path: root/gnqa/paper1_eval/src/data/responses/general/experts/05.json

{
    "created_at": "2024-06-01T14:26:03.986157Z",
    "data": {
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        "answer": "The potential benefits of gene editing technologies like CRISPR-Cas9 include the ability to modify genes for the treatment of diseases, improvement of crop species, and the development of personalized drug or cell therapies. It can also be used for functional screening in the development of therapies and for the study of molecular causes of ageing. However, there are risks associated with these technologies. These include off-target effects, which can lead to unwanted mutations, and the potential for wide-ranging deletions or recombination events. There's also a risk of triggering a P53 response leading to apoptosis in cycling cells, and the potential for subjects to generate antibodies to Cas9, which could limit gene therapies. Furthermore, the long-term safety of CRISPR genome editing in humans is yet to be determined.",
        "context": {
            "03110c8a-1232-40c2-8380-c9feb8b6468c": [
                {
                    "document_id": "03110c8a-1232-40c2-8380-c9feb8b6468c",
                    "text": "\n\nGene editing has gained considerable interest with the identification of the CRISPR-Cas9 system, 27 which allows for a targeted modification in the DNA sequence of an organism.Researchers can utilize their knowledge of the basic biology of the gene and its protein function to precisely change the DNA sequence, thus altering the protein function of the gene and allowing for edits to stay within the species.Researchers at the University of Missouri used the CRISPR-Cas9 system to modify the CD163 gene such that the PRRS virus is not able to replicate inside the pig. 28This slight modification of the swine genome through gene editing keeps the pigs from succumbing to PRRS which has an annual estimated loss to the United States swine industry of over $660 million per year.Despite this benefit, given the public's concerns over food safety, it is likely that approval for such technology is years away in the US, Canada and Europe.However, in some cultures, there is a wide range of non-livestock species that are consumed.Therefore, it is conceivable that these countries and cultures may be open to transgenic/gene edited livestock.They may see the importance of useful gene editing which may lead to approval and consumption of reasonable genetically edited animal products such as those with modifications that are already found in nature or those that offer a substantial welfare benefit to society."
                }
            ],
            "1942712a-a39d-44f7-9b2d-609926374cbd": [
                {
                    "document_id": "1942712a-a39d-44f7-9b2d-609926374cbd",
                    "text": "\n\nAs a researcher who has devoted an entire career since 1994 to the development of genome editing tools and methods, I have been amazed by the rapid progress in the field over the last few years.Considering the widespread use of the tools, I am sure that the pace will continue to accelerate.Indeed, programmable nucleases, may eventually enable humans-products of evolution-to become masters of evolution.delivered preassembled recombinant Cas9-guide RNA ribonucleoproteins (RNPs) into animal embryos 6,9 and plant 11 and mammalian cells [73][74][75] .Indeed, Cas9 RNPs were rapidly turned over in cells 73 , reducing off-target effects and mosaicism in gene-edited organisms 11 .Cas9 RNPs can be delivered into cells by various methods, including microinjection 6,9 , electroporation 73 , lipofection 74 and protein transduction 75 .Importantly-and unlike in conventional gene therapy, where therapeutic genes are delivered via plasmids or viral vectors-Cas9 RNP delivery does not involve the use of exogenous DNA; host innate immune responses against foreign DNA are not elicited, and undesired integration of foreign DNA into the host genome is avoided."
                }
            ],
            "33f1abde-a821-483b-b8b4-785f499db09d": [
                {
                    "document_id": "33f1abde-a821-483b-b8b4-785f499db09d",
                    "text": "\n\nIn comparison to a transgenic approach, a gene editing technique such as CRISPR-Cas9 offers the advantage that gene-edited crops are not considered genetically modified organism (GMO) in some countries, such as the US, where the demand for natural food colorants such as anthocyanins is high.Indeed, the use of GMO crops as a source of natural pigments may be inconsistent with consumer interests.However, carrot cultivars engineered with either the transgenic or gene editing approach have not been reported so far, but their development is possible."
                }
            ],
            "4f709611-ea0b-4bcc-a634-df5d518ccb54": [
                {
                    "document_id": "4f709611-ea0b-4bcc-a634-df5d518ccb54",
                    "text": "\n\nThe notable accuracy and versatility of CRISPR-Cas for genome editing also opened the door to its use in preclinical and translational settings.In the latter case, CRISPR in vivo gene editing has led to several proof-of-concept studies that would have been unachievable without it, as in the first ever correction of inherited pathogenic mutations linked to degenerative disease in a living organism [22] and even shown to be possible in human embryos [23,24].It also has great potential in the field of precision medicine as large-scale population DNA sequencing studies have provided vast amounts of information linking particular diseases with specific genetic mutations which could, in theory, be targeted through CRISPR [25,26].This could be used during the identification and validation of potential DNA targets during the development of personalised drug or cell therapies, which will require the generation of engineered cell lines and/or animal models.Techniques such as HDR-mediated gene targeting are too labour intensive, with low targeting efficiencies and long times necessary for their establishment, and consequently are not ideally suited for drug discovery purposes.Conversely, CRISPR-Cas has been proven to be efficient for editing virtually any kind of cell line, from primary immune cells to induced pluripotent stem cells (iPSCs) [27,28].Additionally, CRISPR can also be used for functional screening in the development of combined inhibitory therapy aimed at strengthening the efficiency of targeted therapeutics.An example of the latter is shown in a study where a variation of the technology known as CRISPR interference (CRISPRi) was used in genome-wide scale to identify different survival pathways used by cancer cells after oncogene inactivation and allowing the identification of successful combination therapies [29].In terms of translational applications, the overall safety of CRISPR genome editing in humans will require long-term scrutiny before its adoption in the clinic.Nonetheless, a number of CRISPR-based clinical trials are currently in progress, including studies focused on targeting patients' own T cells in order to improve the immune response towards some forms of malignant cancer [30,31], and others aimed at correcting pathogenic mutations in the hematopoietic cells of patients with beta-thalassemia and sickle cell disease [32]."
                },
                {
                    "document_id": "4f709611-ea0b-4bcc-a634-df5d518ccb54",
                    "text": "Caveats and Ethical Concerns of CRISPR-Cas Applications\n\nDespite the presence of both a PAM sequence and a specific gRNA, the CRISPR-Cas9 system is not infallible.In fact, DSBs can occur at different sites in the genome, potentially causing so-called \"off-target\" effects.This eventuality remains to date the biggest concern in the field, as possible undesirable modifications must be properly identified and followed in order to guarantee safety for medical purposes.Nevertheless, there is still little evidence of the biological consequence of Cas9 off-target effects.Two recent studies describe new methods to investigate potential off-target effects in both mammals and plants [33,34].In both cases, whole-genome sequencing revealed that selective nucleotide changes, such as conversion of an adenine to a guanine, caused off-target occurrence very rarely, with a frequency comparable to the one of spontaneous mutations.However, substitution of a cytosine with a thymidine was linked to a sizable number of off-target mutations.This newly acquired information adds to the plethora of studies conducted on the safety of CRISPR, which altogether highlight the need for the establishment of clinical standards for the future use of genome-editing techniques in the clinic.Despite this and other technical challenges still ahead for CRISPR genome editing, the pace at which this technology has developed in recent years suggests many of these concerns could be addressed soon, as long as proper ethical guidelines and regulatory mechanisms are established."
                },
                {
                    "document_id": "4f709611-ea0b-4bcc-a634-df5d518ccb54",
                    "text": "Conclusions\n\nThere is no reason to doubt that the development of CRISPR-Cas genome editing represents an unprecedented breakthrough in modern science, as it has potential applications in a wide array of disciplines ranging from agriculture, zoology and renewable energy to biomedicine and synthetic biology.This powerful tool holds promise for further elucidating the molecular causes of ageing by allowing scientists to probe genetic and epigenetic pathways with a level of sophistication that was unattainable just a few years ago.It will allow so in traditional animal and cell models of ageing, but it will also drastically accelerate the generation of refined versions of those models or even allow the development of new research approaches in non-model organisms.Moreover, CRISPR-based genome editing is already having a significant impact in research aiming to understand the cellular and molecular origins of age-related diseases, as well as developing potential treatments against them.The application of CRISPR-Cas gene editing for the treatment of age-related diseases is not over the horizon yet, as it will require the identification of causative genes and their role under a variety of contexts that could be as diverse as the ageing process is across individuals.However, CRISPR-Cas might also hold the key for solving such conundrum, as it has opened the way for achieving true personalised medicine by providing both the precision and scalability required for conducting genome-wide functional screens during the refinement of drug-and cell-based therapies for age-related diseases."
                },
                {
                    "document_id": "4f709611-ea0b-4bcc-a634-df5d518ccb54",
                    "text": "\n\nSince its discovery, CRISPR-Cas technology has ignited a biological revolution by providing a highly versatile platform that allows fast and efficient genome editing in an ever-growing list of organisms.In this chapter we will first describe the most recent advances in the development and application of the CRISPR-Cas platform in biomedical research.Then we will discuss the most recent and notable basic research applications of this technology in the study of the molecular causes of ageing.Finally, we will review how CRISPR-Cas has been used for creating new models for the study of age-related diseases, as well as for manipulating diseaseassociated gene pathways."
                }
            ],
            "50c72e55-b5fe-42a6-b837-64c28620a4c0": [
                {
                    "document_id": "50c72e55-b5fe-42a6-b837-64c28620a4c0",
                    "text": "Caveats of advanced genome editing tools\n\nOff-target effects.The DNA-binding domains of ZFNs and TALENs need to be very specific for the target site to avoid off-target cleavage, which results in unwanted mutations and potentially cytotoxic effects [27].CRISPR/Cas9 is also known to generate off-target alterations, albeit apparently at low incidence [28,29], since mispairing is allowed between the guide RNA and the genomic DNA.Nonetheless, caution is required in their design and use.Some strategies involving the optimization of the guide RNA/Cas9 include using of software tools to predict potential off-target sites (http://omictools.com/crispr-cas9-Figure1: Genome editing methodologies which can be applied to human pluripotent stem cells.Homologous recombination (HR), or the more advanced tools such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system can be applied to human pluripotent stem cells (hPSCs) either to 1) create naturally occurring mutations or 2) repair a mutation to generate isogenic controls in hPSCs, to understand the function of a gene of interest.c1268-p1.html),truncating the guide RNA (<20 nucleotides) to decrease off-target mutagenesis [30], lowering the dosage of guide RNA and Cas9 plasmids, and decreasing the number of mismatches between the guide RNA and the genomic DNA.A \"double nick\" system with Cas9 nickase, which contains a single inactive catalytic domain, may also be used [31e33]."
                }
            ],
            "52480703-5353-4e55-a06b-110fd59db3a6": [
                {
                    "document_id": "52480703-5353-4e55-a06b-110fd59db3a6",
                    "text": "CRISPR screening technologies\n\nThe discovery of CRISPR-Cas9 as a sequence-specific programmable nuclease democratized gene editing and fueled progress in forward genetic screening [20 , 66] .Genetic screens using Cas9 with a pooled singleguide RNA (sgRNA) library allow the interrogation of seemingly all genes in a genome in a single experiment [96 , 97] [null] .Engineered Cas9 variants further extend the versatility of forward genetic screening.Catalytically inactive Cas9 (dCas9) fused with chromatin effector domains permit specific activation (CRISPRa) or inhibition (CRISPRi) of gene expression [37 , 54] .Recently developed and emerging technologies -base editors, prime editors, and Cas transposases -are beginning to enable new types of genetic screens with directed, controlled, and on demand mutations by allowing the creation of user specified modifications, such as single base conversion, deletions, and insertions [4 , 42 , 58] ."
                }
            ],
            "801c9288-70c9-4d14-b8bc-13ee6708803a": [
                {
                    "document_id": "801c9288-70c9-4d14-b8bc-13ee6708803a",
                    "text": "\n\nComing on the heels of engineered nucleases, CRISPR-Cas9 tools have accelerated the pace of genomic research by permitting highly efficient knockouts or edits of virtually any gene in cells or model organisms.Multiple CRISPR-Cas9-based clinical trials are in progress or are expected to begin soon.Although Cas9engineered cells haven't yet demonstrated efficacy at scale, early trial results suggest that such cells are stable and don't cause acute adverse reactions in humans.Long-term safety is yet to be determined.Current applications largely focus on single-gene disorders for which gene editing can be carried out ex vivo on appropriate cells, such as bone marrow hematopoietic stem cells in the case of sickle cell anemia.Exploration is under way to develop delivery systems that can target the gene-editing apparatus to the appropriate tissue in vivo."
                },
                {
                    "document_id": "801c9288-70c9-4d14-b8bc-13ee6708803a",
                    "text": "\n\nOver the past 8 years, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) technologies have emerged as accessible and adaptable tools for studying and altering genomes. 5RISPR-Cas9 can be used to induce genome edits by creating targeted DNA breaks that trigger site-specific DNA repair.In nextgeneration formats, it can also control the transcriptional output of genes or alter genome sequences using a process of nucleotide base editing that does not require repair of DNA breaks.As these technologies continue to mature, it will become increasingly possible to alter cellular genomes efficiently and accurately."
                }
            ],
            "a7f21808-dce3-4110-8e7c-ceb2437e72ff": [
                {
                    "document_id": "a7f21808-dce3-4110-8e7c-ceb2437e72ff",
                    "text": "\n\nThe type II CRISPR-Cas9 systems, repurposed from prokaryotic adaptive immune responses, are now widely used for targeted genome modifications in plants, animals, and human cells (Kim et al. 2014;Woo et al. 2015;Zuris et al. 2015).In particular, Cas9 nucleases have shown promise for gene and cell therapy (Maeder and Gersbach 2016).Typically, these nucleases are expressed or delivered in vivo using plasmid DNA or viruses (Yin et al. 2014;Ran et al. 2015).However, plasmid DNA delivery is often inefficient, especially in vivo, and can cause integration of small plasmid fragments degraded by endogenous nucleases at on-target and offtarget sites in the genome (Kim et al. 2014).Viral delivery of Cas9 can be highly efficient in vivo (Ran et al. 2015;Long et al. 2016;Nelson et al. 2016;Tabebordbar et al. 2016), but may be hampered by antibodies or T cells induced against the protein (Shankar et al. 2007;Calcedo et al. 2015;Chew et al. 2016).We and others have shown that preassembled Cas9 ribonucleoproteins (RNPs) can be delivered to human primary and stem cells and mice to modify target genes (Kim et al. 2014;Schumann et al. 2015;Zuris et al. 2015).Cas9 RNPs are rapidly turned over in cells, reducing off-target effects.Furthermore, Cas9 RNPs are unlikely to be limited by host immune systems because they function and disappear before the generation of antibodies and T cells directed against them.Currently, despite these advantages of RNPs, the difficult delivery of Cas9 RNPs in vivo limits its utility for therapeutic applications (Zuris et al. 2015).Here, we show that in vivo genome editing of an wild-type gene, whose up-regulation is responsible for pathogenesis, could be a new therapeutic modality for the treatment of nongenetic degenerative diseases.Our ultimate goal is to harness Cas9 RNPs for a clinical application of therapeutic genome surgery in patients with AMD."
                }
            ],
            "ac00c552-7514-49d4-9e90-ab01c22472ae": [
                {
                    "document_id": "ac00c552-7514-49d4-9e90-ab01c22472ae",
                    "text": "\n\nClustered regularly interspaced short palindromic repeat (CRISPR)-Cas nucleases have revolutionized the field of gene editing and have tremendous application in the field of molecular medicine [98][99][100][101][102].Despite a significant surge in CRISPR/Cas9mediated genome editing in various disease models, the progress in the field of AD has lagged behind substantially.We believe that genome editing can significantly improve the development of AD models and also create novel opportunities for the development of the next generation precision targeted AD gene and stem cell therapies.Since there are several excellent review articles on CRISPR/Cas9-mediated genome editing, here we will limit our focus on select recent articles that are noteworthy.CRISPR/Cas9 system can be engineered to either activate transcription (gain-of-function) or achieve gene silencing (Loss-of-function).Dahlman et al. have developed a CRISPR-based system that uses catalytically active Cas9 and distinct single guide (sgRNA) constructs to activate and knockout different genes in the same cell [103].Konermann et al. have used structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci [104].Using crystallographic studies, they have engineered a combination of sgRNA2.0,NLS-dCas9-VP64 and MS2-p65-HSF1 to develop one of the most effective transcription activation system."
                }
            ],
            "b72eb0d1-50e3-4def-94bc-abf77891f519": [
                {
                    "document_id": "b72eb0d1-50e3-4def-94bc-abf77891f519",
                    "text": "Limitations of CRISPR-Cas9\n\nCRISPR provides a simple and easy tool not only for in vitro use but potentially also for in vivo genome editing.However, there are limitations and downsides to this approach.First, and despite considerable improvements in the technology, the risk of the offtarget effect remains and must be considered carefully.Second, DSB may lead to wide-ranging deletions or recombination events involving the on-target site (204).Third, in cycling cells, DNA double strand breaks caused by Cas9 cleavage may trigger a P53 response leading to apoptosis and enrichment for potentially oncogenic P53-deficient cells (205,206).Fourth, subjects may generate antibodies to Cas9, potentially limiting gene therapies (207,208)."
                },
                {
                    "document_id": "b72eb0d1-50e3-4def-94bc-abf77891f519",
                    "text": "\n\nGenome editing tools that target the desired genomic region and allow for variants to be altered (e.g. from risk to protective), or for more substantial changes to be made (e.g. the deletion of a longer stretch of DNA harbouring a number of variants) and can help to answer each of these questions.These technologies are evolving rapidly (Figure 1 and Table 2).The most recently developed of these, Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technology, originally developed by Doudna, Charpentier and their colleagues (72,73) and Zhang and his colleagues (50) has become a widely used tool for this purpose.Engineered CRISPR/Cas9 technology uses a guide RNA (gRNA) to direct CRISPR-associated endonuclease (Cas) to the target DNA and generate a double strand DNA break.Correction of a mutation or variant in the target DNA sequence can then be carried out by homology-directed DNA repair (HDR) with a donor template.Since its discovery eight years ago, CRISPR technology has evolved quickly to be a critical part of the molecular biologist's toolbox."
                }
            ],
            "c3ae2186-ef48-46a5-b214-dc944366df8f": [
                {
                    "document_id": "c3ae2186-ef48-46a5-b214-dc944366df8f",
                    "text": "INTRODUCTION\n\nGenome editing technologies based on the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 enable rapid and efficient modification of endogenous genes in a variety of cell types, allowing for analysis of gene function in many organs in vivo.CRISPR-Cas9 induces DNA double strand breaks (DSBs) at single-guide RNA (sgRNA)-specific loci in the genome, which are repaired through either non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathways.While NHEJ introduces unpredictable pattern of insertion or deletion (indel) mutations, HDR directs a precise recombination event between a homologous DNA donor template and the damaged DNA site (Cong et al., 2013;Cox et al., 2015;Doudna and Charpentier, 2014;Heidenreich and Zhang, 2016;Jinek et al., 2012;Mali et al., 2013;Sander and Joung, 2014;Wang et al., 2013;Yang et al., 2013).Thus, HDR can be used to precisely introduce sequence insertions, deletions or mutations by encoding the desired changes in the donor template DNA."
                }
            ],
            "d14e93b5-01de-4208-8255-baae7898a7bb": [
                {
                    "document_id": "d14e93b5-01de-4208-8255-baae7898a7bb",
                    "text": "\nCRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development.What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA.Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions.For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene.In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening.In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening."
                },
                {
                    "document_id": "d14e93b5-01de-4208-8255-baae7898a7bb",
                    "text": "\n\nCRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development.What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA.Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions.For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene.In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening.In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening."
                },
                {
                    "document_id": "d14e93b5-01de-4208-8255-baae7898a7bb",
                    "text": "\n\nThe recent development of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 for experimental purposes has dismantled the perception that genome editing technology is off-limits for screening in mammalian systems (Heintze et al., 2013).Since this system employs the basic principle of Watson-Crick base pairing for gene targeting, generation of libraries with whole-genome target coverage is relatively easy and cost-effective.For instance, simple protocols are available to synthesize pooled lentiviral libraries by in silico design of oligonucleotides, which can then be cloned, packaged and delivered to cells by viral transduction (Paddison et al., 2004;LeProust et al., 2010).Similarly, the generation of arrayed libraries can be achieved by following protocols originally developed for arrayed shRNA library production that have been in use for a number of years (Moffat et al., 2006).All in all, the stage is set for CRISPR to make an enormous impact on genomic screening and thus scientific discovery in the coming years, and recent demonstrations of this system have shown great promise (Shalem et al., 2015).However, a number of technical challenges must be addressed in order to maximize the benefit of this technology.In this review, we will discuss current applications of CRISPR in functional genomics and provide a perspective on future developments in this area."
                }
            ],
            "e5cf067c-8be0-4b0a-b376-7882cdc9d96c": [
                {
                    "document_id": "e5cf067c-8be0-4b0a-b376-7882cdc9d96c",
                    "text": "Genome editing for crop improvement\n\nReports of CRISPR-Cas9-based genome editing first appeared in 2013 (Cong et al., 2013;Feng et al., 2013;Mao et al., 2013).Since then, genome editing technologies have proven to be powerful and efficient tools for the improvement of many crop species.At present, genome editing has been widely used to introduce/modify agronomically important traits, such as increased yield, improved nutritional quality, and resistance to biotic and abiotic stresses, in multiple crops, including rice, wheat, maize, tomato, and potato (Lu et al., 2017;Soyk et al., 2017;Tang et al., 2017;D'Ambrosio et al., 2018;Ye et al., 2018;Miao et al., 2019;Zhang et al., 2019;Zhong et al., 2019;Butt et al., 2020;Zhang et al., 2020c;Li et al., 2021b;Zhan et al., 2021).CRISPR-Cas-based genome editing has been extended to targeted mutagenesis, base editing, and precisely targeted gene/allele replacement or tagging in plants.mportantly, using CRISPR-Cas9 technology, transgenes present in the genomes of genome-edited plants can be removed by chromosomal segregation via a simple self-pollination or hybridization step.Gene editing technologies continue to be developed and utilized (Mao et al., 2013;Lu and Zhu, 2017;Lu et al., 2020)."
                }
            ]
        },
        "data_source": [
            {
                "document_id": "b72eb0d1-50e3-4def-94bc-abf77891f519",
                "section_type": "main",
                "text": "Limitations of CRISPR-Cas9\n\nCRISPR provides a simple and easy tool not only for in vitro use but potentially also for in vivo genome editing.However, there are limitations and downsides to this approach.First, and despite considerable improvements in the technology, the risk of the offtarget effect remains and must be considered carefully.Second, DSB may lead to wide-ranging deletions or recombination events involving the on-target site (204).Third, in cycling cells, DNA double strand breaks caused by Cas9 cleavage may trigger a P53 response leading to apoptosis and enrichment for potentially oncogenic P53-deficient cells (205,206).Fourth, subjects may generate antibodies to Cas9, potentially limiting gene therapies (207,208)."
            },
            {
                "document_id": "801c9288-70c9-4d14-b8bc-13ee6708803a",
                "section_type": "main",
                "text": "\n\nComing on the heels of engineered nucleases, CRISPR-Cas9 tools have accelerated the pace of genomic research by permitting highly efficient knockouts or edits of virtually any gene in cells or model organisms.Multiple CRISPR-Cas9-based clinical trials are in progress or are expected to begin soon.Although Cas9engineered cells haven't yet demonstrated efficacy at scale, early trial results suggest that such cells are stable and don't cause acute adverse reactions in humans.Long-term safety is yet to be determined.Current applications largely focus on single-gene disorders for which gene editing can be carried out ex vivo on appropriate cells, such as bone marrow hematopoietic stem cells in the case of sickle cell anemia.Exploration is under way to develop delivery systems that can target the gene-editing apparatus to the appropriate tissue in vivo."
            },
            {
                "document_id": "4f709611-ea0b-4bcc-a634-df5d518ccb54",
                "section_type": "main",
                "text": "Caveats and Ethical Concerns of CRISPR-Cas Applications\n\nDespite the presence of both a PAM sequence and a specific gRNA, the CRISPR-Cas9 system is not infallible.In fact, DSBs can occur at different sites in the genome, potentially causing so-called \"off-target\" effects.This eventuality remains to date the biggest concern in the field, as possible undesirable modifications must be properly identified and followed in order to guarantee safety for medical purposes.Nevertheless, there is still little evidence of the biological consequence of Cas9 off-target effects.Two recent studies describe new methods to investigate potential off-target effects in both mammals and plants [33,34].In both cases, whole-genome sequencing revealed that selective nucleotide changes, such as conversion of an adenine to a guanine, caused off-target occurrence very rarely, with a frequency comparable to the one of spontaneous mutations.However, substitution of a cytosine with a thymidine was linked to a sizable number of off-target mutations.This newly acquired information adds to the plethora of studies conducted on the safety of CRISPR, which altogether highlight the need for the establishment of clinical standards for the future use of genome-editing techniques in the clinic.Despite this and other technical challenges still ahead for CRISPR genome editing, the pace at which this technology has developed in recent years suggests many of these concerns could be addressed soon, as long as proper ethical guidelines and regulatory mechanisms are established."
            },
            {
                "document_id": "03110c8a-1232-40c2-8380-c9feb8b6468c",
                "section_type": "main",
                "text": "\n\nGene editing has gained considerable interest with the identification of the CRISPR-Cas9 system, 27 which allows for a targeted modification in the DNA sequence of an organism.Researchers can utilize their knowledge of the basic biology of the gene and its protein function to precisely change the DNA sequence, thus altering the protein function of the gene and allowing for edits to stay within the species.Researchers at the University of Missouri used the CRISPR-Cas9 system to modify the CD163 gene such that the PRRS virus is not able to replicate inside the pig. 28This slight modification of the swine genome through gene editing keeps the pigs from succumbing to PRRS which has an annual estimated loss to the United States swine industry of over $660 million per year.Despite this benefit, given the public's concerns over food safety, it is likely that approval for such technology is years away in the US, Canada and Europe.However, in some cultures, there is a wide range of non-livestock species that are consumed.Therefore, it is conceivable that these countries and cultures may be open to transgenic/gene edited livestock.They may see the importance of useful gene editing which may lead to approval and consumption of reasonable genetically edited animal products such as those with modifications that are already found in nature or those that offer a substantial welfare benefit to society."
            },
            {
                "document_id": "801c9288-70c9-4d14-b8bc-13ee6708803a",
                "section_type": "main",
                "text": "\n\nOver the past 8 years, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) technologies have emerged as accessible and adaptable tools for studying and altering genomes. 5RISPR-Cas9 can be used to induce genome edits by creating targeted DNA breaks that trigger site-specific DNA repair.In nextgeneration formats, it can also control the transcriptional output of genes or alter genome sequences using a process of nucleotide base editing that does not require repair of DNA breaks.As these technologies continue to mature, it will become increasingly possible to alter cellular genomes efficiently and accurately."
            },
            {
                "document_id": "ac00c552-7514-49d4-9e90-ab01c22472ae",
                "section_type": "main",
                "text": "\n\nClustered regularly interspaced short palindromic repeat (CRISPR)-Cas nucleases have revolutionized the field of gene editing and have tremendous application in the field of molecular medicine [98][99][100][101][102].Despite a significant surge in CRISPR/Cas9mediated genome editing in various disease models, the progress in the field of AD has lagged behind substantially.We believe that genome editing can significantly improve the development of AD models and also create novel opportunities for the development of the next generation precision targeted AD gene and stem cell therapies.Since there are several excellent review articles on CRISPR/Cas9-mediated genome editing, here we will limit our focus on select recent articles that are noteworthy.CRISPR/Cas9 system can be engineered to either activate transcription (gain-of-function) or achieve gene silencing (Loss-of-function).Dahlman et al. have developed a CRISPR-based system that uses catalytically active Cas9 and distinct single guide (sgRNA) constructs to activate and knockout different genes in the same cell [103].Konermann et al. have used structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci [104].Using crystallographic studies, they have engineered a combination of sgRNA2.0,NLS-dCas9-VP64 and MS2-p65-HSF1 to develop one of the most effective transcription activation system."
            },
            {
                "document_id": "d14e93b5-01de-4208-8255-baae7898a7bb",
                "section_type": "abstract",
                "text": "\nCRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development.What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA.Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions.For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene.In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening.In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening."
            },
            {
                "document_id": "d14e93b5-01de-4208-8255-baae7898a7bb",
                "section_type": "main",
                "text": "\n\nCRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development.What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA.Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions.For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene.In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening.In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening."
            },
            {
                "document_id": "e5cf067c-8be0-4b0a-b376-7882cdc9d96c",
                "section_type": "main",
                "text": "Genome editing for crop improvement\n\nReports of CRISPR-Cas9-based genome editing first appeared in 2013 (Cong et al., 2013;Feng et al., 2013;Mao et al., 2013).Since then, genome editing technologies have proven to be powerful and efficient tools for the improvement of many crop species.At present, genome editing has been widely used to introduce/modify agronomically important traits, such as increased yield, improved nutritional quality, and resistance to biotic and abiotic stresses, in multiple crops, including rice, wheat, maize, tomato, and potato (Lu et al., 2017;Soyk et al., 2017;Tang et al., 2017;D'Ambrosio et al., 2018;Ye et al., 2018;Miao et al., 2019;Zhang et al., 2019;Zhong et al., 2019;Butt et al., 2020;Zhang et al., 2020c;Li et al., 2021b;Zhan et al., 2021).CRISPR-Cas-based genome editing has been extended to targeted mutagenesis, base editing, and precisely targeted gene/allele replacement or tagging in plants.mportantly, using CRISPR-Cas9 technology, transgenes present in the genomes of genome-edited plants can be removed by chromosomal segregation via a simple self-pollination or hybridization step.Gene editing technologies continue to be developed and utilized (Mao et al., 2013;Lu and Zhu, 2017;Lu et al., 2020)."
            },
            {
                "document_id": "4f709611-ea0b-4bcc-a634-df5d518ccb54",
                "section_type": "main",
                "text": "Conclusions\n\nThere is no reason to doubt that the development of CRISPR-Cas genome editing represents an unprecedented breakthrough in modern science, as it has potential applications in a wide array of disciplines ranging from agriculture, zoology and renewable energy to biomedicine and synthetic biology.This powerful tool holds promise for further elucidating the molecular causes of ageing by allowing scientists to probe genetic and epigenetic pathways with a level of sophistication that was unattainable just a few years ago.It will allow so in traditional animal and cell models of ageing, but it will also drastically accelerate the generation of refined versions of those models or even allow the development of new research approaches in non-model organisms.Moreover, CRISPR-based genome editing is already having a significant impact in research aiming to understand the cellular and molecular origins of age-related diseases, as well as developing potential treatments against them.The application of CRISPR-Cas gene editing for the treatment of age-related diseases is not over the horizon yet, as it will require the identification of causative genes and their role under a variety of contexts that could be as diverse as the ageing process is across individuals.However, CRISPR-Cas might also hold the key for solving such conundrum, as it has opened the way for achieving true personalised medicine by providing both the precision and scalability required for conducting genome-wide functional screens during the refinement of drug-and cell-based therapies for age-related diseases."
            },
            {
                "document_id": "4f709611-ea0b-4bcc-a634-df5d518ccb54",
                "section_type": "main",
                "text": "\n\nThe notable accuracy and versatility of CRISPR-Cas for genome editing also opened the door to its use in preclinical and translational settings.In the latter case, CRISPR in vivo gene editing has led to several proof-of-concept studies that would have been unachievable without it, as in the first ever correction of inherited pathogenic mutations linked to degenerative disease in a living organism [22] and even shown to be possible in human embryos [23,24].It also has great potential in the field of precision medicine as large-scale population DNA sequencing studies have provided vast amounts of information linking particular diseases with specific genetic mutations which could, in theory, be targeted through CRISPR [25,26].This could be used during the identification and validation of potential DNA targets during the development of personalised drug or cell therapies, which will require the generation of engineered cell lines and/or animal models.Techniques such as HDR-mediated gene targeting are too labour intensive, with low targeting efficiencies and long times necessary for their establishment, and consequently are not ideally suited for drug discovery purposes.Conversely, CRISPR-Cas has been proven to be efficient for editing virtually any kind of cell line, from primary immune cells to induced pluripotent stem cells (iPSCs) [27,28].Additionally, CRISPR can also be used for functional screening in the development of combined inhibitory therapy aimed at strengthening the efficiency of targeted therapeutics.An example of the latter is shown in a study where a variation of the technology known as CRISPR interference (CRISPRi) was used in genome-wide scale to identify different survival pathways used by cancer cells after oncogene inactivation and allowing the identification of successful combination therapies [29].In terms of translational applications, the overall safety of CRISPR genome editing in humans will require long-term scrutiny before its adoption in the clinic.Nonetheless, a number of CRISPR-based clinical trials are currently in progress, including studies focused on targeting patients' own T cells in order to improve the immune response towards some forms of malignant cancer [30,31], and others aimed at correcting pathogenic mutations in the hematopoietic cells of patients with beta-thalassemia and sickle cell disease [32]."
            },
            {
                "document_id": "d14e93b5-01de-4208-8255-baae7898a7bb",
                "section_type": "main",
                "text": "\n\nThe recent development of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 for experimental purposes has dismantled the perception that genome editing technology is off-limits for screening in mammalian systems (Heintze et al., 2013).Since this system employs the basic principle of Watson-Crick base pairing for gene targeting, generation of libraries with whole-genome target coverage is relatively easy and cost-effective.For instance, simple protocols are available to synthesize pooled lentiviral libraries by in silico design of oligonucleotides, which can then be cloned, packaged and delivered to cells by viral transduction (Paddison et al., 2004;LeProust et al., 2010).Similarly, the generation of arrayed libraries can be achieved by following protocols originally developed for arrayed shRNA library production that have been in use for a number of years (Moffat et al., 2006).All in all, the stage is set for CRISPR to make an enormous impact on genomic screening and thus scientific discovery in the coming years, and recent demonstrations of this system have shown great promise (Shalem et al., 2015).However, a number of technical challenges must be addressed in order to maximize the benefit of this technology.In this review, we will discuss current applications of CRISPR in functional genomics and provide a perspective on future developments in this area."
            },
            {
                "document_id": "b72eb0d1-50e3-4def-94bc-abf77891f519",
                "section_type": "main",
                "text": "\n\nGenome editing tools that target the desired genomic region and allow for variants to be altered (e.g. from risk to protective), or for more substantial changes to be made (e.g. the deletion of a longer stretch of DNA harbouring a number of variants) and can help to answer each of these questions.These technologies are evolving rapidly (Figure 1 and Table 2).The most recently developed of these, Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technology, originally developed by Doudna, Charpentier and their colleagues (72,73) and Zhang and his colleagues (50) has become a widely used tool for this purpose.Engineered CRISPR/Cas9 technology uses a guide RNA (gRNA) to direct CRISPR-associated endonuclease (Cas) to the target DNA and generate a double strand DNA break.Correction of a mutation or variant in the target DNA sequence can then be carried out by homology-directed DNA repair (HDR) with a donor template.Since its discovery eight years ago, CRISPR technology has evolved quickly to be a critical part of the molecular biologist's toolbox."
            },
            {
                "document_id": "52480703-5353-4e55-a06b-110fd59db3a6",
                "section_type": "main",
                "text": "CRISPR screening technologies\n\nThe discovery of CRISPR-Cas9 as a sequence-specific programmable nuclease democratized gene editing and fueled progress in forward genetic screening [20 , 66] .Genetic screens using Cas9 with a pooled singleguide RNA (sgRNA) library allow the interrogation of seemingly all genes in a genome in a single experiment [96 , 97] [null] .Engineered Cas9 variants further extend the versatility of forward genetic screening.Catalytically inactive Cas9 (dCas9) fused with chromatin effector domains permit specific activation (CRISPRa) or inhibition (CRISPRi) of gene expression [37 , 54] .Recently developed and emerging technologies -base editors, prime editors, and Cas transposases -are beginning to enable new types of genetic screens with directed, controlled, and on demand mutations by allowing the creation of user specified modifications, such as single base conversion, deletions, and insertions [4 , 42 , 58] ."
            },
            {
                "document_id": "c3ae2186-ef48-46a5-b214-dc944366df8f",
                "section_type": "main",
                "text": "INTRODUCTION\n\nGenome editing technologies based on the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 enable rapid and efficient modification of endogenous genes in a variety of cell types, allowing for analysis of gene function in many organs in vivo.CRISPR-Cas9 induces DNA double strand breaks (DSBs) at single-guide RNA (sgRNA)-specific loci in the genome, which are repaired through either non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathways.While NHEJ introduces unpredictable pattern of insertion or deletion (indel) mutations, HDR directs a precise recombination event between a homologous DNA donor template and the damaged DNA site (Cong et al., 2013;Cox et al., 2015;Doudna and Charpentier, 2014;Heidenreich and Zhang, 2016;Jinek et al., 2012;Mali et al., 2013;Sander and Joung, 2014;Wang et al., 2013;Yang et al., 2013).Thus, HDR can be used to precisely introduce sequence insertions, deletions or mutations by encoding the desired changes in the donor template DNA."
            },
            {
                "document_id": "4f709611-ea0b-4bcc-a634-df5d518ccb54",
                "section_type": "main",
                "text": "\n\nSince its discovery, CRISPR-Cas technology has ignited a biological revolution by providing a highly versatile platform that allows fast and efficient genome editing in an ever-growing list of organisms.In this chapter we will first describe the most recent advances in the development and application of the CRISPR-Cas platform in biomedical research.Then we will discuss the most recent and notable basic research applications of this technology in the study of the molecular causes of ageing.Finally, we will review how CRISPR-Cas has been used for creating new models for the study of age-related diseases, as well as for manipulating diseaseassociated gene pathways."
            },
            {
                "document_id": "1942712a-a39d-44f7-9b2d-609926374cbd",
                "section_type": "main",
                "text": "\n\nAs a researcher who has devoted an entire career since 1994 to the development of genome editing tools and methods, I have been amazed by the rapid progress in the field over the last few years.Considering the widespread use of the tools, I am sure that the pace will continue to accelerate.Indeed, programmable nucleases, may eventually enable humans-products of evolution-to become masters of evolution.delivered preassembled recombinant Cas9-guide RNA ribonucleoproteins (RNPs) into animal embryos 6,9 and plant 11 and mammalian cells [73][74][75] .Indeed, Cas9 RNPs were rapidly turned over in cells 73 , reducing off-target effects and mosaicism in gene-edited organisms 11 .Cas9 RNPs can be delivered into cells by various methods, including microinjection 6,9 , electroporation 73 , lipofection 74 and protein transduction 75 .Importantly-and unlike in conventional gene therapy, where therapeutic genes are delivered via plasmids or viral vectors-Cas9 RNP delivery does not involve the use of exogenous DNA; host innate immune responses against foreign DNA are not elicited, and undesired integration of foreign DNA into the host genome is avoided."
            },
            {
                "document_id": "50c72e55-b5fe-42a6-b837-64c28620a4c0",
                "section_type": "main",
                "text": "Caveats of advanced genome editing tools\n\nOff-target effects.The DNA-binding domains of ZFNs and TALENs need to be very specific for the target site to avoid off-target cleavage, which results in unwanted mutations and potentially cytotoxic effects [27].CRISPR/Cas9 is also known to generate off-target alterations, albeit apparently at low incidence [28,29], since mispairing is allowed between the guide RNA and the genomic DNA.Nonetheless, caution is required in their design and use.Some strategies involving the optimization of the guide RNA/Cas9 include using of software tools to predict potential off-target sites (http://omictools.com/crispr-cas9-Figure1: Genome editing methodologies which can be applied to human pluripotent stem cells.Homologous recombination (HR), or the more advanced tools such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system can be applied to human pluripotent stem cells (hPSCs) either to 1) create naturally occurring mutations or 2) repair a mutation to generate isogenic controls in hPSCs, to understand the function of a gene of interest.c1268-p1.html),truncating the guide RNA (<20 nucleotides) to decrease off-target mutagenesis [30], lowering the dosage of guide RNA and Cas9 plasmids, and decreasing the number of mismatches between the guide RNA and the genomic DNA.A \"double nick\" system with Cas9 nickase, which contains a single inactive catalytic domain, may also be used [31e33]."
            },
            {
                "document_id": "b72eb0d1-50e3-4def-94bc-abf77891f519",
                "section_type": "main",
                "text": "\n\nIn addition to gene knock-out and HDR repair, genome-wide pooled CRISPR-Cas9 libraries have been used to systematically delete genes responsible for diverse phenotypes.Recent studies have shown that such loss-of-function screens using libraries comprising tens of thousands of sgRNAs can be used to identify genes involved in tumour growth and metastasis (171).In the diabetes field, similar approaches have also been used recently to identify key insulin gene regulators (172) and the genes involving in auto-immune killing of b cell transplants (173).Screens based on transcriptional interference (CRISPRi) and activation (CRISPRa) have also harnessed Cas9-based technologies for use in genome-wide studies (59,174).In addition, recent improvements in lentiviral library generation and propagation, as well as large-scale DNA and RNA synthesis, have allowed CRISPR-Cas9 technology to be exploited across multiple model platforms (59,(175)(176)(177)(178)."
            },
            {
                "document_id": "a7f21808-dce3-4110-8e7c-ceb2437e72ff",
                "section_type": "main",
                "text": "\n\nThe type II CRISPR-Cas9 systems, repurposed from prokaryotic adaptive immune responses, are now widely used for targeted genome modifications in plants, animals, and human cells (Kim et al. 2014;Woo et al. 2015;Zuris et al. 2015).In particular, Cas9 nucleases have shown promise for gene and cell therapy (Maeder and Gersbach 2016).Typically, these nucleases are expressed or delivered in vivo using plasmid DNA or viruses (Yin et al. 2014;Ran et al. 2015).However, plasmid DNA delivery is often inefficient, especially in vivo, and can cause integration of small plasmid fragments degraded by endogenous nucleases at on-target and offtarget sites in the genome (Kim et al. 2014).Viral delivery of Cas9 can be highly efficient in vivo (Ran et al. 2015;Long et al. 2016;Nelson et al. 2016;Tabebordbar et al. 2016), but may be hampered by antibodies or T cells induced against the protein (Shankar et al. 2007;Calcedo et al. 2015;Chew et al. 2016).We and others have shown that preassembled Cas9 ribonucleoproteins (RNPs) can be delivered to human primary and stem cells and mice to modify target genes (Kim et al. 2014;Schumann et al. 2015;Zuris et al. 2015).Cas9 RNPs are rapidly turned over in cells, reducing off-target effects.Furthermore, Cas9 RNPs are unlikely to be limited by host immune systems because they function and disappear before the generation of antibodies and T cells directed against them.Currently, despite these advantages of RNPs, the difficult delivery of Cas9 RNPs in vivo limits its utility for therapeutic applications (Zuris et al. 2015).Here, we show that in vivo genome editing of an wild-type gene, whose up-regulation is responsible for pathogenesis, could be a new therapeutic modality for the treatment of nongenetic degenerative diseases.Our ultimate goal is to harness Cas9 RNPs for a clinical application of therapeutic genome surgery in patients with AMD."
            },
            {
                "document_id": "1942712a-a39d-44f7-9b2d-609926374cbd",
                "section_type": "main",
                "text": "Genome editing comes of age\n\nJin-Soo Kim 1,2 Genome editing harnesses programmable nucleases to cut and paste genetic information in a targeted manner in living cells and organisms.Here, I review the development of programmable nucleases, including zinc finger nucleases (ZFNs), TAL (transcription-activator-like) effector nucleases (TALENs) and CRISPR (cluster of regularly interspaced palindromic repeats)-Cas9 (CRISPR-associated protein 9) RNA-guided endonucleases (RGENs).I specifically highlight the key advances that set the foundation for the rapid and widespread implementation of CRISPR-Cas9 genome editing approaches that has revolutionized the field."
            },
            {
                "document_id": "1942712a-a39d-44f7-9b2d-609926374cbd",
                "section_type": "main",
                "text": "\n\nThe ability to genetically modify living cells and organisms is a fundamental tool for biological research, but achieving highly specific targeted changes has been technically demanding.Genome editing has been recently democratized by the development of RGENs (see Glossary in Box 1), repurposed from the type II CRISPR-Cas9 prokaryotic adaptive immune system 1 .Unlike other programmable nucleases, namely ZFNs and TALENs, whose target specificities are determined by modifying their DNA-binding domains, CRISPR-Cas9 can be customized by replacing guide RNAs, making the system much more affordable and scalable.Cas9 nucleases have been successfully used for modifying genomes in human cells [2][3][4][5] , animals [6][7][8][9] and plants 10,11 , heralding the age of genome editing.Furthermore, Cas9 or guide RNAs have been linked to various effector proteins to enable targeted gene regulation 12,13 and epigenome modifications 14,15 .It is worth noting, however, that many of these feats had been demonstrated previously using other nucleases or DNA-binding proteins 1,16 .In this Perspective, I shed light on early genome editing platforms that laid the groundwork for the widespread use of CRISPR-Cas9 in research and medicine (Fig. 1)."
            },
            {
                "document_id": "4f709611-ea0b-4bcc-a634-df5d518ccb54",
                "section_type": "main",
                "text": "\n\nGenome editing has always been a challenging area to provide more efficient ways to create a meaningful change in the genome.Today, the CRISPR (clustered regularly interspaced short palindromic repeat) restoration system is considered as one of the suitable and promising options for genome editing.Compared to the previous systems, CRISPR can deactivate or eliminate a gene without interfering with intracellular mechanisms.The system could be used in the treatment of diseases and in related research by identifying the performance of defective genes in these diseases.CRISPR seems to have more potential and applications compared to previous systems.Among these applications, we can note the use of CRISPR in understanding complex genetic and epigenetic conditions such as aging or cancer.The complex interactions between several genetic and epigenetic mechanisms that characterize aging pose significant challenges to scientists attempting to understand this phenomenon and its causes and still constitute a barrier to a better understanding of aging and the ability to develop effective application of CRISPR-cas to aging research."
            },
            {
                "document_id": "b72eb0d1-50e3-4def-94bc-abf77891f519",
                "section_type": "main",
                "text": "Cas9\n\nDue to its simplicity and adaptability, CRISPR has rapidly become the most popular genome editing tool available for the mammalian genome (50,63).Because NHEJ DNA repair often introduces unwanted indels at the Cas9 cutting site, CRISPR has been used to knock-out genes by introducing frameshift mutations, resulting in protein depletion (156,157).In the diabetes field, CRISPR has also been adopted to study several genes in b cell lines and in human ES-derived b cells (21,151,158,159) as well as in animals (160,161)."
            },
            {
                "document_id": "e2d1d559-d48f-4e57-8372-04d31f0f9da3",
                "section_type": "main",
                "text": "\n\nSome believe genome editing tools provide the best imaginable technology for mutating the germline.Indeed it is hard at the moment to imagine what could be better.Nevertheless there are remaining challenges.We need to improve efficiency of editing within a given population of cells (destined for SCNT) and in the zygote and overcome mosaicism.In our work with zygotes we regularly achieve 30 % editing frequency with delivery of editors-ZFN, TALEN and CRISPR/ Cas9-to the cytoplasm of livestock.We should aspire to at least [50 % and why not frequencies approaching or even achieving 100 %."
            },
            {
                "document_id": "4f709611-ea0b-4bcc-a634-df5d518ccb54",
                "section_type": "main",
                "text": "\n\nThe application of CRISPR-Cas in epigenome editing is currently in its infancy.However, the technique holds significant promise for providing clarity to the myriad of epigenetic mechanisms that may impact on the ageing process.In this regard, it must be noted that in comparison to other hallmarks of ageing, the range of discrepancies observed across model species is the broadest in the case of some epigenetic alterations.This prevents the use of many of the most time-cost efficient in vivo models of ageing such as yeast, worms and flies, as they can even lack some of those alterations.Therefore, the use of CRISPR-Cas for the fast and efficient generation of in vitro and in vivo models of higher species will prove invaluable for studying epigenetic mechanisms of ageing that are of relevance to humans."
            },
            {
                "document_id": "33f1abde-a821-483b-b8b4-785f499db09d",
                "section_type": "main",
                "text": "\n\nIn comparison to a transgenic approach, a gene editing technique such as CRISPR-Cas9 offers the advantage that gene-edited crops are not considered genetically modified organism (GMO) in some countries, such as the US, where the demand for natural food colorants such as anthocyanins is high.Indeed, the use of GMO crops as a source of natural pigments may be inconsistent with consumer interests.However, carrot cultivars engineered with either the transgenic or gene editing approach have not been reported so far, but their development is possible."
            },
            {
                "document_id": "b72eb0d1-50e3-4def-94bc-abf77891f519",
                "section_type": "main",
                "text": "\n\nApplications of gene/genome editing tools."
            },
            {
                "document_id": "d14e93b5-01de-4208-8255-baae7898a7bb",
                "section_type": "main",
                "text": "\n\nKey characteristics in CRISPR and siRNA technologies."
            },
            {
                "document_id": "d14e93b5-01de-4208-8255-baae7898a7bb",
                "section_type": "main",
                "text": "CRISPR/Cas9 Screening\n\nA growing number of published studies have utilized CRISPR technology for screening (see Table 3 for a comparison).CRISPR  For an overview of key differences between siRNA and CRISPR technologies, please see Taylor and Woodcock (2015)."
            },
            {
                "document_id": "d14e93b5-01de-4208-8255-baae7898a7bb",
                "section_type": "main",
                "text": "\n\nFinally, CRISPR screening has become a possibility in 3D models, tissues and whole organisms (Platt et al., 2014;Chen et al., 2015).The generation of a Cre-dependent Cas9 knockin mouse enables the manipulation of genes in specific tissues, for instance by viral or non-viral delivery of sgRNA to the brain or other tissues.Importantly, this technology for the first time enables complex studies of acute modulation of brainspecific phenotypes, which will be key to develop a more thorough understanding of neuronal diseases.Using tissuespecific expression systems, it is thus possible to target a functionalized protein to any location within a whole organism.This truly is a new age in functional genomics."
            },
            {
                "document_id": "4f709611-ea0b-4bcc-a634-df5d518ccb54",
                "section_type": "main",
                "text": "The Molecular Basis of CRISPR-Cas Technology and Its Variants\n\nDuring the last decade, a more robust system in terms of targeting efficiency and ease of design was developed and rapidly became the most widely used gene-editing technique in the life sciences.CRISPR stands for clustered regularly interspaced short palindromic repeat DNA sequences.These short repeat elements were first observed in E. coli in 1987 and were later determined to be part of the bacterial adaptive immune system [9].However, the first concrete experimental evidence of the potential widespread application of CRISPR came with the demonstration that following viral infection, bacteria could integrate specific sequences of the viral genome into their own.These sequences would then be used by bacteria to produce short RNAs able to recognise the viral DNA in subsequent infections and guide the Cas9 nuclease to it.The RNA/Cas9 complex would then induce a DSB in the viral DNA, disabling it [10].This defence mechanism can be easily exploited in an experimental set-up, where short RNA sequences (around 20 base pairs), named gRNA (guide RNA), can be designed to bind any determined DNA sequence in virtually any kind of cell.gRNAs then become complexed to the Cas9 enzyme and will dictate the specificity of its enzymatic action, which in turn will lead to the generation of a DSB in the targeted genome."
            },
            {
                "document_id": "b72eb0d1-50e3-4def-94bc-abf77891f519",
                "section_type": "main",
                "text": "\n\nIn view of the above, genome editing tools need to be carefully selected.The newly developed nCas9-RT holds great potential: 1.The nCas9 nicks the DNA rather than induces DSB and therefore avoids indel formation at the cutting site; 2. The use of pegRNA, which is a combination of gRNA, reverse transcription template and primer-binding sites, increases the specificity of target DNA binding hence reduces off-targets (62); 3.While multiplex pegRNAs could target various variants including SNPs, deletions or insertions without separating DNA donors as templates, it is possible the nCas9-RT will be able to convert all variants at once.This new technique, however, is still in early development, and its editing efficiency and sideeffects remain to be seen."
            },
            {
                "document_id": "f28111d5-fe88-4668-8699-f02f907af80a",
                "section_type": "main",
                "text": "\n\n146 Genome studies and molecular genetics Features of Cas9-mediated gene editing methods in wheat.The top graph shows the percentages of on-target mutagenesis using the different methods across independent experiments as defined by their target genes or genotypes [39 ,42 ,43].Features include the method of delivery of the Cas9 and gRNA, the stable or transient expression of the system and the possibility of nuclear DNA integration.The target genes used in each method are shown, although the detailed homoeolog specificity of each experiment is not always reported.The number of different hexaploid (6x) bread wheat and tetraploid (4x) pasta wheat varieties used for each method is also shown in parenthesis.Abbreviations: Transiently expressing CRISPR/Cas9 DNA (TECCDNA) or RNA (TECCRNA), ribonucleoprotein (RNP).Original publications for stable transformation [39 ,42 ], TECCDNA [42 ,43], TECCRNA [42 ] and RNP [43].15, 2017, 367-378.effectively between disciplines and appreciate the potential of genomics and field-based research to complement each other. 'Reaping the benefits' [57] of the latest genomic developments will ultimately depend on our success in translating this knowledge into improved wheat cultivars for farmers and consumers."
            },
            {
                "document_id": "d14e93b5-01de-4208-8255-baae7898a7bb",
                "section_type": "main",
                "text": "Conclusion and Outlook\n\nOverall, the use of CRISPR based methods in high-throughput functional genomics screening is still in its infancy.The first pooled libraries show encouraging results, but many technical considerations need to be explored for the development of arrayed libraries.The generation of large-scale libraries is possible not only for human and mouse, but virtually any organism.In the past, siRNA libraries have mostly focused on Drosophila, C. elegans, human, mouse, and rat genomes, though in principle has always been possible to design and produce libraries for other organisms as well.It is uncertain which model organisms will be targeted with whole genome or focused libraries using CRISPR as the availability of whole-genome sequence information expands."
            },
            {
                "document_id": "429abfc1-f628-48ff-bfe8-f7be6d1419a8",
                "section_type": "main",
                "text": "Effective protocols that exist for gene editing use\nguide RNA in combination with the enzyme recombinase\n(CRISPR/Cas9), zinc finger nuclease, or zinc finger protein in combination with a nuclease (TALEN) (Gupta and\nMusunuru, 2014).  Although these methods can be applied\neasily to livestock species, the mouse still has the unequal\nadvantage of a short generation interval, which shortens the\ntime for testing the effects of the introduced mutation several\nfold."
            },
            {
                "document_id": "ac00c552-7514-49d4-9e90-ab01c22472ae",
                "section_type": "main",
                "text": "\n\nAs compared to the complete gene knockout mouse models, CRISPR/Cas9 based gene editing provides only a partial knockout in a fraction of targeted cells.Therefore, it is important to improve the current gene editing efficiency of the CRISPR/Cas9 systems.Another caveat is that using a ubiquitous promoter to drive the expression of CRISPR/Cas9 can lead to gene editing in the non-target cells.This can be achieved using a highly tissue specific promoter to drive the expression of CRISPR/Cas9.However, despite utilizing a tissue specific promoter, it is still not desirable to have constitutive expression of CRISPR/Cas9 as it may lead to off-target effects.This potential drawback can be overcome by utilizing a conditional expression system wherein the expression levels as well as the duration of CRISPR/Cas9 can be tightly regulated.This has been recently demonstrated by de Solis et al. who have developed a doxycycline-inducible AAV based system for gene editing [118].Their strategy involved generating two separate AAV/DJ vectors such that the vector harbors a TRE Tight promoter driving the expression of CRISPR/Cas9 while the second vector contains a U6 promoter driving Tet2 sgRNA and a CMV promoter driving the expression of rtTA (Tet-On Advanced and an IRES driven GFP.Surprisingly, their results indicate doxycycline-inducible expression of CRISPR but Tet2 gene editing in a doxycycline independent manner due to leakiness.To overcome the issue of leakiness, they have significantly modified their vectors by utilizing a combination of hybrid H1/TO promoter to drive the expression of Tet2-sgRNA and a CMV promoter controlling the expression of TetR in frame with a self-cleaving P2A sequence followed by a GFP ORF fused to a KASH domain.In this system in the absence of doxycycline, TetR binds to H1/TO promoter and represses the gRNA transcription.However, addition of doxycycline inhibits TetR binding and induces gRNA expression.This system allowed doxycycline dependent genome editing of Tet2 in N2A cells in vitro.Besides, doxycycline inducible system there are several other inducible systems available including rapamycin, mifepristone, tamoxifen, and ecdysone inducible systems that can be engineered to overcome the leakiness of the dinducible system."
            },
            {
                "document_id": "d14e93b5-01de-4208-8255-baae7898a7bb",
                "section_type": "main",
                "text": "From Genome Editing to Genome Functionalization\n\nThe Cas9 protein has been engineered to obtain various properties that range from transcriptional repression to endogenous gene tagging (Table 1).In a more simplified view, Cas9 can be seen as the adaptor between the target sequence and a variety of functions.This reveals the most powerful concept of CRISPR technology: the ability to target a function to an exact genomic position.With this view in mind, it is conceivable to be able to design a minimal Cas9 protein with all extraneous regions deleted so that the protein simply binds the target DNA, and this would provide the most basic possible template for protein engineering.To date, successful CRISPR-based genome functionalization techniques have been based on fusing one or several functional domain to full-length catalytically inactive Cas9 (dCas9), which binds to the target locus but does not cleave the DNA.An important experimental consideration to take into account when following these approaches is that different sgRNAs must be designed for each functionalization in order to target the correct genomic features and achieve the desired output.For instance, transcriptional regulation requires sgRNAs that target promoter or regulatory regions, whereas sgRNAs used for knockouts most commonly target exons.Furthermore, the location of targeting within an individual gene can have a significant impact on the functional effect of the resulting mutation.For example, when using wild-type Cas9, targeting a coding region corresponding to a functional protein domain has been shown to be result in loss-of-function even for in-frame mutations, compared to exclusively targeting early exon regions, which often require frameshift mutations to achieve loss-of-function (Shi et al., 2015).Deliberately targeting certain gene regions can be used for achieving specific outcomes, such as knocking out a specific splice variant."
            },
            {
                "document_id": "9a12db75-1efa-46b1-9da4-d2fc8d828f42",
                "section_type": "main",
                "text": "\n\nIn addition to DNA-targeting CRISPR systems, RNA-targeting Cas9 enzymes are also available, such as CasRx which showed robust knockdown of gene expression (Konermann et al., 2018).Interestingly, CasRx can also be used to target pre-mRNA to manipulate alternative splicing et al., 2018).Deregulation of alternative splicing has been implicated in the aging process (Li et al., 2017) and observed in several ageassociated diseases such as amyotrophic lateral sclerosis and Alzheimer's disease (Lin et al., 1998;Spillantini et al., 1998;Glatz et al., 2006).In regards to AMD, Allikmets et al. (1997) have shown that a point mutation (G5196A) in the Stargardt disease gene ABCA4, eliminates a 5 donor splice site and increases the risk of AMD.However, a subsequent GWAS study with larger cohorts could not confirm this association between ABCA4 and AMD (Fritsche et al., 2016).Overall, the association of AMD pathophysiology with alternative splicing regulation remains unclear and CasRx technology could facilitate research in this understudied area.In summary, recent development of CRISPR/Cas technology has greatly expanded the toolbox to carry out functional study of AMD-associated genes, providing new tools that can modulate gene expression by targeting at the DNA level, RNA level as well as the splicing variants."
            },
            {
                "document_id": "b72eb0d1-50e3-4def-94bc-abf77891f519",
                "section_type": "main",
                "text": "\n\nManipulation of hESC/iPSC cells via CRISPR-Cas9 technology provides a platform for the correction of genomic mutations not only in diabetes but in other disease fields as well (276)(277)(278)(279).Through CRISPR-mediated HDR and base editing, it is possible to correct the vast majority of genetic variants, if not all.Conversion of GWAS-identified non-coding variants has not been conducted/documented in the diabetes field, but it seems inevitable that such work will be carried out in the near future given its importance in basic research and potential clinical application.Variants identified by GWAS are often clustered in the genome (134).Although an individual variant may change transcription factor binding on its own, neighbouring risk variants might cooperate to change the transcriptional landscape of local chromatin and thus the activity of the enhancer cluster leading to changes in the expression of multiple genes whose aggregate effect is to impair b cell function.Hence, multiplex genome-editing needs to be carried out to convert multiple risk variants into protective (non-risk) variants in hESC or iPSC cells.In this case, the off-target effects brought by multiplex gRNAs may have a large impact on the rest of the genome and raise major concerns."
            }
        ],
        "document_id": "38E097866214E3EEFE346FB836ABF345",
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        "focus": "api",
        "keywords": [
            "CRISPR-Cas9",
            "gene&editing",
            "off-target&effects",
            "genome",
            "clinical&trials",
            "agriculture",
            "biomedicine",
            "precision&medicine",
            "transgenic",
            "ethical&concerns"
        ],
        "metadata": [
            {
                "object": "Genome-wide association analyses in 22,981 participants 2280 shingles cases from the electronic Medical Records and Genomics Network identified a genomic region in the combined and European ancestry groups that has an age of onset effect reaching genome-wide significance region tags the non-coding gene HCP5 HLA Complex P5.",
                "predicate": "http://www.w3.org/2000/01/rdf-schema#comment",
                "subject": "ndd791caee50643ad90a986f563d2a0dab319975"
            },
            {
                "object": "Study show that CXCL9 and CXCL10 are overexpressed in skin of HPV16 E7 transgenic mice when compared with non-transgenic animals. Immune cell infiltration to E7 transgenic skin is a consequence of epidermal hyperplasia, and that hyperplasia induces CXCL9 and CXCL10 production to recruit a subset of CXCR3+ T cells, promoting rejection of grafted E7 transgenic skin depleted of immunosuppressive lymphocytes.",
                "predicate": "http://www.w3.org/2000/01/rdf-schema#comment",
                "subject": "ndd791caee50643ad90a986f563d2a0dab749071"
            },
            {
                "object": "shRNAs targeting MSTN were expressed in muscles of transgenic sheep. MSTN expression was inhibited in muscle tissues of transgenics compared with controls. Moreover, transgenic sheep showed a tendency to faster increase in body weight than control sheep.",
                "predicate": "http://www.w3.org/2000/01/rdf-schema#comment",
                "subject": "ndd791caee50643ad90a986f563d2a0dab705362"
            },
            {
                "object": "ompounding a previously described Bmi1-transgene and Pten-deficiency prostate cancer mouse model with the Ezh2 transgene did not enhance tumour progression or drive metastasis formation. In conclusion, we here report the generation of a wildtype Ezh2 overexpression mouse model that allows for intravital surveillance of tissues with activated transgene",
                "predicate": "http://www.w3.org/2000/01/rdf-schema#comment",
                "subject": "ndd791caee50643ad90a986f563d2a0dab546432"
            },
            {
                "object": "Using a series of transgenic constructs with various Alk1 genomic fragments joined to a reporter, it seems a 9.2-kb genomic fragment including the 2.7-kb promoter region & the whole intron 2 is sufficient for arterial endothelium-specific expression.",
                "predicate": "http://www.w3.org/2000/01/rdf-schema#comment",
                "subject": "ndd791caee50643ad90a986f563d2a0dab988637"
            },
            {
                "object": "Data including data from studies using transgenic plants or cells from transgenic plants suggest crucial role for UreG in nickel delivery for urease multimerization/activation. These studies were conducted using recombinant Arabidopsis thaliana proteins expressed in transgenic Nicotiana benthamiana cloned cells or hydroponic plants. UreG = urease accessory protein UreG",
                "predicate": "http://www.w3.org/2000/01/rdf-schema#comment",
                "subject": "ndd791caee50643ad90a986f563d2a0dab173373"
            },
            {
                "object": "TaWRKY33 transgenic Arabidopsis lines showed lower rates of water loss than TaWRKY1 transgenic Arabidopsis lines and wild type plants during dehydration. Most importantly, TaWRKY33 transgenic lines exhibited enhanced tolerance to heat stress. [WRKY33]",
                "predicate": "http://www.w3.org/2000/01/rdf-schema#comment",
                "subject": "ndd791caee50643ad90a986f563d2a0dab766040"
            },
            {
                "object": "Study tracked the post-weaning development of a motor phenotype that arose in mice after random insertion of a transgene into the genome. The transgene was later found to have disrupted the beta-IV spectrin gene, which was confirmed by reduced expression of betaIV spectrin protein in brain homogenates. The motor phenotype was recessive, occurring in 20% of L25+/-xL25+/- progeny.",
                "predicate": "http://www.w3.org/2000/01/rdf-schema#comment",
                "subject": "ndd791caee50643ad90a986f563d2a0dab752806"
            },
            {
                "object": "eight of 11 Sox10 genomic elements direct reporter gene expression in transgenic zebrafish similar to patterns observed in transgenic mice, despite an absence of observable sequence conservation between mice and zebrafish.",
                "predicate": "http://www.w3.org/2000/01/rdf-schema#comment",
                "subject": "ndd791caee50643ad90a986f563d2a0dab252134"
            },
            {
                "object": "More oligodendrocytes and less demyelination were observed after SCI in p35 transgenic mice than in controls which did not carry the p35 transgene. Motor function recovered more in the cre/p35 transgenic mice than in the control cre mice.",
                "predicate": "http://www.w3.org/2000/01/rdf-schema#comment",
                "subject": "ndd791caee50643ad90a986f563d2a0dab501036"
            }
        ],
        "question": "What are the potential benefits and risks associated with gene editing technologies like CRISPR-Cas9?",
        "subquestions": null,
        "task_id": "38E097866214E3EEFE346FB836ABF345",
        "usage": {
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