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

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

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

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

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

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

scholarly journals Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient

2021 ◽  
Vol 22 (19) ◽  
pp. 10570
Author(s):  
Yuya Kato ◽  
Hirotaka Tabata ◽  
Kumiko Sato ◽  
Mariko Nakamura ◽  
Izumu Saito ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Genome Editing ◽  
Adenovirus Vector ◽  
Target Sequence ◽  
Guide Rna ◽  
Guide Rnas ◽  
B Virus ◽  
X Gene

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.

scholarly journals Gene editing and CRISPR in the clinic: current and future perspectives

2020 ◽  
Vol 40 (4) ◽  
Author(s):  
Matthew P. Hirakawa ◽  
Raga Krishnakumar ◽  
Jerilyn A. Timlin ◽  
James P. Carney ◽  
Kimberly S. Butler
Keyword(s):  
Clinical Trials ◽  
Genome Editing ◽  
Dna Sequences ◽  
Gene Editing ◽  
Ex Vivo ◽  
Scientific Basis ◽  
Current Status ◽  
Zinc Finger Nucleases ◽  
Transcription Activator

Abstract Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application.

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

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

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

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

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

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

scholarly journals In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 2153 ◽  
Author(s):  
Cia-Hin Lau ◽  
Yousin Suh
Keyword(s):  
Clinical Trials ◽  
Genome Editing ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Guide Rna ◽  
Routes Of Administration ◽  
Cas9 Nuclease ◽  
Wide Range ◽  
Short Palindromic Repeat

Adeno-associated virus (AAV) has shown promising therapeutic efficacy with a good safety profile in a wide range of animal models and human clinical trials. With the advent of clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome-editing technologies, AAV provides one of the most suitable viral vectors to package, deliver, and express CRISPR components for targeted gene editing. Recent discoveries of smaller Cas9 orthologues have enabled the packaging of Cas9 nuclease and its chimeric guide RNA into a single AAV delivery vehicle for robust in vivo genome editing. Here, we discuss how the combined use of small Cas9 orthologues, tissue-specific minimal promoters, AAV serotypes, and different routes of administration has advanced the development of efficient and precise in vivo genome editing and comprehensively review the various AAV-CRISPR systems that have been effectively used in animals. We then discuss the clinical implications and potential strategies to overcome off-target effects, immunogenicity, and toxicity associated with CRISPR components and AAV delivery vehicles. Finally, we discuss ongoing non-viral-based ex vivo gene therapy clinical trials to underscore the current challenges and future prospects of CRISPR/Cas9 delivery for human therapeutics.

scholarly journals A systematic evaluation of data processing and problem formulation of CRISPR off-target site prediction

2021 ◽  
Author(s):  
Ofir Yaish ◽  
Maor Asif ◽  
Yaron Orenstein
Keyword(s):  
Data Processing ◽  
Gene Editing ◽  
Target Site ◽  
Systematic Evaluation ◽  
Guide Rna ◽  
Guide Rnas ◽  
Target Sites ◽  
Evaluation Of Data

AbstractCRISPR/Cas9 system is widely used in a broad range of gene-editing applications. While this gene-editing technique is quite accurate in the target region, there may be many unplanned off-target edited sites. Consequently, a plethora of computational methods have been developed to predict off-target cleavage sites given a guide RNA and a reference genome. However, these methods are based on small-scale datasets (only tens to hundreds of off-target sites) produced by experimental techniques to detect off-target sites with a low signal-to-noise ratio. Recently, CHANGE-seq, a new in vitro experimental technique to detect off-target sites, was used to produce a dataset of unprecedented scale and quality (more than 200,000 off-target sites over 110 guide RNAs). In addition, the same study included GUIDE-seq experiments for 58 of the guide RNAs to produce in vivo measurements of off-target sites. Here, we fill the gap in previous computational methods by utilizing these data to perform a systematic evaluation of data processing and formulation of the CRISPR off-target site prediction problem. Our evaluations show that data transformation as a pre-processing phase is critical prior to model training. Moreover, we demonstrate the improvement gained by adding potential inactive off-target sites to the training datasets. Furthermore, our results point to the importance of adding the number of mismatches between the guide RNA and the off-target site as a feature. Finally, we present predictive off-target in vivo models based on transfer learning from in vitro. Our conclusions will be instrumental to any future development of an off-target predictor based on high-throughput datasets.

scholarly journals A stable DNA-free screening system for CRISPR/RNPs-mediated gene editing in hot and sweet cultivars of Capsicum annuum

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Hyeran Kim ◽  
Jisun Choi ◽  
Kang-Hee Won
Keyword(s):  
Genome Editing ◽  
Capsicum Annuum ◽  
Gene Editing ◽  
Critical Time ◽  
Sweet Pepper ◽  
Hot Pepper ◽  
Sequencing Analysis ◽  
Guide Rna ◽  
Guide Rnas

Abstract Background DNA-free, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) ribonucleoprotein (RNP)-based genome editing is a simple, convincing, and promising tool for precision crop breeding. The efficacy of designed CRISPR-based genome editing tools is a critical prerequisite for successful precision gene editing in crops. Results This study demonstrates that soil-grown leaf- or callus-derived pepper protoplasts are a useful system for screening of efficient guide RNAs for CRISPR/Cas9 or CRISPR/Cas12a (Cpf1). CRISPR/Cas9 or Cpf1 were delivered as CRISPR/RNP complexes of purified endonucleases mixed with the designed single guide RNA, which can edit the target gene, CaMLO2 in two pepper cultivars with whole genome sequenced, Capsicum annuum ‘CM334’ and C. annuum ‘Dempsey’. The designed guide RNAs (sgRNAs for Cas9 or crRNAs for Cpf1) are conserved for CaMLO2 in both CM334 and Dempsey and cleave CaMLO2 in vitro. CRISPR/Cas9- or /Cpf1-RNP complexes were transfected into purely isolated protoplasts of the hot pepper CM334 and sweet pepper Dempsey by PEG-mediated delivery. Targeted deep sequencing analysis indicated that the targeted CaMLO2 gene was differentially edited in both cultivars, depending on the applied CRISPR/RNPs. Conclusions Pepper protoplast-based CRISPR guide-RNA selection is a robust method to check the efficacy of designed CRISPR tools and is a prerequisite for regenerating edited plants, which is a critical time-limiting procedure. The rapid and convincing selection of guide RNA against a target genome reduces the laborious efforts for tissue culture and facilitates effective gene editing for pepper improvement.

scholarly journals Programming PAM antennae for efficient CRISPR-Cas9 DNA editing

2020 ◽  
Vol 6 (19) ◽  
pp. eaay9948
Author(s):  
Fei Wang ◽  
Yaya Hao ◽  
Qian Li ◽  
Jiang Li ◽  
Honglu Zhang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Super Resolution ◽  
Single Molecule Level ◽  
Guide Rna ◽  
Guide Rnas ◽  
Protospacer Adjacent Motif ◽  
Target Dna

Bacterial CRISPR-Cas9 nucleases have been repurposed as powerful genome editing tools. Whereas engineering guide RNAs or Cas nucleases have proven to improve the efficiency of CRISPR editing, modulation of protospacer-adjacent motif (PAM), indispensable for CRISPR, has been less explored. Here, we develop a DNA origami–based platform to program a PAM antenna microenvironment and address its performance at the single-molecule level with submolecular resolution. To mimic spatially controlled in vivo PAM distribution as may occur in chromatin, we investigate the effect of PAM antennae surrounding target DNA. We find that PAM antennae effectively sensitize the DNA cleavage by recruiting Cas9 molecules. Super-resolution tracking of single single-guide RNA/Cas9s reveals localized translocation of Cas9 among spatially proximal PAMs. We find that the introduction of the PAM antennae effectively modulates the microenvironment for enhanced target cleavage (up to ~50%). These results provide insight into factors that promote more efficient genome editing.

scholarly journals A temporally resolved, multiplex molecular recorder based on sequential genome editing

2021 ◽  
Author(s):  
Junhong Choi ◽  
Wei Chen ◽  
Anna Minkina ◽  
Florence M Chardon ◽  
Chase C Suiter ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Lineage ◽  
General System ◽  
Text Messages ◽  
Tandem Array ◽  
Digital Medium ◽  
Short Text ◽  
Guide Rna ◽  
Guide Rnas

DNA is naturally well-suited to serve as a digital medium for in vivo molecular recording. However, DNA-based memory devices described to date are constrained in terms of the number of distinct signals that can be concurrently recorded and/or by a failure to capture the precise order of recorded events. Here we describe DNA Ticker Tape, a general system for in vivo molecular recording that largely overcomes these limitations. Blank DNA Ticker Tape consists of a tandem array of partial CRISPR-Cas9 target sites, with all but the first site truncated at their 5' ends, and therefore inactive. Signals of interest are coupled to the expression of specific prime editing guide RNAs. Editing events are insertional, and record the identity of the guide RNA mediating the insertion while also shifting the position of the "write head" by one unit along the tandem array, i.e. sequential genome editing. In this proof-of-concept of DNA Ticker Tape, we demonstrate the recording and decoding of complex event histories or short text messages; evaluate the performance of dozens of orthogonal tapes; and construct "long tape" potentially capable of recording the order of as many as 20 serial events. Finally, we demonstrate how DNA Ticker Tape simplifies the decoding of cell lineage histories.

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