Cpf1 nucleases were recently reported to be highly specific and programmable nucleases with efficiencies comparable to those of SpCas9. AsCpf1 and LbCpf1 require a single crRNA and recognize a 5′-TTTN-3′ protospacer adjacent motif (PAM) at the 5′ end of the protospacer for genome editing. For widespread application in precision site-specific human genome editing, the range of sequences that AsCpf1 and LbCpf1 can recognize is limited due to the size of this PAM. To address this limitation, we sought to identify a novel Cpf1 nuclease with simpler PAM requirements. Specifically, here we sought to test and engineer FnCpf1, one reported Cpf1 nuclease (FnCpf1) only requires 5′-TTN-3′ as a PAM but does not exhibit detectable levels of nuclease-induced indels at certain locus in human cells. Surprisingly, we found that FnCpf1 possesses DNA cleavage activity in human cells at multiple loci. We also comprehensively and quantitatively examined various FnCpf1 parameters in human cells, including spacer sequence, direct repeat sequence and the PAM sequence. Our study identifies FnCpf1 as a new member of the Cpf1 family for human genome editing with distinctive characteristics, which shows promise as a genome editing tool with the potential for both research and therapeutic applications.

Cre-loxP, as one of the site-specific genetic manipulation tools, offers a method to study the spatial and temporal regulation of gene expression/inactivation in order to decipher gene function. CRISPR/Cas9-mediated targeted genome engineering technologies are sparking a new revolution in biological research. Whether the traditional site-specific genetic manipulation tool and CRISPR/Cas9 could be combined to create a novel genetic tool for highly specific gene editing is not clear. Here, we successfully generated a CRISPR/Cas9-loxP system to perform gene editing in human cells, providing the proof of principle that these two technologies can be used together for the first time. We also showed that distinct non-homologous end-joining (NHEJ) patterns from CRISPR/Cas9-mediated gene editing of the targeting sequence locates at the level of plasmids (episomal) and chromosomes. Specially, the CRISPR/Cas9-mediated NHEJ pattern in the nuclear genome favors deletions (64%–68% at the human AAVS1 locus versus 4%–28% plasmid DNA). CRISPR/Cas9-loxP, a novel site-specific genetic manipulation tool, offers a platform for the dissection of gene function and molecular insights into DNA-repair pathways.

Cpf1 has been harnessed as a tool for genome manipulation in various species because of its simplicity and high efficiency. Our recent study demonstrated that FnCpf1 could be utilized for human genome editing with notable advantages for target sequence selection due to the flexibility of the protospacer adjacent motif (PAM) sequence. Multiplex genome editing provides a powerful tool for targeting members of multigene families, dissecting gene networks, modeling multigenic disorders in vivo, and applying gene therapy. However, there are no reports at present that show FnCpf1-mediated multiplex genome editing via a single customized CRISPR RNA (crRNA) array. In the present study, we utilize a single customized crRNA array to simultaneously target multiple genes in human cells. In addition, we also demonstrate that a single customized crRNA array to target multiple sites in one gene could be achieved. Collectively, FnCpf1, a powerful genome-editing tool for multiple genomic targets, can be harnessed for effective manipulation of the human genome.

FnCpf1-mediated genome-editing technologies have enabled a broad range of research and medical applications. Recently, we reported that FnCpf1 possesses activity in human cells and recognizes a more compatible PAM (protospacer adjacent motif, 5 0-KYTV-3 0), compared with the other two commonly used Cpf1 enzymes (AsCpf1 and LbCpf1), which requires a 5 0-TTTN-3 0 PAM. However, due to the efficiency and fidelity, FnCpf1-based clinical and basic applications remain a challenge. The direct repeat (DR) sequence is one of the key elements for FnCpf1-mediated genome editing. In principle, its engineering should influence the corresponding genomeediting activity and fidelity. Here we showed that the DR mutants [G(À9)A and U(À7)A] could modulate FnCpf1 performance in human cells, enabling enhancement of both genome-editing efficiency and fidelity. These newly identified features will facilitate the design and optimization of CRISPR-Cpf1-based genome-editing strategies.

PurposeTo evaluate the long-term outcomes of ciliary sulcus versus capsular bag fixation of intraocular lenses (IOLs) in children after pediatric cataract surgery.MethodsIOL was implanted in the ciliary sulcus in 21 eyes of 14 children, and in the capsular bag in 19 eyes of 12 children for the treatment of pediatric cataract in an institutional setting. Ultrasound biomicroscopy (UBM) was performed. Main outcome measures included IOL decentration, IOL tilt, anterior chamber depth (ACD), angle-opening distance at 500 μm (AOD500), trabecular-iris angle (TIA), best-corrected visual acuity (BCVA), intraocular pressure (IOP), and incidence of postoperative complications.ResultsThe mean follow-up period was 6.81 ± 1.82 years. Comparing to the capsular bag fixation group, the ciliary sulcus fixation group had higher vertical IOL decentration, horizontal IOL tilt, and vertical IOL tilt (p = 0.02, 0.01,0.01, respectively), higher incidence of iris-IOL contact and peripheral anterior synechia (p = 0.001, 0.03, respectively), smaller ACD, AOD500, and TIA (p = 0.02, 0.03, 0.04, respectively), higher mean IOP (17.10 ±6.06 mmHg vs.14.15± 4.74 mmHg, p = 0.01), and higher incidence of secondary glaucoma (28.57% vs. 10.53%, p = 0.007).There was no significant difference between the two groups with regard to the BCVA, refractive errors, incidence of myopic shift, nystagmus, strabismus, and visual axis opacity.ConclusionsCiliary sulcus fixation of IOLs in pediatric eyes may increase IOL malposition and crowding of the anterior segment, and may associate with a higher risk of secondary glaucoma compared to capsular bag fixation of IOLs.

X-linked juvenile retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding retinoschisin (RS1), which leads to a significant proportion of visual impairment and blindness. To develop personalized genome editing based gene therapy, knock-in animal disease models that have the exact mutation identified in the patients is extremely crucial, and that the way which genome editing in knock-in animals could be easily transferred to the patients. Here we recruited a family diagnosed with XLRS and identified the causative mutation (RS1, p.Y65X), then a knock-in mouse model harboring this disease-causative mutation was generated via TALEN (transcription activator-like effector nucleases). We found that the b-wave amplitude of the ERG of the RS1-KI mice was significantly decreased. Moreover, we observed that the structure of retina in RS1-KI mice has become disordered, including the disarray of inner nuclear layer and outer nuclear layer, chaos of outer plexiform layer, decreased inner segments of photoreceptor and the loss of outer segments. The novel knock-in mice (RS1-KI) harboring patient-specific mutation will be valuable for development of treatment via genome editing mediated gene correction.

Purpose Only a few studies have reported the role of FOXS1, a transcriptional factor, in the tumor development process. In this article, we investigate the function of FOXS1 in distinct neoplastic development and the tumor immune microenvironment (TIME). Patients and Methods The latent roles of FOXS1 in various tumors were prospected based on TCGA, GTEx, CCLE, GEPIA2, cBioPortal, TIMER, ImmuCellAI databases, GSVA datasets, GSEA datasets, and R packages. The expression difference, gene alteration, clinical characteristics, prognostic values, biological mechanism, potential pathways, tumor microenvironment, and immune cell infiltration related to FOXS1 were appraised. Results FOXS1 was strongly expressed in pan-cancer, and this gene was associated with low survival rates. FOXS1 was linked to many pathways that are cancer-promoting and immune-related. The expression of this transcriptional factor in cancers was positively related to immune cell infiltration, especially M2-like macrophages and Treg cells. In addition to that, FOXS1 demonstrated a positive relationship with many immune-suppression genes, such as TGFB1 and ARORA2A . Conclusion Our study identified an oncogenic effect of FOXS1, which may play a vital role as a prognosticative biological marker in pan-cancer. Exorbitant expression of FOXS1 is associated with high TAMs and Treg cells infiltration. These cells have an immunosuppressive function and promote the development of the immunosuppressive tumor microenvironment. The research of FOXS1 provided a potential drug target for tumor immunotherapy.

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