Effect of Physical Exercise-Based Rehabilitation on Long COVID: A Systematic Review and Meta-analysis.

PURPOSE: The number of persons living with post-coronavirus disease 2019 (COVID-19) conditions or long COVID continues to rise worldwide; however, the etiology and the treatment of long COVID remain nebulous. Therefore, efficient, feasible, and cost-effective therapeutic strategies for a large population with long COVID remain warranted. Physical exercise-based rehabilitation is a promising strategy for long COVID, although its therapeutic effects remain to be determined. This systematic review and meta-analysis aimed to examine the effects of physical exercise-based rehabilitation on long COVID. METHODS: The electronic databases Medline, Embase, Global Health (Ovid), CINAHL (EBSCO), Web of Science, WHO Global Research Database on COVID-19, LitCovid, and Google Scholar were searched from their inception to November 2022. The identified articles were independently screened by three reviewers, and a random-effects model was used to determine the mean differences in the meta-analysis. RESULTS: Twenty-three studies involving 1579 individuals who had COVID-19 (752 women) were included. Physical exercise-based rehabilitation showed beneficial effects on long COVID-related symptoms characterized by dyspnea, fatigue, and depression, as well as on the 6-min walk test, forced expiratory volume in 1 s/forced vital capacity, and quality of life in people who had COVID-19. CONCLUSIONS: Physical exercise-based rehabilitation is a potential therapeutic strategy against long COVID and can be applied as a routine clinical practice in people who have recovered from COVID-19. However, customized physical exercise-based rehabilitation programs and their effects on specific types of long COVID require future large-scale studies.

Distinct roles of core autophagy-related genes in zebrafish definitive hematopoiesis.

Despite the well-described discrepancy between ATG (macroautophagy/autophagy-related) genes in the regulation of hematopoiesis, varying essentiality of core ATG proteins in vertebrate definitive hematopoiesis remains largely unclear. Here, we employed zebrafish (Danio rerio) to compare the functions of six core atg genes, including atg13, becn1 (beclin1), atg9a, atg2a, atg5, and atg3, in vertebrate definitive hematopoiesis via clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoprotein and morpholino targeting. Zebrafish with various atg mutations showed autophagic deficiency and presented partially consistent hematopoietic abnormalities during early development. All six atg mutations led to a declined number of spi1b+ (Spi-1 proto-oncogene b) myeloid progenitor cells. However, only becn1 mutation resulted in the expansion of myb+ (v-myb avian myeloblastosis viral oncogene homolog) hematopoietic stem and progenitor cells (HSPCs) and transiently increased coro1a+ (coronin, actin binding protein, 1A) leukocytes, whereas atg3 mutation decreased the number of HSPCs and leukocytes. Proteomic analysis of caudal hematopoietic tissue identified sin3aa (SIN3 transcription regulator family member Aa) as a potential modulator of atg13- and becn1-regulated definitive hematopoiesis. Disruption of sin3aa rescued the expansion of HSPCs and leukocytes in becn1 mutants and exacerbated the decrease of HSPCs in atg13 mutants. Double mutations were also performed to examine alternative functions of various atg genes in definitive hematopoiesis. Notably, becn1 mutation failed to induce HSPCs expansion with one of the other five atg mutations. These findings demonstrated the distinct roles of atg genes and their interplays in zebrafish definitive hematopoiesis, thereby suggesting that the vertebrate definitive hematopoiesis is regulated in an atg gene-dependent manner.Abbreviations: AGM: aorta-gonad-mesonephros; AO: acridine orange; atg: autophagy related; becn1: beclin 1, autophagy related; CHT: caudal hematopoietic tissue; CKO: conditional knockout; coro1a: coronin, actin binding protein, 1A; CQ: chloroquine; CRISPR: clustered regularly interspaced short palindromic repeats; dpf: days post fertilization; FACS: fluorescence-activated cell sorting; hbae1.1: hemoglobin, alpha embryonic 1.1; HSCs: hematopoietic stem cells; HSPCs: hematopoietic stem and progenitor cells; KD: knockdown; KO: knockout; map1lc3/lc3: microtubule-associated protein 1 light chain 3; MO: morpholino; mpeg1.1: macrophage expressed 1, tandem duplicate 1; mpx: myeloid-specific peroxidase; myb: v-myb avian myeloblastosis viral oncogene homolog; PE: phosphatidylethanolamine; p-H3: phospho-H3 histone; PtdIns3K: class 3 phosphatidylinositol 3-kinase; rag1: recombination activating 1; rb1cc1/fip200: RB1-inducible coiled-coil 1; RFLP: restriction fragment length polymorphism; RNP: ribonucleoprotein; sin3aa: SIN3 transcription regulator family member Aa; spi1b: Spi-1 proto-oncogene b; ulk: unc-51 like autophagy activating kinase; vtg1: vitellogenin 1; WISH: whole-mount in situ hybridization.

Transgenic IDH2R172K and IDH2R140Q zebrafish models recapitulated features of human acute myeloid leukemia.

Isocitrate dehydrogenase 2 (IDH2) mutations occur in more than 15% of cytogenetically normal acute myeloid leukemia (CN-AML) but comparative studies of their roles in leukemogenesis have been scarce. We generated zebrafish models of IDH2R172K and IDH2R140Q AML and reported their pathologic, functional and transcriptomic features and therapeutic responses to target therapies. Transgenic embryos co-expressing FLT3ITD and IDH2 mutations showed accentuation of myelopoiesis. As these embryos were raised to adulthood, full-blown leukemia ensued with multi-lineage dysplasia, increase in myeloblasts and marrow cellularity and splenomegaly. The leukemia cells were transplantable into primary and secondary recipients and resulted in more aggressive disease. Tg(Runx1:FLT3ITDIDH2R172K) but not Tg(Runx1:FLT3ITDIDH2R140Q) zebrafish showed an increase in T-cell development at embryonic and adult stages. Single-cell transcriptomic analysis revealed increased myeloid skewing, differentiation blockade and enrichment of leukemia-associated gene signatures in both zebrafish models. Tg(Runx1:FLT3ITDIDH2R172K) but not Tg(Runx1:FLT3ITDIDH2R140Q) zebrafish showed an increase in interferon signals at the adult stage. Leukemic phenotypes in both zebrafish could be ameliorated by quizartinib and enasidenib. In conclusion, the zebrafish models of IDH2 mutated AML recapitulated the morphologic, clinical, functional and transcriptomic characteristics of human diseases, and provided the prototype for developing zebrafish leukemia models of other genotypes that would become a platform for high throughput drug screening.

Moderate-vigorous physical activity attenuates premature senescence of immune cells in sedentary adults with obesity: a pilot randomized controlled trial.

Despite the well-known senolytic effects of physical exercise on immune cells in older adults, the effect of physical activity (PA) on premature immune senescence in sedentary adults with obesity remains largely unknown. This pilot study aimed to investigate the role of objectively measured physical behaviors and Fitbit watch-based free-living PA intervention in premature senescence of immune cells in sedentary adults with obesity. Forty-five participants were recruited in the cross-sectional analysis, and forty of them further participated in the randomized controlled trial. We found that objectively measured moderate-vigorous PA was independently and inversely correlated with the expression of p16INK4a and p21Cip1 in the peripheral blood mononuclear cell (PBMCs) of adults with obesity; however, chronological age, body mass index, body fat, maximal oxygen consumption, light PA, sedentary behaviors, and sleep duration were not. More importantly, the 12-week PA intervention mitigated the elevated p16INK4a levels in PBMCs, though it showed no effect on p21Cip1 and senescence-associated secretory phenotypes. Taken together, physical inactivity is an independent determinant of premature senescence in immune cells, while the 12-week PA intervention is a promising strategy to alleviate premature immune senescence in adults with obesity.

Modeling leukemia with zebrafish (Danio rerio): Towards precision medicine.

Leukemia is a type of blood cancer characterized by high genetic heterogeneity and fatality. While chemotherapy remains the primary form of treatment for leukemia, its effectiveness was profoundly diminished by the genetic heterogeneity and cytogenetic abnormalities of leukemic cells. Therefore, there is an unmet need to develop precision medicine for leukemia with distinct genetic backgrounds. Zebrafish (Danio rerio), a freshwater fish with exceptional feasibility in genome editing, is a powerful tool for rapid human cancer modeling. In the past decades, zebrafish have been adopted in modeling human leukemia, exploring the molecular mechanisms of underlying genetic abnormalities, and discovering novel therapeutic agents. Although many recurrent mutations of leukemia have been modeled in zebrafish for pathological study and drug discovery, its great potential in leukemia modeling was not yet fully exploited, particularly in precision medicine. In this review, we evaluated the current zebrafish models of leukemia/pre-leukemia and genetic techniques and discussed the potential of zebrafish models with novel techniques, which may contribute to the development of zebrafish as a disease model for precision medicine in treating leukemia.

Leukocyte invasion of the brain after peripheral trauma in zebrafish (Danio rerio).

Despite well-known systemic immune reactions in peripheral trauma, little is known about their roles in posttraumatic neurological disorders, such as anxiety, sickness, and cognitive impairment. Leukocyte invasion of the brain, a common denominator of systemic inflammation, is involved in neurological disorders that occur in peripheral inflammatory diseases, whereas the influences of peripheral leukocytes on the brain after peripheral trauma remain largely unclear. In this study, we found that leukocytes, largely macrophages, transiently invaded the brain of zebrafish larvae after peripheral trauma through vasculature-independent migration, which was a part of the systemic inflammation and was mediated by interleukin-1b (il1b). Notably, myeloid cells in the brain that consist of microglia and invading macrophages were implicated in posttraumatic anxiety-like behaviors, such as hyperactivity (restlessness) and thigmotaxis (avoidance), while a reduction in systemic inflammation or myeloid cells can rescue these behaviors. In addition, invading leukocytes together with microglia were found to be responsible for the clearance of apoptotic cells in the brain; however, they also removed the nonapoptotic cells, which suggested that phagocytes have dual roles in the brain after peripheral trauma. More importantly, a category of conserved proteins between zebrafish and humans or rodents that has been featured in systemic inflammation and neurological disorders was determined in the zebrafish brain after peripheral trauma, which supported that zebrafish is a translational model of posttraumatic neurological disorders. These findings depicted leukocyte invasion of the brain during systemic inflammation after peripheral trauma and its influences on the brain through il1b-dependent mechanisms.

Does the gut microbiota contribute to the antiobesity effect of exercise? A systematic review and meta-analysis.

OBJECTIVE: The aim of this study was to assess gut microbiota modifications after exercise in humans and animal models with obesity or type 2 diabetes and their role in exercise-induced weight loss. METHODS: A systematic search of six databases was conducted on July 31, 2021. The extracted data on body fat or body weight from human and animal studies were analyzed using random-effects meta-analysis. RESULTS: A total of 28 studies were included, with all studies reporting exercise-induced gut microbiota modifications; however, the modified taxa varied among studies. Proteobacteria was the only taxa reported to be altered by exercise in more than one human and one animal study. Taxa belonging to Firmicutes were the most responsive to exercise in humans and mice, whereas Proteobacteria taxa were the most responsive to exercise in rats. A meta-analysis was conducted to examine the weight-lowering effect of exercise based on data subgrouped by altered or unaltered α-diversity or ß-diversity. The association between the weight-lowering effect of exercise and altered ß-diversity was observed in humans with obesity but not in animals. CONCLUSIONS: These findings suggest that gut microbiota modifications contribute to exercise-induced weight loss in obesity; however, their precise contributions, especially those of taxon-level variations, remain to be investigated.

Metformin accelerates zebrafish heart regeneration by inducing autophagy.

Metformin is one of the most widely used drugs for type 2 diabetes and it also exhibits cardiovascular protective activity. However, the underlying mechanism of its action is not well understood. Here, we used an adult zebrafish model of heart cryoinjury, which mimics myocardial infarction in humans, and demonstrated that autophagy was significantly induced in the injured area. Through a systematic evaluation of the multiple cell types related to cardiac regeneration, we found that metformin enhanced the autophagic flux and improved epicardial, endocardial and vascular endothelial regeneration, accelerated transient collagen deposition and resolution, and induced cardiomyocyte proliferation. Whereas, when the autophagic flux was blocked, then all these processes were delayed. We also showed that metformin transiently enhanced the systolic function of the heart. Taken together, our results indicate that autophagy is positively involved in the metformin-induced acceleration of heart regeneration in zebrafish and suggest that this well-known diabetic drug has clinical value for the prevention and amelioration of myocardial infarction.

Is exercise a senolytic medicine? A systematic review.

Cellular senescence, a state of irreversible growth arrest triggered by various stressors, engages in a category of pathological processes, whereby senescent cells accumulate in mitotic tissues. Senolytics as novel medicine against aging and various diseases through the elimination of senescent cells has emerged rapidly in recent years. Exercise is a potent anti-aging and anti-chronic disease medicine, which has shown the capacity to lower the markers of cellular senescence over the past decade. However, whether exercise is a senolytic medicine for aging and various diseases remains unclear. Here, we have conducted a systematic review of the published literature studying the senolytic effects of exercise or physical activity on senescent cells under various states in both human and animal models. Exercise can reduce the markers of senescent cells in healthy humans, while it lowered the markers of senescent cells in obese but not healthy animals. The discrepancy between human and animal studies may be due to the relatively small volume of research and the variations in markers of senescent cells, types of cells/tissues, and health conditions. These findings suggest that exercise has senolytic properties under certain conditions, which warrant further investigations.

1-phenyl 2-thiourea (PTU) activates autophagy in zebrafish embryos.

1-phenyl 2-thiourea (PTU) is a Tyr (tyrosinase) inhibitor that is extensively used to block pigmentation and improve optical transparency in zebrafish (Danio rerio) embryo. Here, we reported a previously undescribed effect of PTU on macroautophagy/autophagy in zebrafish embryos. Upon 0.003% PTU treatment, aberrant autophagosome and autolysosome formation, accumulation of lysosomes, and elevated autophagic flux were observed in various tissues and organs of zebrafish embryos, such as skin, brain, and muscle. Similar to PTU treatment, autophagic activation and lysosomal accumulation were also observed in the somatic tyr mutant zebrafish embryos, which suggest that Tyr inhibition may contribute to PTU-induced autophagic activation. Furthermore, we demonstrated that autophagy contributes to pigmentation inhibition, but is not essential to the PTU-induced pigmentation inhibition. With the involvement of autophagy in a wide range of physiological and pathological processes and the routine use of PTU in zebrafish research of autophagy-related processes, these observations raise a novel concern in autophagy-related studies using PTU-treated zebrafish embryos.Abbreviations: 3-MA: 3-methyladenine; Atg: autophagy-related; BSA: bovine serum albumin; CHT: caudal hematopoietic tissue; CQ: chloroquine; GFP: green fluorescent protein; hpf: hour-post-fertilization; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NGS: normal goat serum; PtdIns3K: class III phosphatidylinositol 3-kinase; PTU: 1-phenyl 2-thiourea; RFP: red fluorescent protein; Sqstm1: sequestosome 1; tyr: tyrosinase.

Deletion of interleukin enhancer binding factor 2 (ILF2) resulted in defective biliary development and bile flow blockage.

PURPOSE: Biliary atresia (BA) is a devastating obstructive bile duct disease of newborns. BA has the highest incidence in Asians (1/5000), and its pathogenesis is unclear. We identified BA-private rare copy number variants (CNVs; 22 duplications and 6 deletions). ILF2 gene locates in the chromosome region (Chr1:153410347-153,634,058) which was deleted in a nonsyndromic BA patient. However, it is still not known whether ILF2 plays a role in hepatobiliary development and its deletion impacts on the bile duct development. METHODS: To investigate if ILF2 is required for biliary development, we knock-out the zebrafish homologs of ILF2 by CRISPR/Cas9 approach, and discover that deletion of ILF2 causes a defective biliary development and a lack of bile flow from the liver to the gall bladder in zebrafish, which is a resemblance of phenotypes of BA. RESULTS: Our data indicate that ILF2 gene is required for biliary development; deletion of ILF2 impairs bile duct development and could contribute to BA pathogenesis. This will be the first study to functionally evaluate the genes interfered by BA-private CNVs in hepatobiliary development and in BA pathogenesis. CONCLUSIONS: Such functional study may reveal the potential value of these BA-private CNVs in the disease pathogenesis for BA. LEVEL OF EVIDENCE: N/A (animal and laboratory study).

Monoallelic Mutations in CC2D1A Suggest a Novel Role in Human Heterotaxy and Ciliary Dysfunction.

BACKGROUND: Human heterotaxy is a group of congenital disorders characterized by misplacement of one or more organs according to the left-right axis. The genetic causes of human heterotaxy are highly heterogeneous. METHODS: We performed exome sequencing in a cohort of 26 probands with heterotaxy followed by gene burden analysis for the enrichment of novel rare damaging mutations. Transcription activator-like effector nuclease was used to generate somatic loss-of-function mutants in a zebrafish model. Ciliary defects were examined by whole-mount immunostaining of acetylated α-tubulin. RESULTS: We identified a significant enrichment of novel rare damaging mutations in the CC2D1A gene. Seven occurrences of CC2D1A mutations were found to affect 4 highly conserved amino acid residues of the protein. Functional analyses in the transcription activator-like effector nuclease-mediated zebrafish knockout models were performed, and heterotaxy phenotypes of the cardiovascular and gastrointestinal systems in both somatic and germline mutants were observed. Defective cilia were demonstrated by whole-mount immunostaining of acetylated α-tubulin. These abnormalities were rescued by wild-type cc2d1a mRNA but not cc2d1a mutant mRNA, strongly suggesting a loss-of-function mechanism. On the other hand, overexpression of cc2d1a orthologous mutations cc2d1a P559L and cc2d1a G808V (orthologous to human CC2D1A P532L and CC2D1A G781V) did not affect embryonic development. CONCLUSIONS: Using a zebrafish model, we were able to establish a novel association of CC2D1A with heterotaxy and ciliary dysfunction in the F2 generation via a loss-of-function mechanism. Future mechanistic studies are needed for a better understanding of the role of CC2D1A in left-right patterning and ciliary dysfunction.

Function of Arl4aa in the Initiation of Hematopoiesis in Zebrafish by Maintaining Golgi Complex Integrity in Hemogenic Endothelium.

ADP-ribosylation factor-like 4aa (Arl4aa) is a member of the ADP-ribosylation factor family. It is expressed in hematopoietic tissue during embryonic development, but its function was unknown. Zebrafish arl4aa is preferentially expressed in the ventral wall of the dorsal aorta (VDA) at 24 and 36 hpf and in caudal hematopoietic tissue at 48 hpf. Morpholino knockdown and transcription activator-like effector nuclease (TALEN) knockout of arl4aa significantly reduced expression of genes associated with definitive hematopoietic stem cells (HSCs). Golgi complex integrity in VDA was disrupted as shown by transmission electron microscopy and immunostaining of Golgi membrane Giantin. Mechanistically, arl4aa knockdown reduced Notch signaling in the VDA and its target gene expression. Protein expression of NICD was also reduced. Effects of arl4aa knockdown on definitive hematopoiesis could be restored by NICD expression. This study identified arl4aa as a factor regulating initiation of definitive HSCs by maintaining the integrity of Golgi complex and, secondarily, maturation of the Notch receptor.

Follistatin is a novel therapeutic target and biomarker in FLT3/ITD acute myeloid leukemia.

Internal tandem duplication of Fms-like tyrosine kinase 3 (FLT3/ITD) occurs in about 30% of acute myeloid leukemia (AML) and is associated with poor response to conventional treatment and adverse outcome. Here, we reported that human FLT3/ITD expression led to axis duplication and dorsalization in about 50% of zebrafish embryos. The morphologic phenotype was accompanied by ectopic expression of a morphogen follistatin (fst) during early embryonic development. Increase in fst expression also occurred in adult FLT3/ITD-transgenic zebrafish, Flt3/ITD knock-in mice, and human FLT3/ITD AML cells. Overexpression of human FST317 and FST344 isoforms enhanced clonogenicity and leukemia engraftment in xenotransplantation model via RET, IL2RA, and CCL5 upregulation. Specific targeting of FST by shRNA, CRISPR/Cas9, or antisense oligo inhibited leukemic growth in vitro and in vivo. Importantly, serum FST positively correlated with leukemia engraftment in FLT3/ITD AML patient-derived xenograft mice and leukemia blast percentage in primary AML patients. In FLT3/ITD AML patients treated with FLT3 inhibitor quizartinib, serum FST levels correlated with clinical response. These observations supported FST as a novel therapeutic target and biomarker in FLT3/ITD AML.

Robust activation of microhomology-mediated end joining for precision gene editing applications.

One key problem in precision genome editing is the unpredictable plurality of sequence outcomes at the site of targeted DNA double stranded breaks (DSBs). This is due to the typical activation of the versatile Non-homologous End Joining (NHEJ) pathway. Such unpredictability limits the utility of somatic gene editing for applications including gene therapy and functional genomics. For germline editing work, the accurate reproduction of the identical alleles using NHEJ is a labor intensive process. In this study, we propose Microhomology-mediated End Joining (MMEJ) as a viable solution for improving somatic sequence homogeneity in vivo, capable of generating a single predictable allele at high rates (56% ~ 86% of the entire mutant allele pool). Using a combined dataset from zebrafish (Danio rerio) in vivo and human HeLa cell in vitro, we identified specific contextual sequence determinants surrounding genomic DSBs for robust MMEJ pathway activation. We then applied our observation to prospectively design MMEJ-inducing sgRNAs against a variety of proof-of-principle genes and demonstrated high levels of mutant allele homogeneity. MMEJ-based DNA repair at these target loci successfully generated F0 mutant zebrafish embryos and larvae that faithfully recapitulated previously reported, recessive, loss-of-function phenotypes. We also tested the generalizability of our approach in cultured human cells. Finally, we provide a novel algorithm, MENTHU (http://genesculpt.org/menthu/), for improved and facile prediction of candidate MMEJ loci. We believe that this MMEJ-centric approach will have a broader impact on genome engineering and its applications. For example, whereas somatic mosaicism hinders efficient recreation of knockout mutant allele at base pair resolution via the standard NHEJ-based approach, we demonstrate that F0 founders transmitted the identical MMEJ allele of interest at high rates. Most importantly, the ability to directly dictate the reading frame of an endogenous target will have important implications for gene therapy applications in human genetic diseases.

Integrating Functional Analysis in the Next-Generation Sequencing Diagnostic Pipeline of RASopathies.

RASopathies are a group of heterogeneous conditions caused by germline mutations in RAS/MAPK signalling pathway genes. With next-generation sequencing (NGS), sequencing capacity is no longer a limitation to molecular diagnosis. Instead, the rising number of variants of unknown significance (VUSs) poses challenges to clinical interpretation and genetic counselling. We investigated the potential of an integrated pipeline combining NGS and the functional assessment of variants for the diagnosis of RASopathies. We included 63 Chinese patients with RASopathies that had previously tested negative for PTPN11 and HRAS mutations. In these patients, we performed a genetic analysis of genes associated with RASopathies using a multigene NGS panel and Sanger sequencing. For the VUSs, we evaluated evidence from genetic, bioinformatic and functional data. Twenty disease-causing mutations were identified in the 63 patients, providing a primary diagnostic yield of 31.7%. Four VUSs were identified in five patients. The functional assessment supported the pathogenicity of the RAF1 and RIT1 VUSs, while the significance of two VUSs in A2ML1 remained unclear. In summary, functional analysis improved the diagnostic yield from 31.7% to 36.5%. Although technically demanding and time-consuming, a functional genetic diagnostic analysis can ease the clinical translation of these findings to aid bedside interpretation.

A Zebrafish Model for Evaluating the Function of Human Leukemic Gene IDH1 and Its Mutation.

The recent advent of next-generation sequencing (NGS) has greatly accelerated identification of gene mutations in myeloid malignancies at unprecedented speed that will soon outpace their functional validation by conventional laboratory techniques and animal models. A high-throughput whole-organism model is useful for the functional validation of new mutations. We recently reported the use of zebrafish to evaluate the hematopoietic function of isocitrate dehydrogenase 1 (IDH1) and the effects of expressing human IDH1-R132H that is frequently identified in human acute myeloid leukemia (AML), in myelopoiesis, with a view to develop zebrafish as a model of AML. Here, we use IDH1 as an example to describe a comprehensive approach to evaluate hematopoietic gene function and the effects of mutations using zebrafish as a model.

TALEN-Mediated Mutagenesis and Genome Editing.

Transcription activator-like effectors (TALEs) are important genomic tools with customizable DNA-binding motifs for locus-specific modifications. In particular, TALE nucleases or TALENs have been successfully used in the zebrafish model system to introduce targeted mutations via repair of double-stranded breaks (DSBs) either through nonhomologous end joining (NHEJ) or by homology-directed repair (HDR) and homology-independent repair in the presence of a donor template. Compared with other customizable nucleases, TALENs offer high binding specificity and fewer sequence constraints in targeting the genome, with comparable mutagenic activity. Here, we describe a detailed in silico design tool for zebrafish genome editing for TALENs and CRISPR/Cas9 custom restriction enzymes using Mojo Hand 2.0 software.