Self-Recognition of an Inducible Host lncRNA by RIG-I Feedback Restricts Innate Immune Response.

The innate RNA sensor RIG-I is critical in the initiation of antiviral type I interferons (IFNs) production upon recognition of "non-self" viral RNAs. Here, we identify a host-derived, IFN-inducible long noncoding RNA, lnc-Lsm3b, that can compete with viral RNAs in the binding of RIG-I monomers and feedback inactivate the RIG-I innate function at late stage of innate response. Mechanistically, binding of lnc-Lsm3b restricts RIG-I protein's conformational shift and prevents downstream signaling, thereby terminating type I IFNs production. Multivalent structural motifs and long-stem structure are critical features of lnc-Lsm3b for RIG-I binding and inhibition. These data reveal a non-canonical self-recognition mode in the regulation of immune response and demonstrate an important role of an inducible "self" lncRNA acting as a potent molecular decoy actively saturating RIG-I binding sites to restrict the duration of "non-self" RNA-induced innate immune response and maintaining immune homeostasis, with potential utility in inflammatory disease management.

Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression.

Identifying tumor-induced leukocyte subsets and their derived circulating factors has been instrumental in understanding cancer as a systemic disease. Nevertheless, how primary tumor-induced non-leukocyte populations in distal organs contribute to systemic spread remains poorly defined. Here, we report one population of tumor-inducible, erythroblast-like cells (Ter-cells) deriving from megakaryocyte-erythroid progenitor cells with a unique Ter-119+CD45-CD71+ phenotype. Ter-cells are enriched in the enlarged spleen of hosts bearing advanced tumors and facilitate tumor progression by secreting neurotrophic factor artemin into the blood. Transforming growth factor ß (TGF-ß) and Smad3 activation are important in Ter-cell generation. In vivo blockade of Ter-cell-derived artemin inhibits hepatocellular carcinoma (HCC) growth, and artemin deficiency abolishes Ter-cells' tumor-promoting ability. We confirm the presence of splenic artemin-positive Ter-cells in human HCC patients and show that significantly elevated serum artemin correlates with poor prognosis. We propose that Ter-cells and the secreted artemin play important roles in cancer progression with prognostic and therapeutic implications.

Author Correction: Inducible degradation of lncRNA Sros1 promotes IFN-γ-mediated activation of innate immune responses by stabilizing Stat1 mRNA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The long noncoding RNA Lnczc3h7a promotes a TRIM25-mediated RIG-I antiviral innate immune response.

The helicase RIG-I initiates an antiviral immune response after recognition of pathogenic RNA. TRIM25, an E3 ubiquitin ligase, mediates K63-linked ubiquitination of RIG-I, which is crucial for RIG-I downstream signaling and the antiviral innate immune response. The components and mode of the RIG-I-initiated innate signaling remain to be fully understood. Here we identify a novel long noncoding RNA (Lnczc3h7a) that binds to TRIM25 and promotes RIG-I-mediated antiviral innate immune responses. Depletion of Lnczc3h7a impairs RIG-I signaling and the antiviral innate response to RNA viruses in vitro and in vivo. Mechanistically, Lnczc3h7a binds to both TRIM25 and activated RIG-I, serving as a molecular scaffold for stabilization of the RIG-I-TRIM25 complex at the early stage of viral infection. Lnczc3h7a facilitates TRIM25-mediated K63-linked ubiquitination of RIG-I and thus promotes downstream signaling transduction. Our findings reveal that host RNAs can enhance the response of innate immune sensors to foreign RNAs, ensuring effective antiviral defense.

Inducible degradation of lncRNA Sros1 promotes IFN-γ-mediated activation of innate immune responses by stabilizing Stat1 mRNA.

Interferon-γ (IFN-γ) is essential for the innate immune response to intracellular bacteria. Noncoding RNAs and RNA-binding proteins (RBPs) need to be further considered in studies of regulation of the IFN-γ-activated signaling pathway in macrophages. In the present study, we found that the microRNA miR-1 promoted IFN-γ-mediated clearance of Listeria monocytogenes in macrophages by indirectly stabilizing the Stat1 messenger RNA through the degradation of the cytoplasmic long noncoding RNA Sros1. Inducible degradation or genetic loss of Sros1 led to enhanced IFN-γ-dependent activation of the innate immune response. Mechanistically, Sros1 blocked the binding of Stat1 mRNA to the RBP CAPRIN1, which stabilized the Stat1 mRNA and, consequently, promoted IFN-γ-STAT1-mediated innate immunity. These observations shed light on the complex RNA-RNA regulatory networks involved in cytokine-initiated innate responses in host-pathogen interactions.

Germinal-center development of memory B cells driven by IL-9 from follicular helper T cells.

Germinal centers (GCs) support high-affinity, long-lived humoral immunity. How memory B cells develop in GCs is not clear. Through the use of a cell-cycle-reporting system, we identified GC-derived memory precursor cells (GC-MP cells) that had quit cycling and reached G0 phase while in the GC, exhibited memory-associated phenotypes with signs of affinity maturation and localized toward the GC border. After being transferred into adoptive hosts, GC-MP cells reconstituted a secondary response like genuine memory B cells. GC-MP cells expressed the interleukin 9 (IL-9) receptor and responded to IL-9. Acute treatment with IL-9 or antibody to IL-9 accelerated or retarded the positioning of GC-MP cells toward the GC edge and exit from the GC, and enhanced or inhibited the development of memory B cells, which required B cell-intrinsic responsiveness to IL-9. Follicular helper T cells (TFH cells) produced IL-9, and deletion of IL-9 from T cells or, more specifically, from GC TFH cells led to impaired memory formation of B cells. Therefore, the GC development of memory B cells is promoted by TFH cell-derived IL-9.

Mitochondrial tRNA pseudouridylation governs erythropoiesis.

ABSTRACT: Pseudouridine is the most prevalent RNA modification, and its aberrant function is implicated in various human diseases. However, the specific impact of pseudouridylation on hematopoiesis remains poorly understood. Here, we investigated the role of transfer RNA (tRNA) pseudouridylation in erythropoiesis and its association with mitochondrial myopathy, lactic acidosis, and sideroblastic anemia syndrome (MLASA) pathogenesis. By using patient-specific induced pluripotent stem cells (iPSCs) carrying a genetic pseudouridine synthase 1 (PUS1) mutation and a corresponding mutant mouse model, we demonstrated impaired erythropoiesis in MLASA-iPSCs and anemia in the MLASA mouse model. Both MLASA-iPSCs and mouse erythroblasts exhibited compromised mitochondrial function and impaired protein synthesis. Mechanistically, we revealed that PUS1 deficiency resulted in reduced mitochondrial tRNA levels because of pseudouridylation loss, leading to aberrant mitochondrial translation. Screening of mitochondrial supplements aimed at enhancing respiration or heme synthesis showed limited effect in promoting erythroid differentiation. Interestingly, the mammalian target of rapamycin (mTOR) inhibitor rapamycin facilitated erythroid differentiation in MLASA-iPSCs by suppressing mTOR signaling and protein synthesis, and consistent results were observed in the MLASA mouse model. Importantly, rapamycin treatment partially ameliorated anemia phenotypes in a patient with MLASA. Our findings provide novel insights into the crucial role of mitochondrial tRNA pseudouridylation in governing erythropoiesis and present potential therapeutic strategies for patients with anemia facing challenges related to protein translation.

The Self-Specific Activation Receptor SLAM Family Is Critical for NK Cell Education.

NK cell education, a term describing a process for NK cell acquisition of functional competence, is primarily achieved by self-MHC-I-specific inhibitory receptors. In this study, we have demonstrated that SLAM family receptors (SFRs) redundantly expressed on hematopoietic cells function as self-specific activation receptors critical for NK cell education. To overcome gene redundancy, we generated mice simultaneously lacking seven SFRs, revealing that NK-cell-mediated rejection of semi-allogeneic hematopoietic cells largely depended on the presence of SFRs on target cells. This stimulatory effect was determined by the presence of SFR-coupled adaptors; however, SFR-deficient mice displayed enhanced reactivity to hematopoietic cells. These findings demonstrate that SFRs endow NK cells with an ability to kill hematopoietic cells during the effector phase; however, the sustained engagement of SFRs can desensitize NK cell responses during an education process. Therefore, self-specific activating ligands may be "tolerogens" for NK cells, akin to self-antigens that induce T cell tolerance.

Trim46 knockout impaired neuronal architecture and caused hypoactive behavior in rats.

BACKGROUND: Tripartite motif (TRIM46) is a relatively novel protein that belongs to tripartite motif family. TRIM46 organizes parallel microtubule arrays on the axons, which are important for neuronal polarity and axonal function. TRIM46 is highly expressed in the brain, but its biological function in adults has not yet been determined. RESULTS: Trim46 knockout (KO) rat line was established using CRISPR/cas9. Trim46 KO rats had smaller hippocampus sizes, fewer neuronal dendritic arbors and dendritic spines, and shorter and more distant axon initial segment. Furthermore, the protein interaction between endogenous TRIM46 and FK506 binding protein 5 (FKBP5) in brain tissues was determined; Trim46 KO increased hippocampal FKBP5 protein levels and decreased hippocampal protein kinase B (Akt) phosphorylation, gamma-aminobutyric acid type A receptor subunit alpha1 (GABRA1) and glutamate ionotropic receptor NMDA type subunit 1 (NMDAR1) protein levels. Trim46 KO rats exhibited hypoactive behavioral changes such as reduced spontaneous activity, social interaction, sucrose preference, impaired prepulse inhibition (PPI), and short-term reference memory. CONCLUSIONS: These results demonstrate the significant impact of Trim46 KO on brain structure and behavioral function. This study revealed a novel potential association of TRIM46 with dendritic development and neuropsychiatric behavior, providing new insights into the role of TRIM46 in the brain.

Cis-acting lnc-Cxcl2 restrains neutrophil-mediated lung inflammation by inhibiting epithelial cell CXCL2 expression in virus infection.

Chemokine production by epithelial cells is important for neutrophil recruitment during viral infection, the appropriate regulation of which is critical for restraining inflammation and attenuating subsequent tissue damage. Epithelial cell expression of long noncoding RNAs (lncRNAs), RNA-binding proteins, and their functional interactions during viral infection and inflammation remain to be fully understood. Here, we identified an inducible lncRNA in the Cxcl2 gene locus, lnc-Cxcl2, which could selectively inhibit Cxcl2 expression in mouse lung epithelial cells but not in macrophages. lnc-Cxcl2-deficient mice exhibited increased Cxcl2 expression, enhanced neutrophils recruitment, and more severe inflammation in the lung after influenza virus infection. Mechanistically, nucleus-localized lnc-Cxcl2 bound to Cxcl2 promoter, recruited a ribonucleoprotein La, which inhibited the chromatin accessibility of chemokine promoters, and consequently inhibited Cxcl2 transcription in cis However, unlike mouse lnc-Cxcl2, human lnc-CXCL2-4-1 inhibited multiple immune cytokine expressions including chemokines in human lung epithelial cells. Together, our results demonstrate a self-protecting mechanism within epithelial cells to restrain chemokine and neutrophil-mediated inflammation, providing clues for better understanding chemokine regulation and epithelial cell function in lung viral infection.

LncRNA Malat1 inhibition of TDP43 cleavage suppresses IRF3-initiated antiviral innate immunity.

Long noncoding RNAs (lncRNAs) involved in the regulation of antiviral innate immune responses need to be further identified. By functionally screening the lncRNAs in macrophages, here we identified lncRNA Malat1, abundant in the nucleus but significantly down-regulated after viral infection, as a negative regulator of antiviral type I IFN (IFN-I) production. Malat1 directly bound to the transactive response DNA-binding protein (TDP43) in the nucleus and prevented activation of TDP43 by blocking the activated caspase-3-mediated TDP43 cleavage to TDP35. The cleaved TDP35 increased the nuclear IRF3 protein level by binding and degrading Rbck1 pre-mRNA to prevent IRF3 proteasomal degradation upon viral infection, thus selectively promoting antiviral IFN-I production. Deficiency of Malat1 enhanced antiviral innate responses in vivo, accompanying the increased IFN-I production and reduced viral burden. Importantly, the reduced MALAT1, augmented IRF3, and increased IFNA mRNA were found in peripheral blood mononuclear cells (PBMCs) from systemic lupus erythematosus (SLE) patients. Therefore, the down-regulation of MALAT1 in virus-infected cells or in human cells from autoimmune diseases will increase host resistance against viral infection or lead to autoinflammatory interferonopathies via the increased type I IFN production. Our results demonstrate that the nuclear Malat1 suppresses antiviral innate responses by targeting TDP43 activation via RNA-RBP interactive network, adding insight to the molecular regulation of innate responses and autoimmune pathogenesis.

Uncoupling protein 1 knockout aggravates isoproterenol-induced acute myocardial ischemia via AMPK/mTOR/PPARα pathways in rats.

Uncoupling protein 1 (UCP1) was found exclusively in the inner membranes of the mitochondria of brown adipose tissue (BAT). We found that UCP1 was also expressed in heart tissue and significantly upregulated in isoproterenol (ISO)-induced acute myocardial ischemia (AMI) rat model. The present study is to determine the underlying mechanism involved in the UCP1 upregulation in ISO-induced AMI rat model. The Ucp1-/- rats were generated by CRISPR-Cas9 system and presented decreased BAT volume. 2-months old Sprague Dawley (SD) wild-type (WT) and Ucp1-/- rats were treated with ISO intraperitoneally 30 mg/kg once a day for 3 consecutive days to establish AMI model. In saline group, the echocardiographic parameters, serum markers of myocardial injury cardiac troponin I (cTnI), creatine kinase isoenzyme MB (CK-MB), oxidant malondialdehyde (MDA), antioxidant superoxide dismutase (SOD) or fibrosis were comparable between WT and Ucp1-/- rats. ISO treatment induced worse left ventricle (LV) hypertrophy, myocardial fibrosis, increased higher cTnI, CK-MB and MDA and decreased lower SOD level in Ucp1-/- rats compared with that of WT rats. Ucp1-/- rats also presented lower myocardial phosphocreatine (PCr)/ATP-ratio, which demonstrated worse cardiac energy regulation defect. ISO treatment induced the phosphorylation of AMP-activated protein kinase (AMPK) activation, subsequently the phosphorylation of mammalian target of rapamycin (mTOR) inhibition and peroxisome proliferators-activated receptor α (PPARα) activation in WT rats, whereas activation of AMPK/mTOR/PPARα pathways significantly inhibited in Ucp1-/- rats. To sum up, UCP1 knockout aggravated ISO-induced AMI by inhibiting AMPK/mTOR/PPARα pathways in rats. Increasing UCP1 expression in heart tissue may be a cytoprotective therapeutic strategy for AMI.

RNA-binding protein YTHDF3 suppresses interferon-dependent antiviral responses by promoting FOXO3 translation.

IFN-stimulated genes (ISGs) are essential effectors of the IFN-dependent antiviral immune response. Dysregulation of ISG expression can cause dysfunctional antiviral responses and autoimmune disorders. Epitranscriptomic regulation, such as N6-methyladenosine (m6A) modification of mRNAs, plays key roles in diverse biological processes. Here, we found that the m6A "reader" YT521-B homology domain-containing family 3 (YTHDF3) suppresses ISG expression under basal conditions by promoting translation of the transcription corepressor forkhead box protein O3 (FOXO3). YTHDF3 cooperates with two cofactors, PABP1 and eIF4G2, to promote FOXO3 translation by binding to the translation initiation region of FOXO3 mRNA. Both the YTH and the P/Q/N-rich domains of YTHDF3 were required for FOXO3 RNA-binding capacity, however, METTL3-mediated m6A modification was not involved in the process observed. Moreover, YTHDF3-/- mice had increased ISG levels and were resistant to several viral infections. Our findings uncover the role of YTHDF3 as a negative regulator of antiviral immunity through the translational promotion of FOXO3 mRNA under homeostatic conditions, adding insight into the networks of RNA-binding protein-RNA interactions in homeostatically maintaining host antiviral immune function and preventing inflammatory response.

Myocardial tissue-specific Dnmt1 knockout in rats protects against pathological injury induced by Adriamycin.

Novel molecular mechanisms of the pathophysiology of heart failure (HF) are continuously being discovered, including epigenetic regulation. Among epigenetic marks, the role of DNA hypomethylation in shaping heart morphology and function in vivo and the pathogenesis of cardiomyopathy and/or HF, especially in adults, has not been clearly established. Here we show that the strong expression of DNA methyltransferase 1 (Dnmt1) is obviously downregulated in the WT adult rat heart with age. By contrast, the expression of Dnmt1 is upregulated suddenly in heart tissues from pressure overload-induced HF mice and adriamycin-induced cardiac injury and HF mice, consistent with the increased expression of Dnmt1 observed in familial hypertrophic cardiomyopathy (FHCM) patients. To further assess the role of Dnmt1, we generated myocardium-specific Dnmt1 knockout (Dnmt1 KO) rats using CRISPR-Cas9 technology. Echocardiographic and histopathological examinations demonstrated that Dnmt1 deficiency is associated with resistance to cardiac pathological changes and protection at the global and organization levels in response to pathological stress. Furthermore, Dnmt1 deficiency in the myocardium restricts the expressional reprogramming of genes and activates pathways involved in myocardial protection and anti-apoptosis in response to pathological stress. Transcriptome and genome-wide DNA methylation analyses revealed that these changes in regulation are linked to alterations in the methylation status of genes due to Dnmt1 knockout. The present study is the first to investigate in vivo the impact of genome-wide cardiac DNA methyltransferase deficiency on physiological development and the pathological processes of heart tissues in response to stress. The exploration of the role of epigenetics in the development, modification, and prevention of cardiomyopathy and HF is in a very preliminary stage but has an infinite future.

Nuclear carbonic anhydrase 6B associates with PRMT5 to epigenetically promote IL-12 expression in innate response.

Interleukin-12 (IL-12) is critical for induction of protective immunity against intracellular bacterial infection. However, the mechanisms for efficient induction of IL-12 in innate response remain poorly understood. Here we report that the B type of carbonic anhydrase 6 (Car6-b, which encoded CA-VI B) is essential for host defense against Listeria monocytogenes (LM) infection by epigenetically promoting IL-12 expression independent of its carbonic anhydrase activity. Deficiency of Car6-b attenuated IL-12 production upon LM infection both in vitro and in vivo. Car6-/- mice were more susceptible to LM infection with less production of IL-12. Mechanistically, the nuclear localized CA-VI B selectively promotes IL-12 expression by interaction with protein arginine N-methyltransferase 5 (PRMT5), which reduces symmetric dimethylation of histone H3 arginine 8 modification (H3R8me2s) at Il12 promoters to facilitate chromatin accessibility, selectively enhancing c-Rel binding to the Il12b promoter. Our findings add insights to the epigenetic regulation of IL-12 induction in innate immunity.

The role of Lys2-Cl- -Lys2 salt linkages in oligomeric intermediates of RbsD protein in Escherichia coli.

As a homo-oligomeric protein, the disassembly of Escherichia coli RbsD decamer produces a urea-unfolded oligomeric intermediate structure, as the dissociation speed of the protein is lower than that of the unfolding process. There are five Lys2-Cl- -Lys2 salt linkages to connect these subunits. To explore the role of the salt linkages in these oligomeric intermediates, the Lys2Ala mutated in the N-terminal of E. coli RbsD protein subunit was designed. It was found that the RbsD mutation protein (RbsD:K2A) loses its minor larger oligomers, which exist in RbsD, and displays other several oligomeric states (less than decamers), meanwhile the state of the oligomers depends on the protein concentration. It was also found that compared with RbsD, the crosslinking capability of the subunits of RbsD:K2A is weaker, while the crosslinking rate of dimers is higher, RbsD:K2A needs to substantially adjust its conformation to meet the space requirements when combined with d-ribose. On the basis of these results, we suggest that Lys2-Cl- -Lys2 salt linkages in E. coli RbsD protein play an important role in stabilizing the intermediate products of oligomers and maintaining interaction between the intermediate products of oligomers, which may shed light on the study of these oligomeric proteins.

Hepatitis B Virus-Upregulated LNC-HUR1 Promotes Cell Proliferation and Tumorigenesis by Blocking p53 Activity.

Recent studies have indicated that a number of long noncoding RNAs (lncRNAs) are dysregulated in hepatocellular carcinoma, while their aberrant expressions are associated with tumorigenesis and poor prognosis. To identify hepatitis B virus (HBV)-related lncRNAs, we used RNA deep sequencing to quantify the abundances of lncRNAs in HepG2 cells and HBV transgenic HepG2-4D14 cells. Here, we demonstrate that lnc-HUR1 is significantly upregulated in HepG2-4D14 cells. We found that HBV-encoded hepatitis B x protein can enhance the transcription of lnc-HUR1. Overexpression of lnc-HUR1 promotes cell proliferation, whereas knockdown of lnc-HUR1 inhibits cell growth. We identified that lnc-HUR1 can interact with p53 and inhibit its transcriptional regulation on downstream genes, such as p21 and B cell lymphoma 2-associated X protein. We generated lnc-HUR1 transgenic mice and performed the partial hepatectomy (PHx) to examine liver regeneration. The data showed that the ratio of liver weight to body weight in lnc-HUR1 transgenic mice is higher than that in wild-type (WT) littermates at day 2 and day 3 following hepatectomy. Consistently, the results of bromodeoxyuridine staining on liver sections following hepatectomy indicate that the ratio of bromodeoxyuridine-positive cells in lnc-HUR1 transgenic mice is significantly higher than that in WT mice, suggesting that lnc-HUR1 promotes cell proliferation during liver regeneration. Next, we performed the experiment of diethylnitrosamine-induced tumorigenesis. The data demonstrate that tumor number in lnc-HUR1 transgenic mice is higher compared with control mice, indicating that lnc-HUR1 enhances diethylnitrosamine-induced tumorigenesis. Conclusion: We reveal that HBV-upregulated lnc-HUR1 promotes cell proliferation and tumorigenesis by interacting with p53 to block downstream gene transcription. Our findings suggest that lnc-HUR1 plays an important role in HBV-related hepatocellular carcinoma development and may serve as a therapeutic marker for hepatocellular carcinoma. (Hepatology 2018; 00:000-000).

RNF122 suppresses antiviral type I interferon production by targeting RIG-I CARDs to mediate RIG-I degradation.

The activation of retinoic acid-inducible gene 1 (RIG-I), a cytoplasmic innate sensor for viral RNA, is tightly regulated to maintain immune homeostasis properly and prevent excessive inflammatory reactions other than initiation of antiviral innate response to eliminate RNA virus effectively. Posttranslational modifications, particularly ubiquitination, are crucial for regulation of RIG-I activity. Increasing evidence suggests that E3 ligases play important roles in various cellular processes, including cell proliferation and antiviral innate signaling. Here we identify that E3 ubiquitin ligase RING finger protein 122 (RNF122) directly interacts with mouse RIG-I through MS screening of RIG-I-interacting proteins in RNA virus-infected cells. The transmembrane domain of RNF122 associates with the caspase activation and recruitment domains (CARDs) of RIG-I; this interaction effectively triggers RING finger domain of RNF122 to deliver the Lys-48-linked ubiquitin to the Lys115 and Lys146 residues of RIG-I CARDs and promotes RIG-I degradation, resulting in a marked inhibition of RIG-I downstream signaling. RNF122 is widely expressed in various immune cells, with preferential expression in macrophages. Deficiency of RNF122 selectively increases RIG-I-triggered production of type I IFNs and proinflammatory cytokines in macrophages. RNF122-deficient mice exhibit more resistance against lethal RNA virus infection, with increased production of type I IFNs. Thus, we demonstrate that RNF122 acts as a selective negative regulator of RIG-I-triggered antiviral innate response by targeting CARDs of RIG-I and mediating proteasomal degradation of RIG-I. Our study outlines a way for E3 ligase to regulate innate sensor RIG-I for the control of antiviral innate immunity.

Increasing the efficiency of CRISPR/Cas9-mediated precise genome editing in rats by inhibiting NHEJ and using Cas9 protein.

Precise modifications such as site mutation, codon replacement, insertion or precise targeted deletion are needed for studies of accurate gene function. The CRISPR/Cas9 system has been proved as a powerful tool to generate gene knockout and knockin animals. But the homologous recombination (HR)-directed precise genetic modification mediated by CRISPR/Cas9 is relatively lower compared with nonhomologous end-joining (NHEJ) pathway and extremely expected to be improved. Here, in this study 2 strategies were used to increase the precise genetic modification in rats. Scr7, a DNA ligase IV inhibitor, first identified as an anti-cancer compound, and considered as a potential NHEJ inhibitor, was used to increase the HR-mediated precise genetic modification. Meanwhile, the Cas9 protein instead of mRNA was used to save the mRNA to protein translation step to improve the precise modification efficiency. The Fabp2 and Dbndd1 loci were selected to knockin Cre and CreER(T2), respectively. Our result showed that both Scr7 and Cas9 protein can increase the precise modification.