scholarly journals Targeted Gene Editing in Porcine Spermatogonia

2021 ◽  
Vol 11 ◽  
Author(s):  
Dennis Webster ◽  
Alla Bondareva ◽  
Staci Solin ◽  
Taylor Goldsmith ◽  
Lin Su ◽  
...  
Keyword(s):  
Stem Cell ◽  
Regenerative Medicine ◽  
Human Disease ◽  
Gene Editing ◽  
Disease Modeling ◽  
Germline Stem Cell ◽  
End Joining ◽  
Homology Directed Repair ◽  
Pig Genome ◽  
Targeted Gene Editing

To study the pathophysiology of human diseases, develop innovative treatments, and refine approaches for regenerative medicine require appropriate preclinical models. Pigs share physiologic and anatomic characteristics with humans and are genetically more similar to humans than are mice. Genetically modified pigs are essential where rodent models do not mimic the human disease phenotype. The male germline stem cell or spermatogonial stem cell (SSC) is unique; it is the only cell type in an adult male that divides and contributes genes to future generations, making it an ideal target for genetic modification. Here we report that CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing in porcine spermatogonia that include SSCs is significantly more efficient than previously reported editing with TALENs and allows precise gene editing by homology directed repair (HDR). We also established homology-mediated end joining (HMEJ) as a second approach to targeted gene editing to enable introduction of larger transgenes and/or humanizing parts of the pig genome for disease modeling or regenerative medicine. In summary, the approaches established in the current study result in efficient targeted genome editing in porcine germ cells for precise replication of human disease alleles.

scholarly journals Genome editing in large animals: current status and future prospects

2019 ◽  
Vol 6 (3) ◽  
pp. 402-420 ◽  
Author(s):  
Jianguo Zhao ◽  
Liangxue Lai ◽  
Weizhi Ji ◽  
Qi Zhou
Keyword(s):  
Regenerative Medicine ◽  
Genome Editing ◽  
Biomedical Research ◽  
Human Disease ◽  
Gene Editing ◽  
Disease Modeling ◽  
Current Status ◽  
Future Prospects ◽  
Recent Advances ◽  
Large Animals

AbstractLarge animals (non-human primates, livestock and dogs) are playing important roles in biomedical research, and large livestock animals serve as important sources of meat and milk. The recently developed programmable DNA nucleases have revolutionized the generation of gene-modified large animals that are used for biological and biomedical research. In this review, we briefly introduce the recent advances in nuclease-meditated gene editing tools, and we outline these editing tools’ applications in human disease modeling, regenerative medicine and agriculture. Additionally, we provide perspectives regarding the challenges and prospects of the new genome editing technology.

scholarly journals Gene targeting facilitated by engineered sequence-specific nucleases and its potential for applications in crop improvement

2021 ◽  
Author(s):  
Daisuke Miki ◽  
Rui Wang ◽  
Jing Li ◽  
Dali Kong ◽  
Lei Zhang ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Agronomic Traits ◽  
Higher Plants ◽  
Crop Improvement ◽  
Strand Break ◽  
End Joining ◽  
Homology Directed Repair ◽  
Target Dna ◽  
Targeted Gene Editing ◽  
Non Homologous End Joining

Abstract Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world’s population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining (NHEJ), and are only rarely repaired via error-free homology-directed repair (HDR) if an appropriate donor template is provided. Gene targeting (GT), i.e., the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.

scholarly journals Engineering a far-red light–activated split-Cas9 system for remote-controlled genome editing of internal organs and tumors

2020 ◽  
Vol 6 (28) ◽  
pp. eabb1777 ◽  
Author(s):  
Yuanhuan Yu ◽  
Xin Wu ◽  
Ningzi Guan ◽  
Jiawei Shao ◽  
Huiying Li ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Light Emitting Diode ◽  
Red Light ◽  
Tumor Model ◽  
End Joining ◽  
Internal Organs ◽  
Fast System ◽  
Homology Directed Repair ◽  
Multiple Loci

It is widely understood that CRISPR-Cas9 technology is revolutionary, with well-recognized issues including the potential for off-target edits and the attendant need for spatiotemporal control of editing. Here, we describe a far-red light (FRL)–activated split-Cas9 (FAST) system that can robustly induce gene editing in both mammalian cells and mice. Through light-emitting diode–based FRL illumination, the FAST system can efficiently edit genes, including nonhomologous end joining and homology-directed repair, for multiple loci in human cells. Further, we show that FAST readily achieves FRL-induced editing of internal organs in tdTomato reporter mice. Finally, FAST was demonstrated to achieve FRL-triggered editing of the PLK1 oncogene in a mouse xenograft tumor model. Beyond extending the spectrum of light energies in optogenetic toolbox for CRISPR-Cas9 technologies, this study demonstrates how FAST system can be deployed for programmable deep tissue gene editing in both biological and biomedical contexts toward high precision and spatial specificity.

scholarly journals Stem-cell research and regenerative medicine in China

2016 ◽  
Vol 3 (2) ◽  
pp. 257-261 ◽  
Author(s):  
Jane Qiu
Keyword(s):  
Stem Cell ◽  
Regenerative Medicine ◽  
Stem Cell Research ◽  
Gene Editing ◽  
Associate Editor ◽  
Human Embryos ◽  
Director General ◽  
Chinese Academy Of Sciences ◽  
The World ◽  
Academy Of Sciences

Abstract For stem-cell researchers around the world, 2015 was a roller-coaster year. In April, Junjiu Huang, a biologist at the Sun Yat-sen University in Guangzhou, published the first paper on gene editing in human embryos with CRISPR-cas9. This sparked a global controversy—with many Western media using this as an example of China's lack of ethical standards. Subsequent discussions, which culminated in the summit in Washington, DC, last December, have eased the anxieties to some extent over this study and similar studies have now been proposed or approved in the UK and Sweden. Surprisingly, according to Nature magazine (the same magazine publishing some of the news reports on this study), Huang was one of the 10 scientists in the world that made a difference last year. In a forum chaired by National Science Review's Executive Associate Editor Mu-ming Poo, stem-cell researchers and a bioethicist discussed how they see last year's furore over gene editing, why China should streamline its oversight and regulatory processes, and where the future of the country's stem-cell research and regenerative medicine lies. Duanqing Pei Stem-cell researcher and Director General of Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, in Guangzhou Xiaomei Zhai Bioethicist and Executive Director of the Centre for Bioethics, Peking Union Medical College, in Beijing Qi Zhou Stem-cell researcher at the Institute of Zoology, Chinese Academy of Sciences, in Beijing Jianhong Zhu Neurosurgeon and neuroscientist at Huashan Hospital, Fudan University, in Shanghai Mu-ming Poo (Chair) Neuroscientist and Director of the Institute of Neuroscience, Chinese Academy of Sciences, in Shanghai

scholarly journals The Germline Stem Cell Niche Unit in Mammalian Testes

2012 ◽  
Vol 92 (2) ◽  
pp. 577-595 ◽  
Author(s):  
Jon M. Oatley ◽  
Ralph L. Brinster
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Regenerative Medicine ◽  
Stem Cell Niche ◽  
Germline Stem Cell ◽  
Tissue Specific ◽  
Support Cell ◽  
Age Related ◽  
Cell Niche ◽  
Germline Stem Cell Niche

This review addresses current understanding of the germline stem cell niche unit in mammalian testes. Spermatogenesis is a classic model of tissue-specific stem cell function relying on self-renewal and differentiation of spermatogonial stem cells (SSCs). These fate decisions are influenced by a niche microenvironment composed of a growth factor milieu that is provided by several testis somatic support cell populations. Investigations over the last two decades have identified key determinants of the SSC niche including cytokines that regulate SSC functions and support cells providing these factors, adhesion molecules that influence SSC homing, and developmental heterogeneity of the niche during postnatal aging. Emerging evidence suggests that Sertoli cells are a key support cell population influencing the formation and function of niches by secreting soluble factors and possibly orchestrating contributions of other support cells. Investigations with mice have shown that niche influence on SSC proliferation differs during early postnatal development and adulthood. Moreover, there is mounting evidence of an age-related decline in niche function, which is likely influenced by systemic factors. Defining the attributes of stem cell niches is key to developing methods to utilize these cells for regenerative medicine. The SSC population and associated niche comprise a valuable model system for study that provides fundamental knowledge about the biology of tissue-specific stem cells and their capacity to sustain homeostasis of regenerating tissue lineages. While the stem cell is essential for maintenance of all self-renewing tissues and has received considerable attention, the role of niche cells is at least as important and may prove to be more receptive to modification in regenerative medicine.

scholarly journals Modeling human neurodevelopmental diseases with brain organoids

2022 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiaoxiang Lu ◽  
Jiajie Yang ◽  
Yangfei Xiang
Keyword(s):  
Stem Cells ◽  
Human Brain ◽  
Pluripotent Stem Cells ◽  
Neurodevelopmental Disorders ◽  
Human Disease ◽  
Gene Editing ◽  
Disease Modeling ◽  
Developmental Abnormalities ◽  
The Past ◽  
Underlying Mechanisms

AbstractStudying the etiology of human neurodevelopmental diseases has long been a challenging task due to the brain’s complexity and its limited accessibility. Human pluripotent stem cells (hPSCs)-derived brain organoids are capable of recapitulating various features and functionalities of the human brain, allowing the investigation of intricate pathogenesis of developmental abnormalities. Over the past years, brain organoids have facilitated identifying disease-associated phenotypes and underlying mechanisms for human neurodevelopmental diseases. Integrating with more cutting-edge technologies, particularly gene editing, brain organoids further empower human disease modeling. Here, we review the latest progress in modeling human neurodevelopmental disorders with brain organoids.

Transcriptome Profiling Reveals Degree of Variability in Induced Pluripotent Stem Cell Lines: Impact for Human Disease Modeling

2015 ◽  
Vol 17 (5) ◽  
pp. 327-337 ◽  
Author(s):  
Jens Schuster ◽  
Jonatan Halvardson ◽  
Laureanne Pilar Lorenzo ◽  
Adam Ameur ◽  
Maria Sobol ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Lines ◽  
Human Disease ◽  
Pluripotent Stem Cell ◽  
Disease Modeling ◽  
Induced Pluripotent Stem Cell ◽  
Transcriptome Profiling ◽  
Stem Cell Lines ◽  
Induced Pluripotent

scholarly journals Understanding the diversity of genetic outcomes from CRISPR-Cas generated homology-directed repair

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Brett M. Sansbury ◽  
Amanda M. Hewes ◽  
Eric B. Kmiec
Keyword(s):  
Dna Repair ◽  
Enzymatic Activity ◽  
Broad Spectrum ◽  
Gene Editing ◽  
Cell Free Extract ◽  
End Joining ◽  
Homology Directed Repair ◽  
Editing Activity ◽  
Non Homologous End Joining

AbstractAs CRISPR-Cas systems advance toward clinical application, it is essential to identify all the outcomes of gene-editing activity in human cells. Reports highlighting the remarkable success of homology-directed repair (HDR) in the treatment of inherited diseases may inadvertently underreport the collateral activity of this remarkable technology. We are utilizing an in vitro gene-editing system in which a CRISPR-Cas complex provides the double-stranded cleavage and a mammalian cell-free extract provides the enzymatic activity to promote non-homologous end joining, micro-homology mediated end joining, and homology-directed repair. Here, we detail the broad spectrum of gene-editing reaction outcomes utilizing Cas9 and Cas12a in combination with single-stranded donor templates of the sense and nonsense polarity. This system offers the opportunity to see the range of outcomes of gene-editing reactions in an unbiased fashion, detailing the distribution of DNA repair outcomes as a function of a set of genetic tools.

scholarly journals CRISPR-Cas9: A Genome Editing Tool in Crop Plants: A Review

2021 ◽  
Author(s):  
Varsha Kumari ◽  
Priyanka Kumawat ◽  
Sharanabasappa Yeri ◽  
Shyam Singh Rajput
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Crop Improvement ◽  
End Joining ◽  
Homology Directed Repair ◽  
Ligase Iv ◽  
A Genome ◽  
Target Dna ◽  
Non Homologous End Joining ◽  
Dna Strand

Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR-Cas9) system is a rapid technology for gene editing. CRISPR-Cas9 is an RNA guided gene editing tool where Cas9 acts as endonuclease by cutting the target DNA strand. Double Stranded Breaks (DBS) can be repaired by non-homologous end joining (NHEJ) and homology-directed repair (HDR). The NHEJ employs DNA ligase IV to rejoin the broken ends which cause insertion or deletion mutations, whereas HDR repairs the DSBs based on a homologous complementary template and results in perfect repair of broken ends. CRISPR-Cas9 impart diverse advantageous features in contrast with the conventional methods. In this review article, we have discussed CRISPR-Cas9 based genome editing along with its mechanism of action and role in crop improvement.

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