scholarly journals Heavily and Fully Modified RNAs Guide Efficient SpyCas9-Mediated Genome Editing

2018 ◽  
Author(s):  
Aamir Mir ◽  
Julia F. Alterman ◽  
Matthew R. Hassler ◽  
Alexandre J. Debacker ◽  
Edward Hudgens ◽  
...  
Keyword(s):  
Chemical Modification ◽  
Genome Editing ◽  
Ex Vivo ◽  
Human Cells ◽  
Chemical Modifications ◽  
Oh Groups ◽  
Guide Rnas ◽  
Chemically Modified

RNA-based drugs depend on chemical modifications to increase potency and nuclease stability, and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. No studies have yet explored chemical modification at all positions of the crRNA guide and tracrRNA cofactor. Here, we have identified several heavily-modified versions of crRNA and tracrRNA that are more potent than their unmodified counterparts. In addition, we describe fully chemically modified crRNAs and tracrRNAs (containing no 2’-OH groups) that are functional in human cells. These designs demonstrate a significant breakthrough for Cas9-based therapeutics since heavily modified RNAs tend to be more stable in vivo (thus increasing potency). We anticipate that our designs will improve the use of Cas9 via RNP and mRNA delivery for in vivo and ex vivo purposes.

scholarly journals Retron Editing for Precise Genome Editing without Exogenous Donor DNA in Human Cells

2021 ◽  
Author(s):  
Xiangfeng Kong ◽  
Zikang Wang ◽  
Yingsi Zhou ◽  
Xing Wang ◽  
Linyu Shi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Human Cells ◽  
Exogenous Dna ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Non Coding Rna ◽  
Bacterial Genetic ◽  
Low Efficiency

CRISPR-Cas9 mediated seamless genome editing can be achieved by incorporating donor DNA into the CRISPR-Cas9 target loci via homology-directed repair (HDR), albeit with relative low efficiency due to the inefficient delivery of exogenous DNA. Retrons are bacterial genetic element composed of a non-coding RNA (ncRNA) and reverse transcriptase (RT). Retrons coupled with CRISPR-Cas9 have been shown to enhance precise genome editing via HDR in yeast through fusing guide RNA (gRNA) to the 3′ end of retron ncRNA, producing multicopy single-stranded DNA (msDNA) covalently tethered to gRNA. Here, we further engineered retrons by fusing Cas9 with E.coli RT from different clades and joining gRNA at the 5′ end of retron ncRNA, and found that retron editing can achieve precise genome editing efficiently in human cells. By co- expression of Cas9-RT fusions and retron-ncRNA gRNA (rgRNA) in HEK293T cells, we demonstrated the rates of retron editing at endogenous genomic loci was up to 10 %. We expect our retron editing system could aid in advancing the ex vivo and in vivo therapeutic applications of retron.

scholarly journals Synthetic CRISPR RNA-Cas9–guided genome editing in human cells

2015 ◽  
Vol 112 (51) ◽  
pp. E7110-E7117 ◽  
Author(s):  
Meghdad Rahdar ◽  
Moira A. McMahon ◽  
Thazha P. Prakash ◽  
Eric E. Swayze ◽  
C. Frank Bennett ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Disruption ◽  
Nuclease Activity ◽  
Human Cells ◽  
Chemical Modifications ◽  
Guide Rna ◽  
Chemically Modified ◽  
System Disease ◽  
Cas9 Nuclease ◽  
Nuclease Digestion

Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA–RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA. Taken together, these findings provide a platform for therapeutic applications, especially for nervous system disease, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 nuclease activity.

Genome editing with CRISPR-Cas9: A budding biological contrivance for colorectal carcinoma research and its perspective in molecular medicine

2020 ◽  
Vol 20 ◽  
Author(s):  
Suman Kumar Ray ◽  
Sukhes Mukherjee
Keyword(s):  
Colon Cancer ◽  
Colorectal Carcinoma ◽  
Genome Editing ◽  
Gene Editing ◽  
Ex Vivo ◽  
Resistance Mechanism ◽  
Molecular Medicine ◽  
Guide Rna ◽  
Guide Rnas

: Genome editing is an addition, deletion, or replacement of a gene for wiping out or initiating explicit and preferred characters in the genome. Utilizing gene editing tools like CRISPR-Cas9 technology could be accomplished either by gene-based methodology or protein based technology that has been under scrutiny for protracted time wherein physical techniques, viral and non-viral strategies have been utilized together. Transplanting ex vivo CRISPR edited cells empowers screening of single guide RNAs with high-throughput and CRISPR based screening in organoids transplantation to validate cancer cells including colorectal carcinoma in various phases of its development and treatment. CRISPR knockout screens have recognized genes driving an interest in the colon cancer develop hallmarks, outstandingly for the disclosure of drug resistance mechanism in some cancer cell lines with single guide RNA. A benefit of this approach is to deal with genomic screening of CRISPR knockout, disrupts gene expression, rather than the partial knockdown which are frequently accomplished with RNA interference and CRISPR-Cas technology. Due to its proficient editing of the target gene, along with CRISPR/Cas system, this technique is used in the treatment of diverse types of cancer. In recent time research showed that CRISPR/Cas gene editing tool potentially reformed expression of long non-coding RNA in colorectal carcinoma. CRISPR/Cas9 technology will positively fuel the advancement of further in vivo gene editing clinical trials in colon cancer for forthcoming days and will have an immense impact in molecular medicine.

scholarly journals Highly Potent GalNAc-Conjugated Tiny LNA Anti-miRNA-122 Antisense Oligonucleotides

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 817
Author(s):  
Tsuyoshi Yamamoto ◽  
Yahiro Mukai ◽  
Fumito Wada ◽  
Chisato Terada ◽  
Yukina Kayaba ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Ex Vivo ◽  
The Body ◽  
Locked Nucleic Acid ◽  
Imaging Studies ◽  
Phosphodiester Bond ◽  
Chemically Modified ◽  
Ex Vivo Imaging ◽  
Mirna Targeting

The development of clinically relevant anti-microRNA antisense oligonucleotides (anti-miRNA ASOs) remains a major challenge. One promising configuration of anti-miRNA ASOs called “tiny LNA (tiny Locked Nucleic Acid)” is an unusually small (~8-mer), highly chemically modified anti-miRNA ASO with high activity and specificity. Within this platform, we achieved a great enhancement of the in vivo activity of miRNA-122-targeting tiny LNA by developing a series of N-acetylgalactosamine (GalNAc)-conjugated tiny LNAs. Specifically, the median effective dose (ED50) of the most potent construct, tL-5G3, was estimated to be ~12 nmol/kg, which is ~300–500 times more potent than the original unconjugated tiny LNA. Through in vivo/ex vivo imaging studies, we have confirmed that the major advantage of GalNAc over tiny LNAs can be ascribed to the improvement of their originally poor pharmacokinetics. We also showed that the GalNAc ligand should be introduced into its 5′ terminus rather than its 3′ end via a biolabile phosphodiester bond. This result suggests that tiny LNA can unexpectedly be recognized by endogenous nucleases and is required to be digested to liberate the parent tiny LNA at an appropriate time in the body. We believe that our strategy will pave the way for the clinical application of miRNA-targeting small ASO therapy.

scholarly journals Transient non-integrative nuclear reprogramming promotes multifaceted reversal of aging in human cells

2019 ◽  
Author(s):  
Tapash Jay Sarkar ◽  
Marco Quarta ◽  
Shravani Mukherjee ◽  
Alex Colville ◽  
Patrick Paine ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Human Fibroblasts ◽  
Human Cells ◽  
Cellular Aging ◽  
Epigenetic Reprogramming ◽  
Epigenetic Clock ◽  
Nuclear Reprogramming ◽  
Loss Of Function ◽  
Age Related

SummaryAging is characterized by a gradual loss of function occurring at the molecular, cellular, tissue and organismal levels1-3. At the chromatin level, aging is associated with the progressive accumulation of epigenetic errors that eventually lead to aberrant gene regulation, stem cell exhaustion, senescence, and deregulated cell/tissue homeostasis3. The technology of nuclear reprogramming to pluripotency, through over-expression of a small number of transcription factors, can revert both the age and the identity of any cell to that of an embryonic cell by driving epigenetic reprogramming2,4,5. Recent evidence has shown that transient transgenic reprogramming can ameliorate age-associated hallmarks and extend lifespan in progeroid mice6. However, it is unknown how this form of ‘epigenetic rejuvenation’ would apply to physiologically aged cells and, importantly, how it might translate to human cells. Here we show that transient reprogramming, mediated by transient expression of mRNAs, promotes a rapid reversal of both cellular aging and of epigenetic clock in human fibroblasts and endothelial cells, reduces the inflammatory profile in human chondrocytes, and restores youthful regenerative response to aged, human muscle stem cells, in each case without abolishing cellular identity. Our method, that we named Epigenetic Reprogramming of Aging (ERA), paves the way to a novel, potentially translatable strategy for ex vivo cell rejuvenation treatment. In addition, ERA holds promise for in vivo tissue rejuvenation therapies to reverse the physiological manifestations of aging and the risk for the development of age-related diseases.

scholarly journals Reactivation of γ-globin in adult β-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing

Blood ◽  
2018 ◽  
Vol 131 (26) ◽  
pp. 2915-2928 ◽  
Author(s):  
Chang Li ◽  
Nikoletta Psatha ◽  
Pavel Sova ◽  
Sucheol Gil ◽  
Hongjie Wang ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Stem Cell ◽  
Binding Site ◽  
Genome Editing ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Cell Genome ◽  
Hsc Transplantation

Key Points CRISPR/Cas9-mediated disruption of a BCL11A binding site in HSCs of β-YAC mice results in the reactivation of γ-globin in erythrocytes. Our approach for in vivo HSC genome editing that does not require HSC transplantation and myeloablation should simplify HSC gene therapy.

scholarly journals Genome-Editing and Biomedical Cell Products: Current State, Safety and Efficacy

2018 ◽  
Vol 18 (3) ◽  
pp. 140-149 ◽  
Author(s):  
A. A. Goryaev ◽  
M. V. Savkina ◽  
K. M. Mefed ◽  
V. P. Bondarev ◽  
V. A. Merkulov ◽  
...  
Keyword(s):  
T Cells ◽  
Genome Editing ◽  
Ex Vivo ◽  
Rapid Development ◽  
The United States ◽  
Human Cells ◽  
The European Union ◽  
Antigen Receptors ◽  
Current State ◽  
Cd34 Cells

Advances in ex vivo technologies of human genome editing have made it possible to develop new approaches to the treatment of genetic, oncological, infectious and other diseases, which may involve the use of biomedical cell products. However, despite the rapid development of these technologies and a large number of clinical trials conducted in many countries around the world, only 4 products (Strimvelis, Zalmoxis, Kymriah and Yescarta) containing ex vivo genetically modified human cells are authorised for use in the European Union and the United States of America. This paper considers three promising technologies (ZFN, TALEN and CRISPR) that allow for easy and effective editing of the genome at the sites of interest, thereby creating a platform for further development of the genetic engineering of human cells. It describes the technology of engineering chimeric antigen receptors (CARs). It also provides data on the efficacy and safety of the approved products: Strimvelis which contains autologous CD34+ cells transduced ex vivo with a retroviral vector containing adenosine deaminase gene, Zalmoxis which contains modified allogeneic T-cells, and two products: Kymriah and Yescarta which contain autologous T-cells with CARs to CD19 antigen, intended for the treatment of CD19+ hematological malignancies.

scholarly journals In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations

2018 ◽  
Author(s):  
Pinar Akcakaya ◽  
Maggie L. Bobbin ◽  
Jimmy A. Guo ◽  
Jose M. Lopez ◽  
M. Kendell Clement ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Strong Support ◽  
Guide Rna ◽  
Genome Wide ◽  
Highly Sensitive ◽  
Further Development ◽  
Target Effects ◽  
Cas Gene

CRISPR-Cas genome-editing nucleases hold substantial promise for human therapeutics1–5 but identifying unwanted off-target mutations remains an important requirement for clinical translation6, 7. For ex vivo therapeutic applications, previously published cell-based genome-wide methods provide potentially useful strategies to identify and quantify these off-target mutation sites8–12. However, a well-validated method that can reliably identify off-targets in vivo has not been described to date, leaving the question of whether and how frequently these types of mutations occur. Here we describe Verification of In Vivo Off-targets (VIVO), a highly sensitive, unbiased, and generalizable strategy that we show can robustly identify genome-wide CRISPR-Cas nuclease off-target effects in vivo. To our knowledge, these studies provide the first demonstration that CRISPR-Cas nucleases can induce substantial off-target mutations in vivo, a result we obtained using a deliberately promiscuous guide RNA (gRNA). More importantly, we used VIVO to show that appropriately designed gRNAs can direct efficient in vivo editing without inducing detectable off-target mutations. Our findings provide strong support for and should encourage further development of in vivo genome editing therapeutic strategies.

scholarly journals Prime editing primarily induces undesired outcomes in mice

2020 ◽  
Author(s):  
Tomomi Aida ◽  
Jonathan J. Wilde ◽  
Lixin Yang ◽  
Yuanyuan Hou ◽  
Mengqi Li ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Frequency ◽  
Mouse Embryos ◽  
Biomedical Science ◽  
New Approach ◽  
Chemically Modified ◽  
Highly Efficient ◽  
Large Deletions

SummaryGenome editing has transformed biomedical science, but is still unpredictable and often induces undesired outcomes. Prime editing (PE) is a promising new approach due to its proposed flexibility and ability to avoid unwanted indels. Here, we show highly efficient PE-mediated genome editing in mammalian zygotes. Utilizing chemically modified guideRNAs, PE efficiently introduced 10 targeted modifications including substitutions, deletions, and insertions across 6 genes in mouse embryos. However, we unexpectedly observed a high frequency of undesired outcomes such as large deletions and found that these occurred more often than pure intended edits across all of the edits/genes. We show that undesired outcomes result from the double-nicking PE3 strategy, but that omission of the second nick largely ablates PE function. However, sequential double-nicking with PE3b, which is only applicable to a fraction of edits, eliminated undesired outcomes. Overall, our findings demonstrate the promising potential of PE for predictable, flexible, and highly efficient in vivo genome editing, but highlight the need for improved variations of PE before it is ready for widespread use.

Sign in / Sign up

Export Citation Format

Share Document