Zhiquan Liu

All Publications

• TIGRa: An ultra-compact programmable activator enabling multiplexed and efficient gene regulation. Cell stem cell Liu, Z., Chen, S., Wang, W., Zhang, F., Wang, Q., Walkiewicz, G., Hossen, F., Zhou, J., Wang, Y., Sun, Y. 2026

  Abstract

  Programmable gene activation has broad therapeutic potential but remains constrained by the large effector size, limited multiplexing capacity, and challenges in in vivo delivery. Here, we develop the TIGR-TasR-mediated activator (TIGRa), a compact transcriptional activator derived from the tandem interspaced guide RNA (TIGR)-TIGR-associated protein (TasR) system that is mechanistically distinct from CRISPR-based activators. TIGRa is less than half the size of dSpCas9-based activators while achieving comparable or greater activation efficiency. Its native TIGR array architecture enables efficient multiplexed regulation, supporting simultaneous activation of up to 12 endogenous genes from a single compact construct. TIGRa-mediated multi-gene activation efficiently reprogrammed human fibroblasts into induced pluripotent stem cells. In addition, an all-in-one adeno-associated virus (AAV)-TIGRa vector activated endogenous CaMKII in vivo, promoting retinal ganglion cell survival and preserving visual function in a mouse model of N-methyl-D-aspartic (NMDA)-induced retinal injury. These results establish TIGRa as a compact and multiplexable platform for therapeutic gene regulation and in vivo genetic medicine.

  View details for DOI 10.1016/j.stem.2026.07.008

  View details for PubMedID 42575098

• Precise Correction of the Pde6b-L659P Mutation Causing Retinal Degeneration with Minimum Bystander Editing by Advanced Genome Editing Tools. Research (Washington, D.C.) Liu, Z., Chen, S., Sun, Y. 2025; 8: 0770

  Abstract

  Recently developed base editing (BE), prime editing (PE), and click editing (CE) technologies enable precise and efficient genome editing with minimal risk of double-strand breaks and associated toxicity. However, their effectiveness in correcting real disease-causing mutations has not been systematically compared. Here, we aim to evaluate the potential of BE, PE, and CE technologies in rescuing the retinal degeneration-causing Pde6b (c.1976T>C, p.L659P) mutation. This site is prone to bystander effects, making it an ideal model for comparing the editing outcomes of these 3 novel technologies, particularly their editing precision. We optimized BE, PE, and CE systems in vitro using Pde6b-L659P cell models and compared their editing via deep sequencing. BE and PE had similar efficiency, but PE was the most precise, minimizing bystander edits. CE had lower efficiency and higher indel rates, needing further optimization. Using the optimal PE system for in vivo electroporation in Pde6b-L659P mice, we achieved 12.4% targeted repair with high precision, partially rescuing retinal degeneration. This study demonstrates proof of concept for the precise correction of the Pde6b-L659P mutation causing retinal degeneration using BE, PE, and CE tools. The findings offer valuable insights into the future optimization of precision gene editing techniques and their potential translational applications.

  View details for DOI 10.34133/research.0770

  View details for PubMedID 40607323

  View details for PubMedCentralID PMC12220932

• Efficient Rescue of Retinal Degeneration in Pde6a Mice by Engineered Base Editing and Prime Editing. Advanced science (Weinheim, Baden-Wurttemberg, Germany) Liu, Z., Chen, S., Davis, A. E., Lo, C. H., Wang, Q., Li, T., Ning, K., Zhang, Q., Zhao, J., Wang, S., Sun, Y. 2024: e2405628

  Abstract

  Retinitis pigmentosa (RP) is a complex spectrum of inherited retinal diseases marked by the gradual loss of photoreceptor cells, ultimately leading to blindness. Among these, mutations in PDE6A, responsible for encoding a cGMP-specific phosphodiesterase, stand out as pivotal in autosomal recessive RP (RP43). Unfortunately, no effective therapy currently exists for this specific form of RP. However, recent advancements in genome editing, such as base editing (BE) and prime editing (PE), offer a promising avenue for precise and efficient gene therapy. Here, it is illustrated that the engineered BE and PE systems, particularly PE, exhibit high efficiency in rescuing a target point mutation with minimal bystander effects in an RP mouse model carrying the Pde6a (c.2009A > G, p.D670G) mutation. The optimized BE and PE systems are first screened in N2a cells and subsequently assessed in electroporated mouse retinas. Notably, the optimal PE system, delivered via dual adeno-associated virus (AAV), precisely corrects the pathogenic mutation with average 9.4% efficiency, with no detectable bystander editing. This correction restores PDE6A protein expression, preserved photoreceptors, and rescued retinal function in Pde6a mice. Therefore, this study offers a proof-of-concept demonstration for the treatment of Pde6a-related retinal degeneration using BE and PE systems.

  View details for DOI 10.1002/advs.202405628

  View details for PubMedID 39297417

• Inhibition of base editors with anti-deaminases derived from viruses NATURE COMMUNICATIONS Liu, Z., Chen, S., Lai, L., Li, Z. 2022; 13 (1): 597

  Abstract

  Cytosine base editors (CBEs), combining cytidine deaminases with the Cas9 nickase (nCas9), enable targeted C-to-T conversions in genomic DNA and are powerful genome-editing tools used in biotechnology and medicine. However, the overexpression of cytidine deaminases in vivo leads to unexpected potential safety risks, such as Cas9-independent off-target effects. This risk makes the development of deaminase off switches for modulating CBE activity an urgent need. Here, we report the repurpose of four virus-derived anti-deaminases (Ades) that efficiently inhibit APOBEC3 deaminase-CBEs. We demonstrate that they antagonize CBEs by inhibiting the APOBEC3 catalytic domain, relocating the deaminases to the extranuclear region or degrading the whole CBE complex. By rationally engineering the deaminase domain, other frequently used base editors, such as CGBE, A&CBE, A&CGBE, rA1-CBE and ABE8e, can be moderately inhibited by Ades, expanding the scope of their applications. As a proof of concept, the Ades in this study dramatically decrease both Cas9-dependent and Cas9-independent off-target effects of CBEs better than traditional anti-CRISPRs (Acrs). Finally, we report the creation of a cell type-specific CBE-ON switch based on a microRNA-responsive Ade vector, showing its practicality. In summary, these natural deaminase-specific Ades are tools that can be used to regulate the genome-engineering functions of BEs.

  View details for DOI 10.1038/s41467-022-28300-0

  View details for Web of Science ID 000749535300004

  View details for PubMedID 35105899

  View details for PubMedCentralID PMC8807840

• Versatile and efficient <i>in vivo</i> genome editing with compact <i>Streptococcus pasteurianus</i> Cas9 MOLECULAR THERAPY Liu, Z., Chen, S., Xie, W., Song, Y., Li, J., Lai, L., Li, Z. 2022; 30 (1): 256-267

  Abstract

  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.

  View details for DOI 10.1016/j.ymthe.2021.06.013

  View details for Web of Science ID 000744579600007

  View details for PubMedID 34174445

  View details for PubMedCentralID PMC8753289

• Highly efficient RNA-guided base editing in rabbit NATURE COMMUNICATIONS Liu, Z., Chen, M., Chen, S., Deng, J., Song, Y., Lai, L., Li, Z. 2018; 9: 2717

  Abstract

  Cytidine base editors (CBEs) and adenine base editors (ABEs), composed of a cytidine deaminase or an evolved adenine deaminase fused to Cas9 nickase, enable the conversion of C·G to T·A or A·T to G·C base pair in organisms, respectively. Here, we show that BE3 and ABE7.10 systems can achieve a targeted mutation efficiency of 53-88% and 44-100%, respectively, in both blastocysts and Founder (F0) rabbits. Meanwhile, this strategy can be used to precisely mimic human pathologies by efficiently inducing nonsense or missense mutations as well as RNA mis-splicing in rabbit. In addition, the reduced frequencies of indels with higher product purity are also determined in rabbit blastocysts by BE4-Gam, which is an updated version of the BE3 system. Collectively, this work provides a simple and efficient method for targeted point mutations and generation of disease models in rabbit.

  View details for DOI 10.1038/s41467-018-05232-2

  View details for Web of Science ID 000438493800016

  View details for PubMedID 30006570

  View details for PubMedCentralID PMC6045575

• Toward a universal neuroprotective strategy for vision preservation. Molecular therapy : the journal of the American Society of Gene Therapy Liu, Z., Chen, S., Sun, Y. 2026

  View details for DOI 10.1016/j.ymthe.2026.02.038

  View details for PubMedID 41794043

• Compact transcriptional activators: Therapeutic gene regulation in ocular disease. Neural regeneration research Liu, Z., Chuu, L. Y., Chen, S., Sun, Y. 2026

  View details for DOI 10.4103/NRR.NRR-D-25-01001

  View details for PubMedID 41641781

• Matrine Reduces Intraocular Pressure in Corticosteroid-Induced Ocular Hypertensive Mouse Eyes. Investigative ophthalmology & visual science Zhang, Q., Liu, Z., Chen, S., Wang, Q., Lo, C. H., Ning, K., Zhao, J., Shen, Y., Liton, P. B., Sun, Y. 2025; 66 (11): 18

  Abstract

  This study investigates the potential of matrine, a quinolizidine alkaloid, in regulating intraocular pressure (IOP) in normal and corticosteroid-induced ocular hypertension (OHT) mice.Wild-type C57BL/6 mice were randomly divided into normal and OHT groups. The OHT mouse model was established by periocular conjunctival fornix injections of dexamethasone-21-acetate (DEX). IOP was measured at 0.0, 0.5, 1.0, 3.0, and 6.0 hours after matrine treatment in both groups. Aqueous humor (AH) outflow facility was measured using our previously described/validated perfusion system. Anterior segment-optical coherence tomography was used to evaluate morphological changes in the anterior chamber following matrine treatment. Hematoxylin and eosin staining and immunofluorescence staining were used to investigate structural changes.Matrine treatment (50-200 µg/g) decreased the IOP in normal mice in a dose-dependent manner. AH outflow facility in normal mice elevated at 0.5 hours after matrine treatment (100 and 200 µg/g). Additionally, 100 µg/g matrine treatment increased the angle opening distance in the anterior chamber. In DEX-induced OHT mice, matrine (100 and 200 µg/g) reduced the elevated IOP and increased the AH outflow facility. Furthermore, 100 µg/g matrine treatment increased the angle opening distance compared with that of PBS-treated controls. However, matrine (100 µg/g) did not induce significant changes in trabecular meshwork gross morphology or the expression of cell contractility and extracellular matrix markers in OHT mice at 0.5 hours after treatment.Matrine decreased the IOP in the DEX-induced OHT mouse model, highlighting its potential as a therapeutic agent for managing glaucoma, particularly in corticosteroid-associated secondary cases.

  View details for DOI 10.1167/iovs.66.11.18

  View details for PubMedID 40772663

• Pathophysiology of intraoperative floppy iris syndrome: An unsettled debate. Survey of ophthalmology Shen, Y., Frauches, R., Zhao, J., Lo, C. H., Ning, K., Chen, S., Liu, Z., Zhang, F., Sun, Y. 2025

  Abstract

  Intraoperative floppy iris syndrome (IFIS)--characterized by iris blowing, prolapse, and progressive miosis during phacoemulsification surgery--poses significant challenges for eye surgeons. Despite being described almost 2 decades ago, its pathophysiology remains unclear. Initially, IFIS was thought to be a result of sympathetic signal blockage in the iris dilator muscle, since α-blockers such as tamsulosin were found to be a strong predisposing factor; however, many IFIS cases occur even in patients who discontinued α-blockers prior to cataract surgery. Several potential mechanisms through which α-blockers induces chronic changes in the iris - iris dilator atrophy, drug-melanin interaction, and loss of vascular tone - have been proposed as possible mechanisms. We address the prevailing theories on α-receptor-dependent mechanisms for IFIS and the current prophylactic measures undertaken to prevent IFIS-associated intraocular complications.

  View details for DOI 10.1016/j.survophthal.2025.06.002

  View details for PubMedID 40472999

• Matrine Reduces Intraocular Pressure in Corticosteroid-induced Ocular Hypertensive Mouse Eyes Shen, Y., Zhang, Q., Liu, Z., Chen, S., Wang, Q., Lo, C., Ning, K., Zhao, J., Liton, P. B., Sun, Y. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2025

  View details for Web of Science ID 001559176600034

• Gene therapy for ocular hypertension using hfCas13d-mediated mRNA targeting. PNAS nexus Chen, S., Liu, Z., Lo, C. H., Wang, Q., Ning, K., Zhang, Q., Zhao, J., Shen, Y., Sun, Y. 2025; 4 (6): pgaf168

  Abstract

  Glaucoma is a major global cause of irreversible vision loss. It is marked by elevated intraocular pressure (IOP) and the loss of retinal ganglion cells (RGC). While there are medical and surgical therapies for glaucoma aiming to reduce aqueous humor production or enhance its drainage, these treatments are often inadequate for effectively managing the disease. In this study, we developed a targeted therapy for glaucoma by knocking down two genes associated with aqueous humor production (aquaporin 1 [AQP1] and carbonic anhydrase type 2 [CA2]) using Cas13 RNA editing systems. We demonstrate that hfCas13d-mediated knockdown of AQP1 and CA2 significantly lowers IOP in wild-type mice and in a corticosteroid-induced glaucoma mouse model. We show that the lowered IOP results from decreasing aqueous production without affecting the outflow facility; this treatment also significantly promotes RGC survival as compared with untreated control groups. Therefore, CRISPR-Cas-based gene editing may be an effective treatment to lower IOP for glaucomatous optic neuropathy.

  View details for DOI 10.1093/pnasnexus/pgaf168

  View details for PubMedID 40575705

  View details for PubMedCentralID PMC12199245

• Precise Correction of the <i>Pde6b</i>-L659P Mutation Causing Retinal Degeneration via Base and Prime Editing Liu, Z., Chen, S., Zhao, J., Sun, Y. CELL PRESS. 2025

  View details for Web of Science ID 001521604300026

• Effect of Brimonidine on Retinal Ganglion Cell Function by in vivo Calcium Imaging of Optic Nerve Crush in Mice. Experimental eye research Li, T., Kowal, T. J., Zhao, J., Li, L., Wang, Q., Ning, K., Lo, C. H., Liu, Z., Shen, Y., Yu, J., Jin, H., Sun, Y. 2025: 110355

  Abstract

  Brimonidine has shown neuroprotective effects in animal studies, but clinical trials failed to demonstrate effective endpoints. Here, we used a newly developed in vivo calcium imaging method to measure RGC function of brimonidine in mice optic nerve crush (ONC) models. To transduce RGCs in vivo, wild-type C57Bl/6j mice were treated with intravitreal AAV2-mSncg-jGCaMP7s, a live-cell Ca2+ tracer. RGCs are defined as 10 subtypes according to different responses to UV light. Mice were treated with topical brimonidine or placebo three times daily for two weeks after ONC. The calcium signals of live-cell RGCs were measured with the Heidelberg cSLO system. Ganglion cell complex (GCC) thickness and IOP were examined at different timepoints after treatment. RGCs were counted after RBPMS immunostaining. Live calcium imaging showed ONC significantly decreased RGC number at 14 days post-ONC compared to controls. The topical brimonidine administration changed calcium signal responses of RGC to UV light in ONC mice. It showed brimonidine partly prevented the decrease of survival ON-RGCs percent after ONC. Single RGC analysis showed a lower conversion percent of ON-RGCs to OFF-RGCs with brimonidine administration after ONC. However, no significant differences in RGC survival, IOP or GCC thickness were noted between eyes treated with brimonidine or placebo. In the acute ONC mice model, in vivo calcium imaging revealed that brimonidine maintained the Ca2+ activation of ON-RGCs to UV stimulation, inhibiting the conversion of survival ON-RGCs to OFF-RGCs. This indicates that ON-RGCs may be more resilient to acute optic nerve injury based on the calcium imaging method.

  View details for DOI 10.1016/j.exer.2025.110355

  View details for PubMedID 40127747

• dCasMINI-mediated therapy rescues photoreceptors degeneration in a mouse model of retinitis pigmentosa. Science advances Wang, Q., Xu, X., Chen, S., Lu, R., Li, L., Lo, C. H., Liu, Z., Ning, K., Li, T., Kowal, T. J., Wang, B., Hartnett, M. E., Wang, S., Qi, L. S., Sun, Y. 2024; 10 (51): eadn7540

  Abstract

  Retinitis pigmentosa (RP) is characterized by degeneration of rod and cone photoreceptors that progresses to irreversible blindness. Now, there are no mutation-agnostic approaches to treat RP. Here, we utilized a single adeno-associated virus (AAV)-based CRISPR activation system to activate phosphodiesterase 6B (Pde6b) to mitigate the severe degeneration in Pde6anmf363 mice. We demonstrate that transcriptional activation of Pde6b can rescue the loss of Pde6a, with preservation of retinal structure, restoration of electroretinography responses, and improvement of visual function as assessed by optokinetic response and looming-induced escape behaviors. These findings demonstrate the therapeutic potential of a dCasMINI-mediated activation strategy that provides a mutation-independent treatment for retinal degeneration. This study offers a promising therapeutic approach for RP and potentially other forms of genetic diseases.

  View details for DOI 10.1126/sciadv.adn7540

  View details for PubMedID 39693439

  View details for PubMedCentralID PMC11654696

• All-in-one AAV-mediated Nrl gene inactivation rescues retinal degeneration in Pde6a mice. JCI insight Liu, Z., Chen, S., Lo, C. H., Wang, Q., Sun, Y. 2024

  Abstract

  Retinitis pigmentosa (RP) is a complex group of inherited retinal diseases characterized by progressive death of photoreceptor cells and eventual blindness. Pde6a, which encodes a cGMP-specific phosphodiesterase, is a crucial pathogenic gene for autosomal recessive RP (RP43); there is no effective therapy for this form of RP. The compact CRISPR/SaCas9 system, which can be packaged into a single adeno-associated virus, holds promise for simplifying effective gene therapy. Here, we demonstrated that all-in-one AAV-SaCas9-mediated Nrl gene inactivation can efficiently prevent retinal degeneration in a RP mouse model with Pde6anmf363/nmf363 mutation. We screened single guide RNAs (sgRNAs) capable of efficiently editing mouse Nrl gene in N2a cells and then achieved effective gene editing by using a single AAV to co-deliver SaCas9 and an optimal Nrl-sg2 into the mouse retina. Excitingly, in vivo inactivation of Nrl improved photoreceptor cell survival and rescued retinal function in treated Pde6a deficient mice. Thus, we showed that a practical, gene-independent method, AAV-SaCas9-mediated Nrl inactivation, holds promise for future therapeutic applications in patients with RP.

  View details for DOI 10.1172/jci.insight.178159

  View details for PubMedID 39499900

• Defective Neurogenesis in Lowe Syndrome is Caused by Mitochondria Loss and Cilia-related Sonic Hedgehog Defects. bioRxiv : the preprint server for biology Lo, C. H., Chen, S., Zhao, J., Liu, Z., Wang, B., Wang, Q., Kowal, T. J., Sun, Y. 2024

  Abstract

  Human brain development is a complex process that requires intricate coordination of multiple cellular and developmental events. Dysfunction of lipid metabolism can lead to neurodevelopmental disorders. Lowe syndrome (LS) is a recessive X-linked disorder associated with proximal tubular renal disease, congenital cataracts and glaucoma, and central nervous system developmental delays. Mutations in OCRL, which encodes an inositol polyphosphate 5-phosphatase, lead to the development of LS. The cellular mechanism responsible for neuronal dysfunction in LS is unknown. Here we show depletion of mitochondrial DNA and decrease in mitochondrial activities result in neuronal differentiation defects. Increased astrocytes, which are secondary responders to neurodegeneration, are observed in neuronal (iN) cells differentiated from Lowe patient-derived iPSCs and an LS mouse model. Inactivation of cilia-related sonic hedgehog signaling, which organizes the pattern of cellular neuronal differentiation, is observed in an OCRL knockout, iN cells differentiated from Lowe patient-derived iPSCs, and an LS mouse model. Taken together, our findings indicate that mitochondrial dysfunction and impairment of the ciliary sonic hedgehog signaling pathway represent a novel pathogenic mechanism underlying the disrupted neuronal differentiation observed in LS.

  View details for DOI 10.1101/2024.11.01.621496

  View details for PubMedID 39553960

  View details for PubMedCentralID PMC11565974

• Primary cilia formation requires the Leigh syndrome-associated mitochondrial protein NDUFAF2. The Journal of clinical investigation Lo, C. H., Liu, Z., Chen, S., Lin, F., Berneshawi, A. R., Yu, C. Q., Koo, E. B., Kowal, T. J., Ning, K., Hu, Y., Wang, W. J., Liao, Y. J., Sun, Y. 2024; 134 (13)

  Abstract

  Mitochondria-related neurodegenerative diseases have been implicated in the disruption of primary cilia function. Mutation in an intrinsic mitochondrial complex I component NDUFAF2 has been identified in Leigh syndrome, a severe inherited mitochondriopathy. Mutations in ARMC9, which encodes a basal body protein, cause Joubert syndrome, a ciliopathy with defects in the brain, kidney, and eye. Here, we report a mechanistic link between mitochondria metabolism and primary cilia signaling. We discovered that loss of NDUFAF2 caused both mitochondrial and ciliary defects in vitro and in vivo and identified NDUFAF2 as a binding partner for ARMC9. We also found that NDUFAF2 was both necessary and sufficient for cilia formation and that exogenous expression of NDUFAF2 rescued the ciliary and mitochondrial defects observed in cells from patients with known ARMC9 deficiency. NAD+ supplementation restored mitochondrial and ciliary dysfunction in ARMC9-deficient cells and zebrafish and ameliorated the ocular motility and motor deficits of a patient with ARMC9 deficiency. The present results provide a compelling mechanistic link, supported by evidence from human studies, between primary cilia and mitochondrial signaling. Importantly, our findings have significant implications for the development of therapeutic approaches targeting ciliopathies.

  View details for DOI 10.1172/JCI175560

  View details for PubMedID 38949024

• Gene Therapy for Glaucoma using CRISPR/Cas13d in mice Chen, S., Liu, Z., Lo, C., Sun, Y. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2024

  View details for Web of Science ID 001313316200302

• Employing plasmid-based CRISPR-hyperdCas12, CasRx, and prime editing systems for in vivo gene manipulation in the mouse retina Li, L., Hamed, L., Liu, Z., Deng, B., Sun, Y., Wang, S. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2024

  View details for Web of Science ID 001313316207295

• Efficient prevention of retinal degeneration in <i>Pde6a</i> mice by all-inone AAV-mediated <i>Nrl</i> gene editing Liu, Z., Chen, S., Lo, C., Wang, Q., Sun, Y. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2024

  View details for Web of Science ID 001312227704183

• Base editing correction of OCRL in Lowe syndrome: ABE-mediated functional rescue in patient-derived fibroblasts. Human molecular genetics Chen, S., Lo, C. H., Liu, Z., Wang, Q., Ning, K., Li, T., Sun, Y. 2024

  Abstract

  Lowe syndrome, a rare X-linked multisystem disorder presenting with major abnormalities in the eyes, kidneys, and central nervous system, is caused by mutations in OCRL gene (NG_008638.1). Encoding an inositol polyphosphate 5-phosphatase, OCRL catalyzes the hydrolysis of PI(4,5)P2 into PI4P. There are no effective targeted treatments for Lowe syndrome. Here, we demonstrate a novel gene therapy for Lowe syndrome in patient fibroblasts using an adenine base editor (ABE) that can efficiently correct pathogenic point mutations. We show that ABE8e-NG-based correction of a disease-causing mutation in a Lowe patient-derived fibroblast line containing R844X mutation in OCRL gene, restores OCRL expression at mRNA and protein levels. It also restores cellular abnormalities that are hallmarks of OCRL dysfunction, including defects in ciliogenesis, microtubule anchoring, α-actinin distribution, and F-actin network. The study indicates that ABE-mediated gene therapy is a feasible treatment for Lowe syndrome, laying the foundation for therapeutic application of ABE in the currently incurable disease.

  View details for DOI 10.1093/hmg/ddae045

  View details for PubMedID 38557732

• A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9 BMC BIOLOGY Qian, Y., Wang, D., Niu, W., Zhao, D., Li, J., Liu, Z., Gao, X., Han, Y., Lai, L., Li, Z. 2023; 21 (1): 155

  Abstract

  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.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.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.

  View details for DOI 10.1186/s12915-023-01644-9

  View details for Web of Science ID 001026673800001

  View details for PubMedID 37434184

  View details for PubMedCentralID PMC10337206

• Targeted mutagenesis in rabbit using an engineered BhCas12b variant JOURNAL OF MOLECULAR CELL BIOLOGY Jia, Y., Wang, T., Zhao, D., Liu, Z., Sui, T., Chen, S., Li, J., Lai, L., Li, Z. 2023; 14 (12)

  View details for DOI 10.1093/jmcb/mjac076

  View details for Web of Science ID 000978876000001

  View details for PubMedID 36572401

  View details for PubMedCentralID PMC10129383

• Versatile and efficient genome editing with <i>Neisseria cinerea</i> Cas9 COMMUNICATIONS BIOLOGY Liu, Z., Chen, S., Xie, W., Yu, H., Lai, L., Li, Z. 2022; 5 (1): 1296

  Abstract

  The CRISPR/Cas9 system is a versatile genome editing platform in biotechnology and therapeutics. However, the requirement of protospacer adjacent motifs (PAMs) limits the genome targeting scope. To expand this repertoire, we revisited and engineered a compact Cas9 orthologue derived from Neisseria cinerea (NcCas9) for efficient genome editing in mammal cells. We demonstrated that NcCas9 generates genome editing at target sites with N4GYAT (Y = T/C) PAM which cannot be recognized by existing Cas9s. By optimizing the NcCas9 architecture and its spacer length, editing efficacy of NcCas9 was further improved in human cells. In addition, the NcCas9-derived Base editors can efficiently generate base conversions. Six anti-CRISPR (Acr) proteins were identified as off-switches for NcCas9. Moreover, NcCas9 successfully generated efficient editing of mouse embryos by microinjection of NcCas9 mRNA and the corresponding sgRNA. Thus, the NcCas9 holds the potential to broaden the CRISPR/Cas9 toolsets for efficient gene modifications and therapeutic applications.

  View details for DOI 10.1038/s42003-022-04258-z

  View details for Web of Science ID 000889147500004

  View details for PubMedID 36435853

  View details for PubMedCentralID PMC9701194

• Efficient multinucleotide deletions using deaminase-Cas9 fusions in human cells JOURNAL OF GENETICS AND GENOMICS Chen, S., Liu, Z., Yu, H., Lai, L., Li, Z. 2022; 49 (10): 927-933

  Abstract

  CRISPR/Cas9 system is a robust genome editing platform in biotechnology and medicine. However, it generally produces small insertions/deletions (indels, typically 1-3 bp) but rarely induces larger deletions in specific target sites. Here, we report a cytidine deaminase-Cas9 fusion-induced deletion system (C-DEL) and an adenine deaminase-Cas9 fusion-induced deletion system (A-DEL) by combining Cas9 with rat APOBEC1 (rA1) and TadA 8e, respectively. Both C-DEL and A-DEL improve the efficiency of deletions compared with the conventional Cas9 system in human cells. In addition, the C-DEL system generates a considerable fraction of predictable multinucleotide deletions from 5'-deaminated C bases to the Cas9-cleavage site and increases the proportion of larger deletions at the target loci. Taken together, the C-DEL and A-DEL systems provide a practical strategy for producing efficient multinucleotide deletions, expanding the CRISPR/Cas9 toolsets for gene modifications in human cells.

  View details for DOI 10.1016/j.jgg.2022.03.007

  View details for Web of Science ID 000875748500002

  View details for PubMedID 35421582

• Compact Cje3Cas9 for Efficient <i>In Vivo</i> Genome Editing and Adenine Base Editing CRISPR JOURNAL Chen, S., Liu, Z., Xie, W., Yu, H., Lai, L., Li, Z. 2022; 5 (3): 472-486

  Abstract

  Many therapeutic applications of CRISPR-Cas9 gene editing rely on delivery using the highly versatile adeno-associated virus (AAV) vector. The smallest type II Cas9 ortholog-Cje1Cas9, derived from Campylobacter jejuni with <1,000 amino acids-is particularly attractive for AAV delivery. However, the complex protospacer adjacent motif (PAM) of Cje1Cas9 (N3VRYAC) greatly restricts the density of recognition sequences in human genome. In this study, we identify two compact CjeCas9 orthologs designated as Cje2Cas9 and Cje3Cas9, whose PAM-interacting residues are different from those of the well-known Cje1Cas9. They can induce efficient genome editing in human cells, and their simpler trinucleotide PAM (N4CYA) requirements expand the scope of targeting. Moreover, Cje3Cas9 efficiently disrupts the Tyr gene in mice after being micro-injected into zygotes with the corresponding sgRNA. It also successfully disrupts the Pcsk9 gene in 8-week-old mouse liver after delivery with an sgRNA using an all-in-one AAV delivery vehicle. The gene-edited mice showed lower cholesterol level than wild-type mice. Notably, the 8e-nCje3-ABE and an sgRNA targeting Pcsk9 were successfully packaged into a single AAV vector for genome editing in adult mouse liver, with editing efficiency up to 12%. Thus, simple PAMs and a compact size enable Cje2/3Cas9 to expand the target scope of CRISPR-Cas9 toolsets, exhibiting considerable potential for therapeutic applications.

  View details for DOI 10.1089/crispr.2021.0143

  View details for Web of Science ID 000886239000013

  View details for PubMedID 35686977

• Efficient C-to-G Base Editing with Improved Target Compatibility Using Engineered Deaminase-nCas9 Fusions CRISPR JOURNAL Chen, S., Liu, Z., Lai, L., Li, Z. 2022; 5 (3): 389-396

  Abstract

  CRISPR-guided DNA base editors (BEs) are potent genome editing tools in biotechnology and medicine. However, conventional cytosine and adenine BEs can only induce base transitions (C-to-T and A-to-G) and cannot induce base transversions. Recently, several C-to-G base editors (CGBEs) were generated and applied in human cells. By comparing them, we found that engineered deaminases rather than additional base excision repair proteins significantly improved the C-to-G efficiency. In addition, significant increase in C-to-G transversions in the GC context were determined by using rationally engineered eAID deaminase. The genome-targeting scope of CGBEs were further expanded by using SpRY Cas9 variant, which then successfully induced stop codon (TAC to TAG) to disrupt Tyr gene in mouse embryos. Taken together, these new CGBEs with engineered deaminase-nCas9 fusions broaden the BE toolsets for efficient gene modification and therapeutic applications.

  View details for DOI 10.1089/crispr.2021.0124

  View details for Web of Science ID 000769501000001

  View details for PubMedID 35238619

• Live imaging of RNA and RNA splicing in mammalian cells via the dcas13a-SunTag-BiFC system BIOSENSORS & BIOELECTRONICS Chen, M., Sui, T., Yang, L., Qian, Y., Liu, Z., Liu, Y., Wang, G., Lai, L., Li, Z. 2022; 204: 114074

  Abstract

  Dynamic tracking of the localization of RNA molecules (nucleus and/or cytoplasm) and RNA splicing in living cells plays an important role in understanding their functions. However, a lack of dynamic imaging and high background fluorescence have been reported in the fluorescence in situ hybridization (FISH). Here, we developed a new tool, the dcas13a-SunTag-BiFC system, which fused the dLwacas13a and SunTag systems. dLwacas13a is used as a tracker to target specific RNAs, while SunTag recruits split Venus fluorescent proteins to label targeted RNAs. Our results showed that 4 × NLS-dCas13a-24 × SunTag-BiFC and 2 × NLS- dCas13a-24 × SunTag-BiFC systems can be used for imaging of endogenous RNA foci in the nucleus (Xist) and cytoplasm (Ppib and stress granules) in living cells, respectively. Compared to 12x MS2-MCP system, the dcas13a-SunTag-BiFC system showed a better performance of mRNA foci tracking in live cells. Furthermore, we confirmed the premature termination codon (PTC)-induced exon skipping of Oxt RNA using the dcas13a-SunTag-BiFC and MS2-MCP systems in the nucleus. Thus, the dcas13a-SunTag-BiFC system will facilitate the study of RNA localization in living cells and provide new insights into RNA translocation and splicing.

  View details for DOI 10.1016/j.bios.2022.114074

  View details for Web of Science ID 000782656800002

  View details for PubMedID 35149451

• Reduced off-target effect of NG-BE4max by using NG-HiFi system MOLECULAR THERAPY NUCLEIC ACIDS Shan, H., Liu, Z., Jia, Y., Chen, S., Chen, M., Song, Y., Sui, T., Lai, L., Li, Z. 2021; 25: 168-172

  Abstract

  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.

  View details for DOI 10.1016/j.omtn.2021.05.012

  View details for Web of Science ID 000697924000015

  View details for PubMedID 34458002

  View details for PubMedCentralID PMC8368781

• Efficient and precise generation of Tay-Sachs disease model in rabbit by prime editing system CELL DISCOVERY Qian, Y., Zhao, D., Sui, T., Chen, M., Liu, Z., Liu, H., Zhang, T., Chen, S., Lai, L., Li, Z. 2021; 7 (1): 50

  View details for DOI 10.1038/s41421-021-00276-z

  View details for Web of Science ID 000672648100002

  View details for PubMedID 34230459

  View details for PubMedCentralID PMC8260710

• Efficient and high-fidelity base editor with expanded PAM compatibility for cytidine dinucleotide SCIENCE CHINA-LIFE SCIENCES Liu, Z., Chen, S., Jia, Y., Shan, H., Chen, M., Song, Y., Lai, L., Li, Z. 2021; 64 (8): 1355-1367

  Abstract

  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.

  View details for DOI 10.1007/s11427-020-1775-2

  View details for Web of Science ID 000606386700009

  View details for PubMedID 33420918

  View details for PubMedCentralID 6961573

• Robustly improved base editing efficiency of Cpf1 base editor using optimized cytidine deaminases CELL DISCOVERY Chen, S., Jia, Y., Liu, Z., Shan, H., Chen, M., Yu, H., Lai, L., Li, Z. 2020; 6 (1)

  View details for DOI 10.1038/s41421-020-00195-5

  View details for Web of Science ID 000572159700001

• CRISPR Start-Loss: A Novel and Practical Alternative for Gene Silencing through Base-Editing-Induced Start Codon Mutations MOLECULAR THERAPY-NUCLEIC ACIDS Chen, S., Xie, W., Liu, Z., Shan, H., Chen, M., Song, Y., Yu, H., Lai, L., Li, Z. 2020; 21: 1062-1073

  Abstract

  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).

  View details for DOI 10.1016/j.omtn.2020.07.037

  View details for Web of Science ID 000569481000001

  View details for PubMedID 32854061

  View details for PubMedCentralID PMC7452150

• Large-Fragment Deletions Induced by Cas9 Cleavage while Not in the BEs System MOLECULAR THERAPY-NUCLEIC ACIDS Song, Y., Liu, Z., Zhang, Y., Chen, M., Sui, T., Lai, L., Li, Z. 2020; 21: 523-526

  Abstract

  CRISPR-Cas9 and base editors (BEs) systems are poised to become the gene-editing tool of choice in clinical contexts; however, large-fragment deletion was found in Cas9-mediated mutation cells and mice. In this study, by analyzing 16 gene-edited rabbit lines (including 112 rabbits) generated using SpCas9, BEs, xCas9, and xCas9-BEs with long-range PCR genotyping and long-read sequencing by the PacBio platform, we show the extension of thousands of base fragment deletions in single-guide RNA/Cas9 and xCas9 system mutation rabbits, but no deletions were found in BE-induced mutation rabbits. Thus, we first validated that no large-fragment deletion was induced by the BEs system, suggesting that BE systems can be beneficial tools for the further development of highly accurate and secure gene therapy for the clinical treatment of human genetic disorders.

  View details for DOI 10.1016/j.omtn.2020.06.019

  View details for Web of Science ID 000569480500003

  View details for PubMedID 32711379

  View details for PubMedCentralID PMC7381496

• Precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions BMC BIOLOGY Liu, Z., Chen, S., Shan, H., Jia, Y., Chen, M., Song, Y., Lai, L., Li, Z. 2020; 18 (1): 111

  Abstract

  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.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.These novel base editors with improved precision and CC context-specificity will expand the toolset for precise gene modification in organisms.

  View details for DOI 10.1186/s12915-020-00849-6

  View details for Web of Science ID 000567875300003

  View details for PubMedID 32867757

  View details for PubMedCentralID PMC7461344

• AcrIIA5 Suppresses Base Editors and Reduces Their Off-Target Effects CELLS Liang, M., Sui, T., Liu, Z., Chen, M., Liu, H., Shan, H., Lai, L., Li, Z. 2020; 9 (8)

  Abstract

  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.

  View details for DOI 10.3390/cells9081786

  View details for Web of Science ID 000565632100001

  View details for PubMedID 32727031

  View details for PubMedCentralID PMC7463901

• Efficient base editing by RNA-guided cytidine base editors (CBEs) in pigs CELLULAR AND MOLECULAR LIFE SCIENCES Yuan, H., Yu, T., Wang, L., Yang, L., Zhang, Y., Liu, H., Li, M., Tang, X., Liu, Z., Li, Z., Lu, C., Chen, X., Pang, D., Ouyang, H. 2020; 77 (4): 719-733

  Abstract

  Cytidine base editors (CBEs) have been demonstrated to be useful for precisely inducing C:G-to-T:A base mutations in various organisms. In this study, we showed that the BE4-Gam system induced the targeted C-to-T base conversion in porcine blastocysts at an efficiency of 66.7-71.4% via the injection of a single sgRNA targeting a xeno-antigen-related gene and BE4-Gam mRNA. Furthermore, the efficiency of simultaneous three gene base conversion via the injection of three targeting sgRNAs and BE4-Gam mRNA into porcine parthenogenetic embryos was 18.1%. We also obtained beta-1,4-N-acetyl-galactosaminyl transferase 2, alpha-1,3-galactosyltransferase, and cytidine monophosphate-N-acetylneuraminic acid hydroxylase deficient pig by somatic cell nuclear transfer, which exhibited significantly decreased activity. In addition, a new CBE version (termed AncBE4max) was used to edit genes in blastocysts and porcine fibroblasts (PFFs) for the first time. While this new version demonstrated a three genes base-editing rate of 71.4% at the porcine GGTA1, B4galNT2, and CMAH loci, it increased the frequency of bystander edits, which ranged from 17.8 to 71.4%. In this study, we efficiently and precisely mutated bases in porcine blastocysts and PFFs using CBEs and successfully generated C-to-T and C-to-G mutations in pigs. These results suggest that CBEs provide a more simple and efficient method for improving economic traits, reducing the breeding cycle, and increasing disease tolerance in pigs, thus aiding in the development of human disease models.

  View details for DOI 10.1007/s00018-019-03205-2

  View details for Web of Science ID 000519371800011

  View details for PubMedID 31302752

  View details for PubMedCentralID PMC11105001

• Efficient base editing with high precision in rabbits using YFE-BE4max CELL DEATH & DISEASE Liu, Z., Chen, S., Shan, H., Jia, Y., Chen, M., Song, Y., Lai, L., Li, Z. 2020; 11 (1): 36

  Abstract

  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.

  View details for DOI 10.1038/s41419-020-2244-3

  View details for Web of Science ID 000511445500004

  View details for PubMedID 31959743

  View details for PubMedCentralID PMC6971250

• Highly efficient base editing with expanded targeting scope using SpCas9-NG in rabbits FASEB JOURNAL Liu, Z., Shan, H., Chen, S., Chen, M., Song, Y., Lai, L., Li, Z. 2020; 34 (1): 588-596

  Abstract

  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.

  View details for DOI 10.1096/fj.201901587R

  View details for Web of Science ID 000507308900040

  View details for PubMedID 31914687

• Efficient base editing with expanded targeting scope using an engineered Spy-mac Cas9 variant CELL DISCOVERY Liu, Z., Shan, H., Chen, S., Chen, M., Song, Y., Lai, L., Li, Z. 2019; 5: 58

  View details for DOI 10.1038/s41421-019-0128-4

  View details for Web of Science ID 000502982600001

  View details for PubMedID 31814995

  View details for PubMedCentralID PMC6888851

• Expanded targeting scope and enhanced base editing efficiency in rabbit using optimized xCas9(3.7) CELLULAR AND MOLECULAR LIFE SCIENCES Liu, Z., Chen, M., Shan, H., Chen, S., Xu, Y., Song, Y., Zhang, Q., Yuan, H., Ouyang, H., Li, Z., Lai, L. 2019; 76 (20): 4155-4164

  Abstract

  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.

  View details for DOI 10.1007/s00018-019-03110-8

  View details for Web of Science ID 000495619800014

  View details for PubMedID 31030226

  View details for PubMedCentralID PMC11105381

• Mutations of <i>GADD45G</i> in rabbits cause cleft lip by the disorder of proliferation, apoptosis and epithelial-mesenchymal transition (EMT) BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE Lu, Y., Liang, M., Zhang, Q., Liu, Z., Song, Y., Lai, L., Li, Z. 2019; 1865 (9): 2356-2367

  Abstract

  The cleft lip with or without cleft palate (CL/P) is one of the most common congenital defects in humans. Genome-wide association studies (GWAS) have been widely used for identifying candidate genes, and different genes or chromosomal regions have shown strong evidence for the presence of causal genes in CL/P. To date, two independent GWAS have identified GADD45G as influencing risk for CL/P. However, there is no animal model evidence about GADD45G related to CL/P. Here, we reported the generation of a novel GADD45G mutated rabbit model by CRISPR/Cas9 and CRISPR-based BE4-Gam systems. The homozygous (GADD45G-/-) while not heterozygous (GADD45G+/-) pups died after birth due to severe craniofacial defects of unilateral or bilateral cleft lip (CL). Moreover, the disorder of proliferation, apoptosis and epithelial-mesenchymal transition (EMT) were also determined in the medial and lateral nasal processes (MNP and LNP) of the embryonic day 13 (E13) GADD45G-/- rabbits, which compared with the normal wild type (WT) rabbits. Thus, our study confirmed for the first time that loss of GADD45G lead to CL at the animal level and provided new insights into the crucial role of GADD45G for upper lip formation and fusion.

  View details for DOI 10.1016/j.bbadis.2019.05.015

  View details for Web of Science ID 000476965200024

  View details for PubMedID 31150757

• Improved base editor for efficient editing in GC contexts in rabbits with an optimized AID-Cas9 fusion FASEB JOURNAL Liu, Z., Shan, H., Chen, S., Chen, M., Zhang, Q., Lai, L., Li, Z. 2019; 33 (8): 9210-9219

  Abstract

  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.

  View details for DOI 10.1096/fj.201900476RR

  View details for Web of Science ID 000478832900041

  View details for PubMedID 31071267

• Efficient and precise base editing in rabbits using human APOBEC3A-nCas9 fusions CELL DISCOVERY Liu, Z., Chen, S., Shan, H., Zhang, Q., Chen, M., Lai, L., Li, Z. 2019; 5: 31

  View details for DOI 10.1038/s41421-019-0099-5

  View details for Web of Science ID 000471115800001

  View details for PubMedID 31231541

  View details for PubMedCentralID PMC6557807

• The disrupted balance between hair follicles and sebaceous glands in <i>Hoxc13</i>-ablated rabbits FASEB JOURNAL Deng, J., Chen, M., Liu, Z., Song, Y., Sui, T., Lai, L., Li, Z. 2019; 33 (1): 1226-1234

  Abstract

  Pure hair and nail ectodermal dysplasia 9 (ECTD-9) is an autosomal recessive genetic disease caused by mutation of HOXC13 and is characterized by hypotrichosis and nail dystrophy in humans. Unlike patients with ECTD-9, Hoxc13-mutated mice and pigs do not faithfully recapitulate the phenotype of hypotrichosis, so there is a limited understanding of the molecular mechanism of Hoxc13-mediated hypotrichosis in animal models and clinically. Here, the homozygous Hoxc13-/- rabbits showed complete loss of hair on the head and dorsum, whereas hypotrichosis in the limbs and tail were determined in the Hoxc13-/- rabbits. In addition, reduced hair follicles (HFs) while the enlarged and increased number of sebaceous glands (SGs) were also found in the Hoxc13-/- rabbits, showing that the disrupted balance between HFs and SGs may respond to hypotrichosis of ECTD-9 in an animal model and clinically. Therefore, our findings demonstrate that Hoxc13-/- rabbits can be used as a model for human ECTD-9, especially to understand the pathologic mechanism of hypotrichosis. Moreover, the disrupted balance between HFs and SGs, especially in the Hoxc13-/- rabbits, can be used as an ideal animal model for dermatology ailments, such as acne and hypotrichosis, in preclinical studies.-Deng, J., Chen, M., Liu, Z., Song, Y., Sui, T., Lai, L., Li, Z. The disrupted balance between hair follicles and sebaceous glands in Hoxc13-ablated rabbits.

  View details for DOI 10.1096/fj.201800928RR

  View details for Web of Science ID 000457401500097

  View details for PubMedID 30125135

• CRISPR-induced exon skipping is dependent on premature termination codon mutations GENOME BIOLOGY Sui, T., Song, Y., Liu, Z., Chen, M., Deng, J., Xu, Y., Lai, L., Li, Z. 2018; 19: 164

  Abstract

  In previous studies, CRISPR/Cas9 was shown to induce unexpected exon skipping; however, the mechanism by which this phenomenon is triggered is controversial. By analyzing 22 gene-edited rabbit lines generated using CRISPR/Cas9, we provide evidence of exon skipping at high frequency in premature termination codon-mutated rabbits but not in the rabbits with a premature termination codon mutation in exon 1 rabbits with non-frameshift or missense mutations. Our results suggest that CRISPR-mediated exon skipping depends on premature termination codon mutation-induced nonsense-associated altered splicing.

  View details for DOI 10.1186/s13059-018-1532-z

  View details for Web of Science ID 000447813100002

  View details for PubMedID 30333044

  View details for PubMedCentralID PMC6193291

• CRISPR/Cas9-mediated mutation of tyrosinase (Tyr) 3′ UTR induce graying in rabbit SCIENTIFIC REPORTS Song, Y., Xu, Y., Deng, J., Chen, M., Lu, Y., Wang, Y., Yao, H., Zhou, L., Liu, Z., Lai, L., Li, Z. 2017; 7: 1569

  Abstract

  The 3' untranslated regions (UTRs), located at the end of mRNA molecules, are believed to play a role in RNA replication and/or protein translation. Mutations in the tyrosinase (Tyr) gene are known to cause recessive albinism in humans and other species. In this study, to test whether the CRISPR/Cas9 system works on the mutation of the UTRs regulatory region in rabbit, the 3' UTR of the rabbit Tyr gene was deleted by a dual sgRNA directed CRISPR/Cas9 system. As expected, gray coat color and reduced melanin in hair follicles and irises was found in the mutated rabbit, thus increasing confidence in the association of the mutation of the Tyr 3' UTR with graying in rabbit. The graying phenotype was also found in the F1 generation, suggesting that the mutated allele can be stably inherited by the offspring. Thus, we provide the first evidence that reduced melanin and graying can be caused by deletion of the Tyr 3' UTR in rabbits. Additionally, CRISPR/Cas9-mediated large fragment deletions can facilitate genotype to phenotype studies of UTRs or non-coding RNAs in future.

  View details for DOI 10.1038/s41598-017-01727-y

  View details for Web of Science ID 000400874500009

  View details for PubMedID 28484254

  View details for PubMedCentralID PMC5431497