International Journal of Molecular Sciences

Editorial

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6 pages, 804 KiB

Open AccessEditorial

On the Origins of Homology Directed Repair in Mammalian Cells

by Brett M. Sansbury and Eric B. Kmiec

Int. J. Mol. Sci. 2021, 22(7), 3348; https://doi.org/10.3390/ijms22073348 - 25 Mar 2021

Cited by 1 | Viewed by 1898

Abstract

Over the course of the last five years, expectations surrounding our capacity to selectively modify the human genome have never been higher. The reduction to practice site-specific nucleases designed to cleave at a unique site within the DNA is now centerstage in the [...] Read more.

Over the course of the last five years, expectations surrounding our capacity to selectively modify the human genome have never been higher. The reduction to practice site-specific nucleases designed to cleave at a unique site within the DNA is now centerstage in the development of effective molecular therapies. Once viewed as being impossible, this technology now has great potential and, while cellular and molecular barriers persist to clinical implementations, there is little doubt that these barriers will be crossed, and human beings will soon be treated with gene editing tools. The most ambitious of these desires is the correction of genetic mutations resident within the human genome that are responsible for oncogenesis and a wide range of inherited diseases. The process by which gene editing activity could act to reverse these mutations to wild-type and restore normal protein function has been generally categorized as homology directed repair. This is a catch-all basket term that includes the insertion of short fragments of DNA, the replacement of long fragments of DNA, and the surgical exchange of single bases in the correction of point mutations. The foundation of homology directed repair lies in pioneering work that unravel the mystery surrounding genetic exchange using single-stranded DNA oligonucleotides as the sole gene editing agent. Single agent gene editing has provided guidance on how to build combinatorial approaches to human gene editing using the remarkable programmable nuclease complexes known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their closely associated (Cas) nucleases. In this manuscript, we outline the historical pathway that has helped evolve the current molecular toolbox being utilized for the genetic re-engineering of the human genome. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms of CRISPR-Directed Gene Repair)

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Research

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11 pages, 9979 KiB

Open AccessArticle

Mismatch Intolerance of 5′-Truncated sgRNAs in CRISPR/Cas9 Enables Efficient Microbial Single-Base Genome Editing

by Ho Joung Lee, Hyun Ju Kim and Sang Jun Lee

Int. J. Mol. Sci. 2021, 22(12), 6457; https://doi.org/10.3390/ijms22126457 - 16 Jun 2021

Cited by 12 | Viewed by 3099

Abstract

The CRISPR/Cas9 system has recently emerged as a useful gene-specific editing tool. However, this approach occasionally results in the digestion of both the DNA target and similar DNA sequences due to mismatch tolerance, which remains a significant drawback of current genome editing technologies. [...] Read more.

The CRISPR/Cas9 system has recently emerged as a useful gene-specific editing tool. However, this approach occasionally results in the digestion of both the DNA target and similar DNA sequences due to mismatch tolerance, which remains a significant drawback of current genome editing technologies. However, our study determined that even single-base mismatches between the target DNA and 5′-truncated sgRNAs inhibited target recognition. These results suggest that a 5′-truncated sgRNA/Cas9 complex could be used to negatively select single-base-edited targets in microbial genomes. Moreover, we demonstrated that the 5′-truncated sgRNA method can be used for simple and effective single-base editing, as it enables the modification of individual bases in the DNA target, near and far from the 5′ end of truncated sgRNAs. Further, 5′-truncated sgRNAs also allowed for efficient single-base editing when using an engineered Cas9 nuclease with an expanded protospacer adjacent motif (PAM; 5′-NG), which may enable whole-genome single-base editing. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms of CRISPR-Directed Gene Repair)

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11 pages, 3496 KiB

Open AccessArticle

Analysis of NHEJ-Based DNA Repair after CRISPR-Mediated DNA Cleavage

by Beomjong Song, Soyeon Yang, Gue-Ho Hwang, Jihyeon Yu and Sangsu Bae

Int. J. Mol. Sci. 2021, 22(12), 6397; https://doi.org/10.3390/ijms22126397 - 15 Jun 2021

Cited by 28 | Viewed by 5992

Abstract

Genome editing using CRISPR-Cas9 nucleases is based on the repair of the DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed repair (HDR), non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) pathways. Among these, it is thought that the [...] Read more.

Genome editing using CRISPR-Cas9 nucleases is based on the repair of the DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed repair (HDR), non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) pathways. Among these, it is thought that the NHEJ pathway is dominant and occurs throughout a cell cycle. NHEJ-based DSB repair is known to be error-prone; however, there are few studies that delve into it deeply in endogenous genes. Here, we quantify the degree of NHEJ-based DSB repair accuracy (termed NHEJ accuracy) in human-originated cells by incorporating exogenous DNA oligonucleotides. Through an analysis of joined sequences between the exogenous DNA and the endogenous target after DSBs occur, we determined that the average value of NHEJ accuracy is approximately 75% in maximum in HEK 293T cells. In a deep analysis, we found that NHEJ accuracy is sequence-dependent and the value at the DSB end proximal to a protospacer adjacent motif (PAM) is relatively lower than that at the DSB end distal to the PAM. In addition, we observed a negative correlation between the insertion mutation ratio and the degree of NHEJ accuracy. Our findings would broaden the understanding of Cas9-mediated genome editing. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms of CRISPR-Directed Gene Repair)

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15 pages, 3649 KiB

Open AccessArticle

CRISPR-Cas9–Mediated TIM3 Knockout in Human Natural Killer Cells Enhances Growth Inhibitory Effects on Human Glioma Cells

by Takayuki Morimoto, Tsutomu Nakazawa, Ryosuke Matsuda, Fumihiko Nishimura, Mitsutoshi Nakamura, Shuichi Yamada, Ichiro Nakagawa, Young-Soo Park, Takahiro Tsujimura and Hiroyuki Nakase

Int. J. Mol. Sci. 2021, 22(7), 3489; https://doi.org/10.3390/ijms22073489 - 28 Mar 2021

Cited by 28 | Viewed by 5134

Abstract

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Natural Killer (NK) cells are potent cytotoxic effector cells against tumor cells inducing GBM cells; therefore, NK cell based- immunotherapy might be a promising target in GBM. T cell [...] Read more.

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Natural Killer (NK) cells are potent cytotoxic effector cells against tumor cells inducing GBM cells; therefore, NK cell based- immunotherapy might be a promising target in GBM. T cell immunoglobulin mucin family member 3 (TIM3), a receptor expressed on NK cells, has been suggested as a marker of dysfunctional NK cells. We established TIM3 knockout in NK cells, using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). Electroporating of TIM3 exon 2- or exon 5-targeting guide RNA- Cas9 protein complexes (RNPs) inhibited TIM3 expression on NK cells with varying efficacy. T7 endonuclease I mutation detection assays showed that both RNPs disrupted the intended genome sites. The expression of other checkpoint receptors, i.e., programmed cell death 1 (PD1), Lymphocyte-activation gene 3 (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and TACTILE (CD96) were unchanged on the TIM3 knockout NK cells. Real time cell growth assays revealed that TIM3 knockout enhanced NK cell–mediated growth inhibition of GBM cells. These results demonstrated that TIM3 knockout enhanced human NK cell mediated cytotoxicity on GBM cells. Future, CRISPR-Cas9 mediated TIM3 knockout in NK cells may prove to be a promising immunotherapeutic alternative in patient with GBM. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms of CRISPR-Directed Gene Repair)

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Review

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18 pages, 1723 KiB

Open AccessReview

Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing

by Christopher E. Denes, Alexander J. Cole, Yagiz Alp Aksoy, Geng Li, Graham Gregory Neely and Daniel Hesselson

Int. J. Mol. Sci. 2021, 22(16), 8571; https://doi.org/10.3390/ijms22168571 - 9 Aug 2021

Cited by 13 | Viewed by 9648

Abstract

Modification of the human genome has immense potential for preventing or treating disease. Modern genome editing techniques based on CRISPR/Cas9 show great promise for altering disease-relevant genes. The efficacy of precision editing at CRISPR/Cas9-induced double-strand breaks is dependent on the relative activities of [...] Read more.

Modification of the human genome has immense potential for preventing or treating disease. Modern genome editing techniques based on CRISPR/Cas9 show great promise for altering disease-relevant genes. The efficacy of precision editing at CRISPR/Cas9-induced double-strand breaks is dependent on the relative activities of nuclear DNA repair pathways, including the homology-directed repair and error-prone non-homologous end-joining pathways. The competition between multiple DNA repair pathways generates mosaic and/or therapeutically undesirable editing outcomes. Importantly, genetic models have validated key DNA repair pathways as druggable targets for increasing editing efficacy. In this review, we highlight approaches that can be used to achieve the desired genome modification, including the latest progress using small molecule modulators and engineered CRISPR/Cas proteins to enhance precision editing. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms of CRISPR-Directed Gene Repair)

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15 pages, 2313 KiB

Open AccessReview

A Consensus Model of Homology-Directed Repair Initiated by CRISPR/Cas Activity

by Kevin Bloh and Natalia Rivera-Torres

Int. J. Mol. Sci. 2021, 22(8), 3834; https://doi.org/10.3390/ijms22083834 - 7 Apr 2021

Cited by 3 | Viewed by 2742

Abstract

The mechanism of action of ssODN-directed gene editing has been a topic of discussion within the field of CRISPR gene editing since its inception. Multiple comparable, but distinct, pathways have been discovered for DNA repair both with and without a repair template oligonucleotide. [...] Read more.

The mechanism of action of ssODN-directed gene editing has been a topic of discussion within the field of CRISPR gene editing since its inception. Multiple comparable, but distinct, pathways have been discovered for DNA repair both with and without a repair template oligonucleotide. We have previously described the ExACT pathway for oligo-driven DNA repair, which consisted of a two-step DNA synthesis-driven repair catalyzed by the simultaneous binding of the repair oligonucleotide (ssODN) upstream and downstream of the double-strand break. In order to better elucidate the mechanism of ExACT-based repair, we have challenged the assumptions of the pathway with those outlines in other similar non-ssODN-based DNA repair mechanisms. This more comprehensive iteration of the ExACT pathway better described the many different ways where DNA repair can occur in the presence of a repair oligonucleotide after CRISPR cleavage, as well as how these previously distinct pathways can overlap and lead to even more unique repair outcomes. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms of CRISPR-Directed Gene Repair)

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42 pages, 8016 KiB

Open AccessReview

Novel CRISPR–Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives

by Sweta Nidhi, Uttpal Anand, Patrik Oleksak, Pooja Tripathi, Jonathan A. Lal, George Thomas, Kamil Kuca and Vijay Tripathi

Int. J. Mol. Sci. 2021, 22(7), 3327; https://doi.org/10.3390/ijms22073327 - 24 Mar 2021

Cited by 107 | Viewed by 24099

Abstract

According to Darwin’s theory, endless evolution leads to a revolution. One such example is the Clustered Regularly Interspaced Palindromic Repeats (CRISPR)–Cas system, an adaptive immunity system in most archaea and many bacteria. Gene editing technology possesses a crucial potential to dramatically impact miscellaneous [...] Read more.

According to Darwin’s theory, endless evolution leads to a revolution. One such example is the Clustered Regularly Interspaced Palindromic Repeats (CRISPR)–Cas system, an adaptive immunity system in most archaea and many bacteria. Gene editing technology possesses a crucial potential to dramatically impact miscellaneous areas of life, and CRISPR–Cas represents the most suitable strategy. The system has ignited a revolution in the field of genetic engineering. The ease, precision, affordability of this system is akin to a Midas touch for researchers editing genomes. Undoubtedly, the applications of this system are endless. The CRISPR–Cas system is extensively employed in the treatment of infectious and genetic diseases, in metabolic disorders, in curing cancer, in developing sustainable methods for fuel production and chemicals, in improving the quality and quantity of food crops, and thus in catering to global food demands. Future applications of CRISPR–Cas will provide benefits for everyone and will save countless lives. The technology is evolving rapidly; therefore, an overview of continuous improvement is important. In this review, we aim to elucidate the current state of the CRISPR–Cas revolution in a tailor-made format from its discovery to exciting breakthroughs at the application level and further upcoming trends related to opportunities and challenges including ethical concerns. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms of CRISPR-Directed Gene Repair)

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22 pages, 1247 KiB

Open AccessReview

CRISPR/Cas Technology in Pig-to-Human Xenotransplantation Research

by Natalia Ryczek, Magdalena Hryhorowicz, Joanna Zeyland, Daniel Lipiński and Ryszard Słomski

Int. J. Mol. Sci. 2021, 22(6), 3196; https://doi.org/10.3390/ijms22063196 - 21 Mar 2021

Cited by 25 | Viewed by 9269

Abstract

CRISPR/Cas (clustered regularly interspaced short palindromic repeats linked to Cas nuclease) technology has revolutionized many aspects of genetic engineering research. Thanks to it, it became possible to study the functions and mechanisms of biology with greater precision, as well as to obtain genetically [...] Read more.

CRISPR/Cas (clustered regularly interspaced short palindromic repeats linked to Cas nuclease) technology has revolutionized many aspects of genetic engineering research. Thanks to it, it became possible to study the functions and mechanisms of biology with greater precision, as well as to obtain genetically modified organisms, both prokaryotic and eukaryotic. The changes introduced by the CRISPR/Cas system are based on the repair paths of the single or double strand DNA breaks that cause insertions, deletions, or precise integrations of donor DNA. These changes are crucial for many fields of science, one of which is the use of animals (pigs) as a reservoir of tissues and organs for xenotransplantation into humans. Non-genetically modified animals cannot be used to save human life and health due to acute immunological reactions resulting from the phylogenetic distance of these two species. This review is intended to collect and summarize the advantages as well as achievements of the CRISPR/Cas system in pig-to-human xenotransplantation research. In addition, it demonstrates barriers and limitations that require careful evaluation before attempting to experiment with this technology. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms of CRISPR-Directed Gene Repair)

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