Highly efficient base editing with expanded targeting scope using SpCas9-NG in rabbits.

Base editors, composed of a cytidine deaminase or an evolved adenine deaminase fused to Cas9 nickase, enable efficient C-to-T or A-to-G conversion in various organisms. However, the NGG protospacer adjacent motif (PAM) requirement of Streptococcus pyogenes Cas9 (SpCas9) substantially limits the target sites suitable for base editing. Quite recently, a new engineered SpCas9-NG variant, which can recognize minimal NG PAMs more efficiently than the present xCas9 variant. Here, we investigated the efficiency and PAM compatibility of SpCas9-NG-assisted cytidine base editors (CBEs) and adenine base editors (ABEs) in rabbits. In this study, we showed that NG-BE4max and NG-ABEmax systems can achieve a targeted mutation efficiency of 75%-100% and 80%-100% with excellent PAM compatibility of NGN PAMs in rabbit embryos, respectively. In addition, both base editors were successfully applied to create new rabbit models with precise point mutations, demonstrating their high efficiency and expanded genome-targeting scope in rabbits. Meanwhile, NG-ABEmax can be used to precisely mimic human Hoxc13 p.Q271R missense mutation in Founder (F0) rabbits, which is arduous for conventional ABEs to achieve due to a NGA PAM requirement. Collectively, NG-BE4max and NG-ABEmax systems provide promising tools to perform efficient base editing with expanded targeting scope in rabbits and enhances its capacity to model human diseases.

Precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions.

BACKGROUND: Cytidine base editors (CBEs), composed of a cytidine deaminase fused to Cas9 nickase (nCas9), enable efficient C-to-T conversion in various organisms. However, current base editors can induce unwanted bystander C-to-T conversions when multiple Cs are present in the ~ 5-nucleotide activity window of cytidine deaminase, which negatively affects their precision. Here, we develop a new base editor which significantly reduces unwanted bystander activities. RESULTS: We used an engineered human APOBEC3G (eA3G) C-terminal catalytic domain with preferential cytidine-deaminase activity in motifs with a hierarchy CCC>CCC>CC (where the preferentially deaminated C is underlined), to develop an eA3G-BE with distinctive CC context-specificity and reduced generation of bystander mutations. Targeted editing efficiencies of 18.3-58.0% and 54.5-92.2% with excellent CC context-specificity were generated in human cells and rabbit embryos, respectively. In addition, a base editor that can further recognize relaxed NG PAMs is achieved by combining hA3G with an engineered SpCas9-NG variant. The A3G-BEs were used to induce accurate single-base substitutions which led to nonsense mutation with an efficiency of 83-100% and few bystander mutations in Founder (F0) rabbits at Tyr loci. CONCLUSIONS: These novel base editors with improved precision and CC context-specificity will expand the toolset for precise gene modification in organisms.

Improved base editor for efficient editing in GC contexts in rabbits with an optimized AID-Cas9 fusion.

Cytidine base editors, which are composed of a cytidine deaminase fused to clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) nickase, enable the efficient conversion of the C·G base pair to T·A in various organisms. However, the currently used rat apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 1(rA1)-based BE3 is often inefficient in target Cs that are immediately downstream of a G (GC context). Here, we observed that, with an 11-nt editing window, an optimized activation-induced cytidine deaminase (AID)-Cas9 fusion can efficiently convert C to T in a variety of sequence contexts in rabbits. Strikingly, the enhanced AID-Cas9 fusion (eAID-BE4max) has significant effectiveness of inducing Tyr p.R299H mutation in GC contexts (from 16.67 to 83.33%) in comparison with BE3 in founder rabbits. Furthermore, the engineered AID-Cas9 variants were produced with reduced bystander activity [eAID (N51G)-BE4max] and increased genome-targeting scope (eAID-NG-BE4max). Overall, this work provides a series of improved tools that were generated using optimized AID-Cas9 fusions and associated engineered variants that can be used for efficient and versatile C-to-T base editing, especially in GC contexts.-Liu, Z., Shan, H., Chen, S., Chen, M., Zhang, Q., Lai, L., Li, Z. Improved base editor for efficient editing in GC contexts in rabbits with an optimized AID-Cas9 fusion.

Expanded targeting scope and enhanced base editing efficiency in rabbit using optimized xCas9(3.7).

Evolved xCas9(3.7) variant with broad PAM compatibility has been reported in cell lines, while its editing efficiency was site-specific. Here, we show that xCas9(3.7) can recognize a broad PAMs including NGG, NGA, and NGT, in both embryos and Founder (F0) rabbits. Furthermore, the codon-optimized xCas9-derived base editors, exBE4 and exABE, can dramatically improve the base editing efficiencies in rabbit embryos. Our results demonstrated that the optimized xCas9 with expanded PAM compatibility and enhanced base editing efficiency could be used for precise gene modifications in organisms.

HOXA3 functions as the on-off switch to regulate the development of hESC-derived third pharyngeal pouch endoderm through EPHB2-mediated Wnt pathway.

Objectives: Normal commitment of the endoderm of the third pharyngeal pouch (3PP) is essential for the development and differentiation of the thymus. The aim of this study was to investigate the role of transcription factor HOXA3 in the development and differentiation of 3PP endoderm (3PPE) from human embryonic stem cells (hESCs). Methods: The 3PPE was differentiated from hESC-derived definitive endoderm (DE) by mimicking developmental queues with Activin A, WNT3A, retinoic acid and BMP4. The function of 3PPE was assessed by further differentiating into functional thymic epithelial cells (TECs). The effect of HOXA3 inhibition on cells of 3PPE was subsequently investigated. Results: A highly efficient approach for differentiating 3PPE cells was developed and these cells expressed 3PPE related genes HOXA3, SIX1, PAX9 as well as EpCAM. 3PPE cells had a strong potential to develop into TECs which expressed both cortical TEC markers K8 and CD205, and medullary TEC markers K5 and AIRE, and also promoted the development and maturation of T cells. More importantly, transcription factor HOXA3 not only regulated the differentiation of 3PPE, but also had a crucial role for the proliferation and migration of 3PPE cells. Our further investigation revealed that HOXA3 controlled the commitment and function of 3PPE through the regulation of Wnt signaling pathway by activating EPHB2. Conclusion: Our results demonstrated that HOXA3 functioned as the on-off switch to regulate the development of hESC-derived 3PPE through EPHB2-mediated Wnt pathway, and our findings will provide new insights into studying the development of 3PP and thymic organ in vitro and in vivo.

The Higher the Life Satisfaction, the Better the Psychological Capital? Life Satisfaction and Psychological Capital: A Moderated Mediation Model.

This study investigates the mediator role of attachment avoidance and the moderator role of rejection sensitivity on the links between life satisfaction and psychological capital (PsyCap). This study uses the Experiences in Close Relationship Scale, Rejection Sensitive Scale, Positive Psychological Capital Scale, and Life Satisfaction Scale among 999 Chinese young adults as subjects. The results presented that life satisfaction had a significant positive predictive effect on PsyCap. Mediation analysis of this study shows that attachment avoidance mediated the association between life satisfaction and PsyCap. Furthermore, moderated mediation analysis indicated that rejection sensitivity moderates the link between life satisfaction and attachment avoidance (first-stage moderation). Compared with individuals with low rejection sensitivity, individuals with high rejection sensitivity show more attachment avoidance under low life satisfaction. This study helps understand the relationship between life satisfaction and PsyCap from the perspective of rejection sensitivity theory and attachment theory and has implications for guiding college students toward strengthening PsyCap and weakening rejection sensitivity.

Reduced off-target effect of NG-BE4max by using NG-HiFi system.

Recently, a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize a minimal NG protospacer adjacent motif (PAM) was reported to expand the targeting scope in genome editing. However, increased genome-wide off-target mutations with this variant compared with SpCas9 were reported in previous studies. In addition, lower base editing frequencies and higher unintended off-target mutations were also found in Hoxc13-ablated rabbits generated by NG-BE4max in our study. Here, a high-fidelity base editor, NG-HiFi, in comparison to NG-BE4max, showed retention of on-target activity while exhibiting significantly decreased off-target activity in Hoxc13-ablated rabbits. Collectively, the improved specificity and reduced off-target effect of SpCas9-NG assisted in cytidine base editing with the NG-HiFi system, providing a promising tool to precisely model human diseases in rabbits.

Efficient and high-fidelity base editor with expanded PAM compatibility for cytidine dinucleotide.

Cytidine base editor (CBE), which is composed of a cytidine deaminase fused to Cas9 nickase, has been widely used to induce C-to-T conversions in a wide range of organisms. However, the targeting scope of current CBEs is largely restricted to protospacer adjacent motif (PAM) sequences containing G, T, or A bases. In this study, we developed a new base editor termed "nNme2-CBE" with excellent PAM compatibility for cytidine dinucleotide, significantly expanding the genome-targeting scope of CBEs. Using nNme2-CBE, targeted editing efficiencies of 29.0%-55.0% and 17.3%-52.5% were generated in human cells and rabbit embryos, respectively. In contrast to conventional nSp-CBE, the nNme2-CBE is a natural high-fidelity base editing platform with minimal DNA off-targeting detected in vivo. Significantly increased efficiency in GC context and precision were determined by combining nNme2Cas9 with rationally engineered cytidine deaminases. In addition, the Founder rabbits with accurate single-base substitutions at Fgf5 gene loci were successfully generated by using the nNme2-CBE system. These novel nNme2-CBEs with expanded PAM compatibility and high fidelity will expand the base editing toolset for efficient gene modification and therapeutic applications.

Efficient base editing with high precision in rabbits using YFE-BE4max.

Cytidine base editors, composed of a cytidine deaminase fused to Cas9 nickase, enable efficient C-to-T conversion in various organisms. However, current base editors suffer from severe trade-off between editing efficiency and precision. Here, based on rationally mutated cytidine deaminase domain, we develop a new base editor, YFE-BE4max, effectively narrow the editing width to as little as approximately three nucleotides while maintaining high efficiency in rabbits. Moreover, YFE-BE4max successfully mediated the Tyr p. Q68Stop and Lmna p. G607G mutation in F0 rabbit with high efficiency and precision, which precisely recapitulates the pathological features of human OCA1 and HGPS, respectively. Collectively, YFE-BE4max system provide promising tools to perform efficient base editing with high precision in rabbits and enhances its capacity to precisely model human diseases.

CRISPR Start-Loss: A Novel and Practical Alternative for Gene Silencing through Base-Editing-Induced Start Codon Mutations.

CRISPR-Cas9-mediated gene knockout and base-editing-associated induction of STOP codons (iSTOP) have been widely used to exterminate the function of a coding gene, while they have been reported to exhibit side effects. In this study, we propose a novel and practical alternative method referred to as CRISPR Start-Loss (CRISPR-SL), which eliminates gene expression by utilizing both adenine base editors (ABEs) and cytidine base editors (CBEs) to disrupt the initiation codon (ATG). CRISPR-SL has been verified to be a feasible strategy on the cellular and embryonic levels (mean editing efficiencies up to 30.67% and 73.50%, respectively) and in two rabbit models mimicking Otc deficiency (Otc gene) and long hair economic traits (Fgf5 gene).

AcrIIA5 Suppresses Base Editors and Reduces Their Off-Target Effects.

The CRISPR/nCas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) are capable of catalyzing C•G to T•A or A•T to G•C conversions, respectively, and have become new, powerful tools for achieving precise genetic changes in a wide range of organisms. These base editors hold great promise for correcting pathogenic mutations and for being used for therapeutic applications. However, the recognition of cognate DNA sequences near their target sites can cause severe off-target effects that greatly limit their clinical applications, and this is an urgent problem that needs to be resolved for base editing systems. The recently discovered phage-derived proteins, anti-CRISPRs, which can suppress the natural CRISPR nuclease activity, may be able to ameliorate the off-target effects of base editing systems. Here, we confirm for the first time that AcrIIA2, AcrIIA4, and AcrIIA5 efficiently inhibit base editing systems in human cells. In particular, AcrIIA5 has a significant inhibitory effect on all base editing variant systems tested in our study. We further show that the off-target effects of BE3 and ABE7.10 were significantly reduced in AcrIIA5 treated cells. This study suggests that AcrIIA5 should be widely used for the precise control of base editing and to thoroughly "shut off" nuclease activity of both CBE and ABE systems.