species, limiting our ability to optimize methods. As a model gene for measuring these parameters of the CRISPR/Cas9 application in a primate species (rhesus monkey), we selected the β-hemoglobin gene (HBB), which also has high relevance to the potential application of gene editing and stem-cell technologies for treating human disease. Our data demonstrate an ability to achieve a high efficiency of gene editing in rhesus monkey zygotes, with no detected off-target effects at selected off-target loci. Considerable genetic mosaicism and variation in the fraction of embryonic cells bearing targeted alleles are observed, and the timing of editing events is revealed using a new model. The uses of Cas9-WT protein combined with optimized concentrations of sgRNAs are two likely areas for further refinement to enhance efficiency while limiting unfavorable outcomes that can be exceedingly costly for application of gene editing in primate species.
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A New Era of Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated Protein 9 Gene Editing Technology in Cardiovascular Diseases: Opportunities, Challenges, and Perspectives.
Cardiovascular diseases (CVDs) remain major causes of global mortality in the world. Genetic approaches have succeeded in the discovery of the molecular basis of an increasing number of cardiac diseases. Genome-editing strategies are one of the most effective methods for assisting therapeutic approaches. Potential therapeutic methods of correcting disease-causing mutations or of knocking out specific genes as approaches for the prevention of CVDs have gained substantial attention using genome-editing techniques. Recently, the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has become the most widely used genome-editing technology in molecular biology due to its benefits such as simple design, high efficiency, good repeatability, short cycle, and cost-effectiveness. In the present review, we discuss the possibilities of applying the CRISPR/Cas9 genome-editing tool in the CVDs.
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Generation of C-to-G transversion in mouse embryos via CG editors.
Base editors (BEs) are efficient and precise tools for generating single base conversions in living organisms. While most BE systems are limited in mediating C-to-T or A-to-G conversions, recently developed C-to-G base editors (CGBEs) could produce C-to-G transversions. CGBEs convert cytosine within the editing window to abasic intermediates, which would be replaced with any base after base excision repair (BER). By far, though the efficiency and editing scope of CGBEs have been investigated in cultured cells via gRNA library and machine-learning, the viability of CGBEs in generating mouse models has not been adequately tested. In this study, we tested the C-to-G transversion efficiency of the CGBE1 and CGBE-XRCC1 systems in mouse embryos. Our results showed that both of the CGBE systems were able to mediate C-to-G transversion on 2 out of 3 targets tested, with up to 20% frequency within the editing window. Notably, most of the groups showed over 40% of other base conversions, predominantly C-to-T. Lastly, we successfully acquired the F1 mouse carrying a disease-causing mutation. In all, our study suggested that CGBEs systems held great potential in generating mouse models and indicated that XRCC1 based system is applicable in mouse embryos.
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Human 8-cell embryos enable efficient induction of disease-preventive mutations without off-target effect by cytosine base editor.
Approximately 140 million people worldwide are homozygous carriers of APOE4 (ε4), a strong genetic risk factor for late onset familial and sporadic Alzheimer's disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and noncarriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects. In particular, 80% of AD-susceptible ε4 allele copies were converted to the AD-neutral ε3 allele in human ε4-carrying embryos. Stringent control measures combined with targeted deep sequencing, whole genome sequencing, and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells. Furthermore, base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage. Finally, we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia. Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos, a potential approach for reducing human susceptibility to AD or other genetic diseases.
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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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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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ScCas9 recognizes NNG protospacer adjacent motif in genome editing of rice.
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