scholarly journals Efficiency of SpCas9 and AsCpf1 (Cas12a) programmable nucleases at genomic safe harbor loci in HEK293 cells

2020 ◽  
Vol 49 ◽  
Author(s):  
S. V. Pavlova ◽  
E. A. Elisaphenko ◽  
L. Sh. Shayakhmetova ◽  
S. P. Medvedev
Keyword(s):  
Genome Engineering ◽  
Hek293 Cells ◽  
Open Chromatin ◽  
Safe Harbor ◽  
Dna Breaks ◽  
Guide Rna ◽  
Nucleosome Density ◽  
Low Efficiency ◽  
Hek293ft Cell

Rationale: The development of eukaryote genome engineering tools based on CRISPR-Cas programmable bacterial nucleases systems opens wide horizons for gene therapies, human disease cell modeling, as well as investigation into manifestation of disease phenotypes and visualization of cellular processes. The safety and approximation of experiments both at the cellular and organismal levels depend on the accuracy of introducing double-stranded breaks into the target DNA regions. The search for new variants of more accurate CRISPR-Cas nucleases and evaluation of their ability to hydrolyze nucleosome DNA in vivo is considered a critical task for the development of the genome engineering technologies.Aim: To analyze the activity of the programmable nuclease AsCpf1 (Cas12a), with low level of off-target activity, in the human genome loci that are safe for the introduction of transgenic constructs (“safe harbor”) and to compare its efficiency with that of the widely used SpCas9 nuclease in HEK293 cells.Materials and methods: We performed the bioinformatics analysis of the association between target regions with nucleosomes and other proteins in the safe harbor loci AAVS1 and GSH-Ch1 and the transcriptionally inactive gene MYBPC3 (cardiac myosin binding protein 3) based on ATAC-seq data for the HEK293FT cells obtained from the NCBI SRA database. Plasmids encoding SpCas9 and AsCpf1 nucleases and guide RNA to the target regions were constructed and transfected into the HEK293FT cells. Events in the target regions of the HEK293FT cell genome were studied in the sequenograms with the TIDE algorithm.Results: The results of the ATAC-seq experiments for HEK293FT cells have shown that the AAVS1 locus can be referred as open chromatin with a low nucleosome density, while the GSH-Ch1 locus can be attributed to closed chromatin. In HEK293FT cells, the cardiac MYBPC3 gene has intermediate chromatin density. Assessment of the efficiency of introducing breaks into the studied HEK293FT cell chromatin loci by nucleases has shown that SpCas9 is able to cope with chromatin of any nucleosome density, while AsCpf1 can effectively introduce DNA breaks only at loci with open chromatin, such as AAVS1 and MYBPC3. Editing events occur at a very low rate at the GSH-Ch1 locus with a high nucleosome density.Conclusion: We have found low efficiency of the AsCpf1 nuclease in the genomic safe harbor locus GSH-Ch1, which is characterized by a high nucleosome density. When planning an experiment on AsCpf1 nuclease genome editing, the epigenetic chromatin landscape and the nucleosome density should be considered, as well as chromatin opening substances should be used.

Prime-editors (nickases), hRad51–Cas9 nickase fusions and dCas9 have the same problem as conventional CRISPR-Cas9 of plasmid/Cas9 integration after making a double stranded break

2019 ◽  
Author(s):  
Sandeep Chakraborty
Keyword(s):  
Cellular Response ◽  
Sequencing Data ◽  
Dna Breaks ◽  
Precise Integration ◽  
Guide Rna ◽  
Double Strand Dna Breaks ◽  
Human Therapy ◽  
P53 Activation ◽  
Programmable Nucleases

‘Prime-editing’ proposes to replace traditional programmable nucleases (CRISPR-Cas9) using a catalytically impaired Cas9 (dCas9) connected to a engineered reverse transcriptase, and a guide RNA encoding both the target site and the desired change. With just a ‘nick’ on one strand, it is hypothe- sized, the negative, uncontrollable effects arising from double-strand DNA breaks (DSBs) - translocations, complex proteins, integrations and p53 activation - will be eliminated. However, sequencing data pro- vided (Accid:PRJNA565979) reveal plasmid integration, indicating that DSBs occur. Also, looking at only 16 off-targets is inadequate to assert that Prime-editing is more precise. Integration of plasmid occurs in all three versions (PE1/2/3). Interestingly, dCas9 which is known to be toxic in E. coli and yeast, is shown to have residual endonuclease activity. This also affects studies that use dCas9, like base- editors and de/methylations systems. Previous work using hRad51–Cas9 nickases also show significant integration in on-targets, as well as off-target integration [1]. Thus, we show that cellular response to nicking involves DSBs, and subsequent plasmid/Cas9 integration. This is an unacceptable outcome for any in vivo application in human therapy.

scholarly journals New human chromosomal safe harbor sites for genome engineering with CRISPR/Cas9, TAL effector and homing endonucleases

2018 ◽  
Author(s):  
Stefan Pellenz ◽  
Michael Phelps ◽  
Weiliang Tang ◽  
Blake T. Hovde ◽  
Ryan B. Sinit ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Fluorescent Proteins ◽  
Population Level ◽  
Safe Harbor ◽  
Homing Endonucleases ◽  
Transgene Integration ◽  
New Genes ◽  
Wide Range

AbstractSafe Harbor Sites (SHS) are genomic locations where new genes or genetic elements can be introduced without disrupting the expression or regulation of adjacent genes. We have identified 35 potential new human SHS in order to substantially expand SHS options beyond the three widely used canonical human SHS,AAVS1, CCR5andhROSA26. All 35 potential new human SHS and the three canonical sites were assessed for SHS potential using 9 different criteria weighted to emphasize safety that were broader and more genomics-based than previous efforts to assess SHS potential. We then systematically compared and rank-ordered our 35 new sites and the widely used humanAAVS1, hROSA26andCCR5sites, then experimentally validated a subset of the highly ranked new SHS together versus the canonicalAAVS1site. These characterizations includedin vitroandin vivocleavage-sensitivity tests; the assessment of population-level sequence variants that might confound SHS targeting or use for genome engineering; homology–dependent and –independent, SHS-targeted transgene integration in different human cell lines; and comparative transgene integration efficiencies at two new SHS versus the canonicalAAVS1site. Stable expression and function of new SHS-integrated transgenes were demonstrated for transgene-encoded fluorescent proteins, selection cassettes and Cas9 variants including a transcription transactivator protein that were shown to drive large deletions in aPAX3/FOXO1fusion oncogene and induce expression of theMYF5gene that is normally silent in human rhabdomyosarcoma cells. We also developed a SHS genome engineering ‘toolkit’ to enable facile use of the most extensively characterized of our new human SHS located on chromosome 4p. We anticipate our newly identified human SHS, located on 16 chromosomes including both arms of the human X chromosome, will be useful in enabling a wide range of basic and more clinically-oriented human gene editing and engineering.

scholarly journals All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo

2018 ◽  
Author(s):  
Raed Ibraheim ◽  
Chun-Qing Song ◽  
Aamir Mir ◽  
Nadia Amrani ◽  
Wen Xue ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Neisseria Meningitidis ◽  
Genome Editing ◽  
Viral Vectors ◽  
Genome Engineering ◽  
Degradation Pathway ◽  
Type I ◽  
Guide Rna ◽  
Hereditary Tyrosinemia

AbstractClustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing. Currently, three Cas9 orthologs have been adapted for in vivo genome engineering applications: SpyCas9, SauCas9 and CjeCas9. However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors, especially those in which Cas9 and sgRNA are combined into a single vector genome. Here, we present an additional in vivo editing platform using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct PAM, making it an excellent candidate for safe gene therapy applications. We find that NmeCas9 can be used to target the Pcsk9 and Hpd genes in mice. Using tail vein hydrodynamic-based delivery of NmeCas9 plasmid to target the Hpd gene, we successfully reprogrammed the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we delivered NmeCas9 with its single-guide RNA in a single recombinant adeno-associated vector (rAAV) to target Pcsk9, resulting in lower cholesterol levels in mice. This all-in-one vector yielded >35% gene modification after two weeks of vector administration, with minimal off-target cleavage in vivo. Our findings indicate that NmeCas9 can facilitate future efforts to correct disease-causing mutations by expanding the targeting scope of RNA-guided nucleases.

scholarly journals A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yaojun Tong ◽  
Tue S. Jørgensen ◽  
Christopher M. Whitford ◽  
Tilmann Weber ◽  
Sang Yup Lee
Keyword(s):  
Genome Engineering ◽  
Genetic Manipulation ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Base Editing ◽  
Guide Rna ◽  
E Coli ◽  
Potential Basis ◽  
Low Efficiency ◽  
Nucleotide Resolution

AbstractCRISPR base editing is a powerful method to engineer bacterial genomes. However, it restricts editing to single-nucleotide substitutions. Here, to address this challenge, we adapt a CRISPR-Prime Editing-based, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes. It can introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli with high fidelity. Notably, under optimal conditions, the efficiency of 1-bp deletions reach up to 40%. Moreover, deletions of up to 97 bp and insertions up to 33 bp were successful with the toolkit in E. coli, however, efficiencies dropped sharply with increased fragment sizes. With a second guide RNA, our toolkit can achieve multiplexed editing albeit with low efficiency. Here we report not only a useful addition to the genome engineering arsenal for E. coli, but also a potential basis for the development of similar toolkits for other bacteria.

scholarly journals In Vivo HSC Gene Therapy with Base Editors Allows for Efficient Reactivation of Fetal Globin in Beta-Yac Mice

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-22
Author(s):  
Chang Li ◽  
Afrodite Georgakopoulou ◽  
Sucheol Gil ◽  
Andre Lieber
Keyword(s):  
Bone Marrow ◽  
Genome Engineering ◽  
Bone Marrow Cells ◽  
Hematological Parameters ◽  
Fetal Hemoglobin ◽  
Optimized Design ◽  
Dna Breaks ◽  
Base Editing ◽  
Gamma Globin

Base editors are capable of installing precise nucleotide mutations at targeted genomic loci and present the advantage of avoiding double-stranded DNA breaks. Here, we aimed to target critical motifs regulating gamma-globin reactivation with base editors delivered via HDAd5/35++ vectors. Through optimized design, we successfully rescued a panel of cytidine and adenine base editors (CBE and ABE) targeting the BCL11A enhancer or recreating naturally occurring Hereditary Persistence of Fetal Hemoglobin (HPFH) mutations in the HBG1/2 promoter. In HUDEP-2 cells, all five tested vectors efficiently installed target base conversion and led to gamma-globin reactivation. We observed significant gamma-globin protein production (~23% over β-globin) by using an ABE vector HDAd-ABE-sgHBG#2 specific to the -113A to G HPFH mutation in HBG1/2 promoter. This vector was therefore chosen for downstream in vivo hematopoietic progenitor/stem cell (HSPC) transduction studies in mice that carry 248kb of the human β-globin locus (β-YAC mice) and thus accurately reflect globin switching. An EF1a-mgmtP140K expression cassette flanked by frt and transposon sites was included in the vector for allowing in vivo selection of transduced cells. After in vivo HSPC transduction with HDAd-ABE-HBG#2 + HDAd-SB and low doses of chemoselection, an average of over 40% HbF-positive cells in peripheral red blood cells was measured. This corresponded to ~21% gamma-globin production over human β-globin. The -113 A to G conversion in total bone marrow cells was on average 20%. Compared to untransduced mice, no alterations in hematological parameters, erythropoiesis and bone marrow cellular composition were observed after treatment, demonstrating a good safety profile of our approach. No detectable editing was found at top-scored potential off-target genomic sites. Bone marrow lineage-negative cells, isolated from primary mice at week 16 after transduction, were capable of reconstituting secondary transplanted mice with stable HbF expression. Importantly, the advantage of base editing over CRISPR/Cas9 was reflected by the markedly lower rates of intergenic 4.9kb deletion and no detectable toxicity in human CD34+ stem cells. Our observations demonstrate that base editors delivered by HDAd5/35++ vectors represent a promising strategy for precise in vivo genome engineering for the treatment of hemoglobinopathies. Disclosures Lieber: Ensoma, Inc: Consultancy, Research Funding.

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 Genome and epigenome engineering CRISPR toolkit for probing in vivo cis-regulatory interactions in the chicken embryo

2017 ◽  
Author(s):  
Ruth M Williams ◽  
Upeka Senanayake ◽  
Mara Artibani ◽  
Gunes Taylor ◽  
Daniel Wells ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Neural Crest ◽  
Chicken Embryo ◽  
Genome Engineering ◽  
Next Generation ◽  
Guide Rna ◽  
Regulatory Interactions ◽  
Gene Regulatory ◽  
Generation Sequencing

AbstractCRISPR-Cas9 genome engineering has revolutionised all aspects of biological research, with epigenome engineering transforming gene regulation studies. Here, we present a highly efficient toolkit enabling genome and epigenome engineering in the chicken embryo, and demonstrate its utility by probing gene regulatory interactions mediated by neural crest enhancers. First, we optimise efficient guide-RNA expression from novel chick U6-mini-vectors, provide a strategy for rapid somatic gene knockout and establish protocol for evaluation of mutational penetrance by targeted next generation sequencing. We show that CRISPR/Cas9-mediated disruption of transcription factors causes a reduction in their cognate enhancer-driven reporter activity. Next, we assess endogenous enhancer function using both enhancer deletion and nuclease-deficient Cas9 (dCas9) effector fusions to modulate enhancer chromatin landscape, thus providing the first report of epigenome engineering in a developing embryo. Finally, we use the synergistic activation mediator (SAM) system to activate an endogenous target promoter. The novel genome and epigenome engineering toolkit developed here enables manipulation of endogenous gene expression and enhancer activity in chicken embryos, facilitating high-resolution analysis of gene regulatory interactions in vivo.Summary StatementWe present an optimised toolkit for efficient genome and epigenome engineering using CRISPR in chicken embryos, with a particular focus on probing gene regulatory interactions during neural crest development.List of AbbreviationsGenome Engineering (GE), Epigenome Engineering (EGE), single guide RNA (sgRNA), Neural Crest (NC), Transcription Factor (TF), Next Generation Sequencing (NGS), somite stage (ss), Hamburger Hamilton (HH).

scholarly journals The biogenesis and function of nucleosome arrays

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ashish Kumar Singh ◽  
Tamás Schauer ◽  
Lena Pfaller ◽  
Tobias Straub ◽  
Felix Mueller-Planitz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Sequence ◽  
Chromatin Remodeling ◽  
Genome Engineering ◽  
Genotoxic Stress ◽  
Eukaryotic Cells ◽  
Nucleosome Density ◽  
And Function

AbstractNumerous chromatin remodeling enzymes position nucleosomes in eukaryotic cells. Aside from these factors, transcription, DNA sequence, and statistical positioning of nucleosomes also shape the nucleosome landscape. The precise contributions of these processes remain unclear due to their functional redundancy in vivo. By incisive genome engineering, we radically decreased their redundancy in Saccharomyces cerevisiae. The transcriptional machinery strongly disrupts evenly spaced nucleosomes. Proper nucleosome density and DNA sequence are critical for their biogenesis. The INO80 remodeling complex helps space nucleosomes in vivo and positions the first nucleosome over genes in an H2A.Z-independent fashion. INO80 requires its Arp8 subunit but unexpectedly not the Nhp10 module for spacing. Cells with irregularly spaced nucleosomes suffer from genotoxic stress including DNA damage, recombination and transpositions. We derive a model of the biogenesis of the nucleosome landscape and suggest that it evolved not only to regulate but also to protect the genome.

scholarly journals Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Steven Lin ◽  
Brett T Staahl ◽  
Ravi K Alla ◽  
Jennifer A Doudna
Keyword(s):  
Genome Engineering ◽  
High Efficiency ◽  
Embryonic Stem ◽  
Human Cells ◽  
Dna Breaks ◽  
Site Specific ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Double Strand Dna Breaks ◽  
Cell Mortality

The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (<xref ref-type="bibr" rid="bib13">Jinek et al., 2013</xref>), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.

scholarly journals A Multiplex CRISPR/Cas9 System for Use as an Anti-BmNPV Therapeutic

2018 ◽  
Author(s):  
Zhanqi Dong ◽  
Qi Qin ◽  
Zhigang Hu ◽  
Peng Chen ◽  
Liang Huang ◽  
...  
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
Sustained Delivery ◽  
Dna Breaks ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Transgenic Silkworms ◽  
Enzyme Targeting ◽  
Multiplex System ◽  
Unique Capability

Clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology guided by a single-guide RNA (sgRNA) has recently opened a new avenue for antiviral therapy. A unique capability of the CRISPR/Cas9 system is multiple genome engineering. However, there are few applications in insect viruses by a single Cas9 enzyme targeting two or more sgRNA at different genomic sites for simultaneous production of multiple DNA breaks. To address the need for multi-gene editing and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a one-vector (pSL1180-Cas9-U6-sgRNA) system to express multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus (BmNPV) in insect cells. Here, ie-1, gp64, lef-11, and dnapol genes were screened and identified as multiple sgRNA editing sites according to the BmNPV system infection and DNA replication mechanism. Furthermore, we constructed a multiplex editing vector sgMultiple to efficiently regulate multiplex gene editing steps and inhibit BmNPV replication after viral infection. This is the first report that describes the application of multiplex CRISPR/Cas9 system inhibiting insect virus replication. This multiplex system can significant enable the potential of CRISPR/Cas9-based multiplex genome engineering in transgenic silkworms.

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