scholarly journals Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos

2021 ◽  
Vol 118 (22) ◽  
pp. e2004832117
Author(s):  
Gregorio Alanis-Lobato ◽  
Jasmin Zohren ◽  
Afshan McCarthy ◽  
Norah M. E. Fogarty ◽  
Nada Kubikova ◽  
...  
Keyword(s):  
Genome Editing ◽  
Loss Of Heterozygosity ◽  
Germ Line ◽  
Basic Research ◽  
Preimplantation Embryos ◽  
Human Embryos ◽  
Gene Correction ◽  
Embryo Cells ◽  
Low Efficiency ◽  
Early Human

CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germ line. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism, and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9–targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss of heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in ∼16% of the human embryo cells analyzed and spanned 4–20 kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.

scholarly journals Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human embryos

2020 ◽  
Author(s):  
Gregorio Alanis-Lobato ◽  
Jasmin Zohren ◽  
Afshan McCarthy ◽  
Norah M.E. Fogarty ◽  
Nada Kubikova ◽  
...  
Keyword(s):  
Genome Editing ◽  
Loss Of Heterozygosity ◽  
Basic Research ◽  
Preimplantation Embryos ◽  
Human Embryos ◽  
Gene Correction ◽  
Embryo Cells ◽  
Low Efficiency ◽  
And Control ◽  
Early Human

AbstractCRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germline. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9-targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss-of-heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in approximately 16% of the human embryo cells analysed and spanned 4 to 20kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.

scholarly journals Comparison of gene expression in fresh and frozen–thawed human preimplantation embryos

Reproduction ◽  
2012 ◽  
Vol 144 (5) ◽  
pp. 569-582 ◽  
Author(s):  
Lisa Shaw ◽  
Sharon F Sneddon ◽  
Daniel R Brison ◽  
Susan J Kimber
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Developmental Expression ◽  
Preimplantation Embryos ◽  
Human Embryos ◽  
Molecular Events ◽  
Reliable Source ◽  
Human Preimplantation Embryos ◽  
Early Human

Identification and characterisation of differentially regulated genes in preimplantation human embryonic development are required to improve embryo quality and pregnancy rates in IVF. In this study, we examined expression of a number of genes known to be critical for early development and compared expression profiles in individual preimplantation human embryos to establish any differences in gene expression in fresh compared to frozen–thawed embryos used routinely in IVF. We analysed expression of 19 genes by cDNA amplification followed by quantitative real-time PCR in a panel of 44 fresh and frozen–thawed human preimplantation embryos. Fresh embryos were obtained from surplus early cleavage stage embryos and frozen–thawed embryos from cryopreserved 2PN embryos. Our aim was to determine differences in gene expression between fresh and frozen–thawed human embryos, but we also identified differences in developmental expression patterns for particular genes. We show that overall gene expression among embryos of the same stage is highly variable and our results indicate that expression levels between groups did differ and differences in expression of individual genes was detected. Our results show that gene expression from frozen–thawed embryos is more consistent when compared with fresh, suggesting that cryopreserved embryos may represent a reliable source for studying the molecular events underpinning early human embryo development.

scholarly journals EDITORIAL NEED OF OUR TIMES: IMPACT OF GENOME EDITING SCIENCES AND REQUISITE PREPAREDNESS FOR MOLECULAR BIOTERRORISM

2020 ◽  
Vol 70 (6) ◽  
pp. 1601-03
Author(s):  
Sikandar Hayat Khan
Keyword(s):  
Genome Editing ◽  
Germ Line ◽  
Life Forms ◽  
Human Embryos ◽  
Question Mark ◽  
Ethical Concerns ◽  
Point Of No Return ◽  
Synthetic Genomics ◽  
Community Struggles ◽  
Intelligent Creature

The pace of human evolution has accelerated at an unprecedented rate in the last couple of decades. Never ever before the mankind could witness a global hostage situation by a tiny invisible RNA creature. While the global community struggles at large finding plausible solutions in the information replete era, there are seriouslessons to be learnt. The tiny RNA monster has exposed the vulnerabilities of one the considered most intelligent creature posing a question mark about how to strike the intricate balance between preventive approaches and acquiring the postexposure immunity. The rapidly improving genome editing methods along with synthetic genomics has emerged as a double-edged weapon where on one side it opens newer therapeutic avenues to cure disease, but also its malicious use could results in disasters of limitless magnitudes.The delicate boundaries nature may face terrorism in newer clothes at the hands of nano technological tools to modify genome and synthesizing newer life forms. Unstoppable if it becomes can create man-made disasters with issues leading to emergence of black markets for cloning, designer humans ethnic-specific nucleotide editing for worse and possibly much more. The fiction we saw yesterday is today’s science and can lead the human race to point of no return. “He Jiankui affair” is still one of the genome editing dilemma widely criticized for ethical concerns emerging from germ line editing two human embryos for HIV using Cluster RegularlyInterspaced Short Palindromic Repeats (CRISPR) Cas technology.

CRISPR Gene Editing on Human Embryos

2020 ◽  
Author(s):  
Fatemeh Sefid ◽  
Saedeh Khadempar ◽  
Roshanak Shamriz ◽  
Nooshin Amjadi
Keyword(s):  
Genome Editing ◽  
Human Embryo ◽  
Genome Engineering ◽  
Human Reproduction ◽  
Human Embryos ◽  
Gene Therapies ◽  
Recent Developments ◽  
Technical Possibility ◽  
Early Human

Background: With the recent development of CRISPR/Cas9 genome editing technology, the possibility to genetically influence the human germline (gametes and embryos) has become a separate technical possibility. As a powerful skill for genome engineering, the CRISPR/Cas9 system has been effectively applied to adjust the genomes of several species. The purpose of this review was to appraise the technology and build concepts for the launch of precise hereditary modifications in early human embryos.   Methods: We conducted a systematic review of the related literatures searched from PubMed, Google scholar, Web of Science up to June 30, 2017 and then we extracted the essential data. In this review, we present the brief history and basic mechanisms of the CRISPR/Cas9 system and significant challenges and advances in the field as a comprehensive practical guide to absorbed users of genome editing technologies. We introduce factors that influence CRISPR/Cas9 efficacy which must be addressed before effective in vivo human embryo therapy can be realized .in this review, we highlight the advancements that have been made using CRISPR/Cas9 in relation to Human Embryo.   Results and Conclusion: The possibility of CRISPR/Cas9 use in the context of human reproduction, to change embryos, germline cells, and pluripotent stem cells are studied created on the writers' expert belief. We discuss recent developments leading to the operation of Human Embryonic gene therapies in clinical trials and consider the predictions for future advances in this rapidly developing field.

scholarly journals The TRACE-Seq method tracks recombination alleles and identifies clonal reconstitution dynamics of gene targeted human hematopoietic stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rajiv Sharma ◽  
Daniel P. Dever ◽  
Ciaran M. Lee ◽  
Armon Azizi ◽  
Yidan Pan ◽  
...  
Keyword(s):  
Genetic Diseases ◽  
Basic Research ◽  
Gene Correction ◽  
Coding Region ◽  
Hematopoietic Stem ◽  
Hematopoietic Reconstitution ◽  
Exon 1 ◽  
Template Library ◽  
Donor Template

AbstractTargeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.

scholarly journals Embryoid research calls for reassessment of legal regulations

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Markus Hengstschläger ◽  
Margit Rosner
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Legal Status ◽  
Embryonic Stem ◽  
Basic Research ◽  
Human Embryos ◽  
Human Being ◽  
Legal Regulations ◽  
Definition Of

AbstractIt is known that in countries, in which basic research on human embryos is in fact prohibited by law, working with imported human embryonic stem cells (hESCs) can still be permitted. As long as hESCs are not capable of development into a complete human being, it might be the case that they do not fulfill all criteria of the local definition of an embryo. Recent research demonstrates that hESCs can be developed into entities, called embryoids, which increasingly could come closer to actual human embryos in future. By discussing the Austrian situation, we want to highlight that current embryoid research could affect the prevailing opinion on the legal status of work with hESCs and therefore calls for reassessment of the regulations in all countries with comparable definitions of the embryo.

scholarly journals AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Liyang Zhang ◽  
John A. Zuris ◽  
Ramya Viswanathan ◽  
Jasmine N. Edelstein ◽  
Rolf Turk ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Genome Editing ◽  
Nk Cell ◽  
Basic Research ◽  
Cell Recognition ◽  
Site Specific ◽  
Editing Efficiency ◽  
Rapid Generation ◽  
Therapeutic Cell

AbstractThough AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor editing efficiency, restricting its overall utility. Here we isolate an engineered variant, “AsCas12a Ultra”, that increased editing efficiency to nearly 100% at all sites examined in HSPCs, iPSCs, T cells, and NK cells. We show that AsCas12a Ultra maintains high on-target specificity thereby mitigating the risk for off-target editing and making it ideal for complex therapeutic genome editing applications. We achieved simultaneous targeting of three clinically relevant genes in T cells at >90% efficiency and demonstrated transgene knock-in efficiencies of up to 60%. We demonstrate site-specific knock-in of a CAR in NK cells, which afforded enhanced anti-tumor NK cell recognition, potentially enabling the next generation of allogeneic cell-based therapies in oncology. AsCas12a Ultra is an advanced CRISPR nuclease with significant advantages in basic research and in the production of gene edited cell medicines.

scholarly journals Fertilization Narratives in the Art of Gustav Klimt, Diego Rivera and Frida Kahlo: Repression, Domination and Eros among Cells

Leonardo ◽  
2011 ◽  
Vol 44 (3) ◽  
pp. 221-227 ◽  
Author(s):  
Scott F. Gilbert ◽  
Sabine Brauckmann
Keyword(s):  
20Th Century ◽  
Human Embryos ◽  
Diego Rivera ◽  
Frida Kahlo ◽  
Embryo Formation ◽  
Human Fertilization ◽  
Gustav Klimt ◽  
The Universe ◽  
Early Human

Fertilization narratives are powerful biological stories that can be used for social ends, and 20th-century artists have used fertilization-based imagery to convey political and social ideas. In Danae, Gustav Klimt used an esoteric stage of early human embryos to indicate successful fertilization and the inability of government repression to stifle creativity. In Man, Controller of the Universe, Diego Rivera painted a mural of a man controlling an ovulating ovary, depicting Trotsky's view that society will rationally regulate human fertilization. His former wife, Frida Kahlo, refuted this view in Moses: Nucleus of Creation, wherein she painted images of fertilization and embryo formation as the ultimate acts of erotic consummation and generation.

scholarly journals Biological and Clinical Significance of Mosaicism in Human Preimplantation Embryos

2021 ◽  
Vol 9 (2) ◽  
pp. 18
Author(s):  
Ioanna Bouba ◽  
Elissavet Hatzi ◽  
Paris Ladias ◽  
Prodromos Sakaloglou ◽  
Charilaos Kostoulas ◽  
...  
Keyword(s):  
Assisted Reproduction ◽  
Basic Research ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
New Approach ◽  
Assisted Reproduction Technology ◽  
Clinical Utilization ◽  
Art Practices ◽  
In Utero Development ◽  
Human Preimplantation Embryos

Applications and indications of assisted reproduction technology are expanding, but every new approach is under scrutiny and thorough consideration. Recently, groups of assisted reproduction experts have presented data that support the clinical use of mosaic preimplantation embryos at the blastocyst stage, previously excluded from transfer. In the light of published contemporary studies, with or without clinical outcomes, there is growing evidence that mosaic embryos have the capacity for further in utero development and live birth. Our in-depth discussion will enable readers to better comprehend current developments. This expansion into the spectrum of ART practices requires further evidence and further theoretical documentation, basic research, and ethical support. Therefore, if strict criteria for selecting competent mosaic preimplantation embryos for further transfer, implantation, fetal growth, and healthy birth are applied, fewer embryos will be excluded, and more live births will be achieved. Our review aims to discuss the recent literature on the transfer of mosaic preimplantation embryos. It also highlights controversies as far as the clinical utilization of preimplantation embryos concerns. Finally, it provides the appropriate background to elucidate and highlight cellular and genetic aspects of this novel direction.

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