scholarly journals Generation and Application of a Versatile CRISPR Toolkit for Mammalian Cell Engineering

2021 ◽  
Author(s):  
Nghi Dang ◽  
Alissa Lance-Byrne ◽  
Angela Tung ◽  
Xiaoge Guo ◽  
Ryan J Cecchi ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Gene Editing ◽  
Genome Engineering ◽  
Gene Knockout ◽  
Transient Transfection ◽  
Cell Engineering ◽  
Genetic Manipulations ◽  
Expression Plasmids

Abstract CRISPR/Cas9 has revolutionized the field of genome engineering. Yet, as the CRISPR toolbox has rapidly expanded, there remains a need for a comprehensive library of CRISPR/Cas9 reagents that allow users to perform complex cellular and genetic manipulations without requiring labor-intensive generation of reagents to meet each user’s unique experimental circumstances. Here we described the creation and validation of a pNAX CRISPR library consisting of 72 different Cas9 and gRNA expression plasmids to allow for efficient multiplex gene editing, activation, and repression in mammalian cells. The toolkit plasmids, which are piggyBac or lentiviral based, provide the means for reliable and rapid delivery of Cas9/gRNA through either transient transfection or stable integration. Using the toolkit, we demonstrate the ease with which users can perform single or multiplex gene editing and modulate the expression of both coding and non-coding genes. We also highlight the use of the comprehensive toolkit to perform combinatorial gene knockout to identify factors that regulate homologous recombination, along with investigating the regulatory role of a 68-kb intronic region associated with human disease.

PML isoform expression and DNA break location relative to PML nuclear bodies impacts the efficiency of homologous recombination

2020 ◽  
Vol 98 (3) ◽  
pp. 314-326 ◽  
Author(s):  
Kathleen M. Attwood ◽  
Jayme Salsman ◽  
Dudley Chung ◽  
Sabateeshan Mathavarajah ◽  
Carter Van Iderstine ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Gene Editing ◽  
Genotoxic Stress ◽  
Nuclear Bodies ◽  
Homology Directed Repair ◽  
Pml Nuclear Bodies ◽  
Promyelocytic Leukemia Nuclear Bodies ◽  
Chromosomal Break ◽  
The Impact

Promyelocytic leukemia nuclear bodies (PML NBs) are nuclear subdomains that respond to genotoxic stress by increasing in number via changes in chromatin structure. However, the role of the PML protein and PML NBs in specific mechanisms of DNA repair has not been fully characterized. Here, we have directly examined the role of PML in homologous recombination (HR) using I-SceI extrachromosomal and chromosome-based homology-directed repair (HDR) assays, and in HDR by CRISPR/Cas9-mediated gene editing. We determined that PML loss can inhibit HR in an extrachromosomal HDR assay but had less of an effect on CRISPR/Cas9-mediated chromosomal HDR. Overexpression of PML also inhibited both CRISPR HDR and I-SceI-induced HDR using a chromosomal reporter, and in an isoform-specific manner. However, the impact of PML overexpression on the chromosomal HDR reporter was dependent on the intranuclear chromosomal positioning of the reporter. Specifically, HDR at the TAP1 gene locus, which is associated with PML NBs, was reduced compared with a locus not associated with a PML NB; yet, HDR could be reduced at the non-PML NB-associated locus by PML overexpression. Thus, both loss and overexpression of PML isoforms can inhibit HDR, and proximity of a chromosomal break to a PML NB can impact HDR efficiency.

scholarly journals Gene editing enables rapid engineering of complex antibiotic assembly lines

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wei Li Thong ◽  
Yingxin Zhang ◽  
Ying Zhuo ◽  
Katherine J. Robins ◽  
Joanna K. Fyans ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Chemical Synthesis ◽  
Gene Editing ◽  
Gene Knockout ◽  
Substrate Selectivity ◽  
Assembly Lines ◽  
Bioactive Natural Products ◽  
Peptide Synthetases ◽  
New Antibiotics ◽  
Lipopeptide Antibiotic

AbstractRe-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, is a powerful route to new antibiotics and other bioactive natural products that are too complex for chemical synthesis. However, engineering megasynthases is very challenging using current methods. Here, we describe how CRISPR-Cas9 gene editing can be exploited to rapidly engineer one of the most complex megasynthase assembly lines in nature, the 2.0 MDa NRPS enzymes that deliver the lipopeptide antibiotic enduracidin. Gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. In contrast, attempts to engineer the same NRPS using a conventional homologous recombination-mediated gene knockout and complementation approach resulted in only traces of new enduracidin variants. In addition to exchanging subdomains within the enduracidin NRPS, subdomains from a range of NRPS enzymes of diverse bacterial origins were also successfully utilized.

scholarly journals New and emerging uses of CRISPR/Cas9 to genetically manipulate apicomplexan parasites

Parasitology ◽  
2018 ◽  
Vol 145 (9) ◽  
pp. 1119-1126 ◽  
Author(s):  
Manlio Di Cristina ◽  
Vern B. Carruthers
Keyword(s):  
Toxoplasma Gondii ◽  
Genome Engineering ◽  
Gene Knockout ◽  
Full Potential ◽  
Whole Genome ◽  
Apicomplexan Parasites ◽  
Genetic Manipulations ◽  
Knockout Mutants ◽  
History Of ◽  
Genome Screening

AbstractAlthough the application of CRISPR/Cas9 genome engineering approaches was first reported in apicomplexan parasites only 3 years ago, this technology has rapidly become an essential component of research on apicomplexan parasites. This review briefly describes the history of CRISPR/Cas9 and the principles behind its use along with documenting its implementation in apicomplexan parasites, especially Plasmodium spp. and Toxoplasma gondii. We also discuss the recent use of CRISPR/Cas9 for whole genome screening of gene knockout mutants in T. gondii and highlight its use for seminal genetic manipulations of Cryptosporidium spp. Finally, we consider new variations of CRISPR/Cas9 that have yet to be implemented in apicomplexans. Whereas CRISPR/Cas9 has already accelerated rapid interrogation of gene function in apicomplexans, the full potential of this technology is yet to be realized as new variations and innovations are integrated into the field.

scholarly journals CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Krzysztof Chylinski ◽  
Maria Hubmann ◽  
Ruth E. Hanna ◽  
Connor Yanchus ◽  
Georg Michlits ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Gene Editing ◽  
Genome Engineering ◽  
Expression Cassette ◽  
Rapid Induction ◽  
Versatile Tool ◽  
Cre Recombination ◽  
Rapid Switching ◽  
Target Effects

AbstractCRISPR-Cas9 is an efficient and versatile tool for genome engineering in many species. However, inducible CRISPR-Cas9 editing systems that regulate Cas9 activity or sgRNA expression often suffer from significant limitations, including reduced editing capacity, off-target effects, or leaky expression. Here, we develop a precisely controlled sgRNA expression cassette that can be combined with widely-used Cre systems, termed CRISPR-Switch (SgRNA With Induction/Termination by Cre Homologous recombination). Switch-ON facilitates controlled, rapid induction of sgRNA activity. In turn, Switch-OFF-mediated termination of editing improves generation of heterozygous genotypes and can limit off-target effects. Furthermore, we design sequential CRISPR-Switch-based editing of two loci in a strictly programmable manner and determined the order of mutagenic events that leads to development of glioblastoma in mice. Thus, CRISPR-Switch substantially increases the versatility of gene editing through precise and rapid switching ON or OFF sgRNA activity, as well as switching OVER to secondary sgRNAs.

Genotype–Phenotype Relationships in the Context of Transcriptional Adaptation and Genetic Robustness

2021 ◽  
Vol 55 (1) ◽  
Author(s):  
Gabrielius Jakutis ◽  
Didier Y.R. Stainier
Keyword(s):  
Genome Engineering ◽  
Annual Review ◽  
Publication Date ◽  
Genetic Manipulations ◽  
Functional Studies ◽  
Genetic Robustness ◽  
Transcriptional Modulation ◽  
Transcriptional Adaptation ◽  
Additional Explanation

Genetic manipulations with a robust and predictable outcome are critical to investigate gene function, as well as for therapeutic genome engineering. For many years, knockdown approaches and reagents including RNA interference and antisense oligonucleotides dominated functional studies; however, with the advent of precise genome editing technologies, CRISPR-based knockout systems have become the state-of-the-art tools for such studies. These technologies have helped decipher the role of thousands of genes in development and disease. Their use has also revealed how limited our understanding of genotype–phenotype relationships is. The recent discovery that certain mutations can trigger the transcriptional modulation of other genes, a phenomenon called transcriptional adaptation, has provided an additional explanation for the contradicting phenotypes observed in knockdown versus knockout models and increased awareness about the use of each of these approaches. In this review, we first cover the strengths and limitations of different gene perturbation strategies. Then we highlight the diverse ways in which the genotype–phenotype relationship can be discordant between these different strategies. Finally, we review the genetic robustness mechanisms that can lead to such discrepancies, paying special attention to the recently discovered phenomenon of transcriptional adaptation. Expected final online publication date for the Annual Review of Genetics, Volume 55 is November 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

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