scholarly journals Cell Synchronization Enhances Nuclear Transformation and Genome Editing via Cas9 Enabling Homologous Recombination in Chlamydomonas reinhardtii

2020 ◽  
Vol 9 (10) ◽  
pp. 2840-2850
Author(s):  
Max Angstenberger ◽  
Francesco de Signori ◽  
Valeria Vecchi ◽  
Luca Dall’Osto ◽  
Roberto Bassi
Keyword(s):  
Homologous Recombination ◽  
Chlamydomonas Reinhardtii ◽  
Genome Editing ◽  
Nuclear Transformation ◽  
Cell Synchronization

The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation

1994 ◽  
Vol 14 (6) ◽  
pp. 4011-4019
Author(s):  
J A Nelson ◽  
P B Savereide ◽  
P A Lefebvre
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Selectable Marker ◽  
Marker Gene ◽  
Small Subunit ◽  
Direct Selection ◽  
Nuclear Transformation ◽  
Ribulose Bisphosphate Carboxylase ◽  
Bisphosphate Carboxylase ◽  
Dominant Selectable Marker ◽  
Cry1 Gene

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.

scholarly journals Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination

2013 ◽  
Vol 41 (20) ◽  
pp. e193-e193 ◽  
Author(s):  
Changchun Chen ◽  
Lorenz A. Fenk ◽  
Mario de Bono
Keyword(s):  
Caenorhabditis Elegans ◽  
Homologous Recombination ◽  
Genome Editing

scholarly journals Using the Endogenous CRISPR-Cas System of Heliobacterium modesticaldum To Delete the Photochemical Reaction Center Core Subunit Gene

2019 ◽  
Vol 85 (23) ◽  
Author(s):  
Patricia L. Baker ◽  
Gregory S. Orf ◽  
Kimberly Kevershan ◽  
Michael E. Pyne ◽  
Taner Bicer ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Editing ◽  
Reaction Center ◽  
Photochemical Reaction ◽  
Genetic Locus ◽  
Gene Replacement ◽  
Type I ◽  
Content Type ◽  
Heliobacterium Modesticaldum

ABSTRACT In Heliobacterium modesticaldum, as in many Firmicutes, deleting genes by homologous recombination using standard techniques has been extremely difficult. The cells tend to integrate the introduced plasmid into the chromosome by a single recombination event rather than perform the double recombination required to replace the targeted locus. Transformation with a vector containing only a homologous recombination template for replacement of the photochemical reaction center gene pshA produced colonies with multiple genotypes, rather than a clean gene replacement. To address this issue, we required an additional means of selection to force a clean gene replacement. In this study, we report the genetic structure of the type I-A and I-E CRISPR-Cas systems from H. modesticaldum, as well as methods to leverage the type I-A system for genome editing. In silico analysis of the CRISPR spacers revealed a potential consensus protospacer adjacent motif (PAM) required for Cas3 recognition, which was then tested using an in vivo interference assay. Introduction of a homologous recombination plasmid that carried a miniature CRISPR array targeting sequences in pshA (downstream of a naturally occurring PAM sequence) produced nonphototrophic transformants with clean replacements of the pshA gene with ∼80% efficiency. Mutants were confirmed by PCR, sequencing, optical spectroscopy, and growth characteristics. This methodology should be applicable to any genetic locus in the H. modesticaldum genome. IMPORTANCE The heliobacteria are the only phototrophic members of the largely Gram-positive phylum Firmicutes, which contains medically and industrially important members, such as Clostridium difficile and Clostridium acetobutylicum. Heliobacteria are of interest in the study of photosynthesis because their photosynthetic system is unique and the simplest known. Since their discovery in the early 1980s, work on the heliobacteria has been hindered by the lack of a genetic transformation system. The problem of introducing foreign DNA into these bacteria has been recently rectified by our group; however, issues still remained for efficient genome editing. The significance of this work is that we have characterized the endogenous type I CRISPR-Cas system in the heliobacteria and leveraged it to assist in genome editing. Using the CRISPR-Cas system allowed us to isolate transformants with precise replacement of the pshA gene encoding the main subunit of the photochemical reaction center.

scholarly journals Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated, homology-independent knock-in system

2017 ◽  
Vol 28 (7) ◽  
pp. 898-906 ◽  
Author(s):  
Yohei Katoh ◽  
Saki Michisaka ◽  
Shohei Nozaki ◽  
Teruki Funabashi ◽  
Tomoaki Hirano ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Editing ◽  
Protein Trafficking ◽  
Target Genes ◽  
Practical Method ◽  
Cell Types ◽  
Recombination Activity ◽  
Ciliary Protein ◽  
Genes Encoding ◽  
Universal Donor

The CRISPR/Cas9 system has revolutionized genome editing in virtually all organisms. Although the CRISPR/Cas9 system enables the targeted cleavage of genomic DNA, its use for gene knock-in remains challenging because levels of homologous recombination activity vary among various cells. In contrast, the efficiency of homology-independent DNA repair is relatively high in most cell types. Therefore the use of a homology-independent repair mechanism is a possible alternative for efficient genome editing. Here we constructed a donor knock-in vector optimized for the CRISPR/Cas9 system and developed a practical system that enables efficient disruption of target genes by exploiting homology-independent repair. Using this practical knock-in system, we successfully disrupted genes encoding proteins involved in ciliary protein trafficking, including IFT88 and IFT20, in hTERT-RPE1 cells, which have low homologous recombination activity. The most critical concern using the CRISPR/Cas9 system is off-target cleavage. To reduce the off-target cleavage frequency and increase the versatility of our knock-in system, we constructed a universal donor vector and an expression vector containing Cas9 with enhanced specificity and tandem sgRNA expression cassettes. We demonstrated that the second version of our system has improved usability.

Improvement of Chloroplast Transformation Using CRISPR/Cas9

2020 ◽  
Vol 14 (3) ◽  
pp. 401-407
Author(s):  
Ning Tang ◽  
Yumei Xia ◽  
Yijie Zhan ◽  
Junhao Dan ◽  
Mulan Yu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Gene Editing ◽  
Transformation Efficiency ◽  
Chloroplast Transformation ◽  
Nuclear Transformation ◽  
Important Research ◽  
Transformation System ◽  
Repair Mechanism ◽  
Damage Repair

Chloroplasts are organelles that contain genetic materials (DNA) in higher plant cells. The special genetic characteristics of chloroplasts mean that plasmid transformation has important research value, so it has become an important research direction second to nuclear transformation. Although the techniques of chloroplast genome modification have been successfully applied in tobacco and extended to other high plants, there are still many limitations. Exogenous genes are integrated into the chloroplast genome through homologous recombination. Therefore, the low efficiency of homologous recombination directly limits transformation efficiency. Gene editing with fixed-point cutting function and DNA damage repair mechanism may effectively improve the efficiency. In the present study, we aimed to use CRISPR/Cas9 to cut the site between two homologous recombinant fragments in chloroplast transformation to improve the efficiency by activating the DNA damage repair mechanism. The Cas9 gene and gRNA were added to the chloroplast transformation system of tobacco by co-transformation or integration into a transformation vector. The acquired resistant plants were screened by multiple selection of spectinomycin and chloroplast DNA was isolated for molecular detection by PCR. The results showed that the efficiency of chloroplast transformation increased by 6–10 times with the addition of gene editing technology. Although the transformation efficiency was still far below the level of nuclear transformation, this study may help to increase the efficiency of the plant chloroplast transformation system, and expand the types of plant receptors.

Stable nuclear transformation of Chlamydomonas reinhardtii with a Streptomyces rimosus gene as the selective marker

Gene ◽  
1996 ◽  
Vol 181 (1-2) ◽  
pp. 13-18 ◽  
Author(s):  
Irina A. Sizova ◽  
Tatyana V. Lapina ◽  
Olga N. Frolova ◽  
Nelly N. Alexandrova ◽  
Konstantin E. Akopiants ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Nuclear Transformation ◽  
Streptomyces Rimosus ◽  
Selective Marker

scholarly journals Inhibition of p53 improves CRISPR/Cas - mediated precision genome editing

2017 ◽  
Author(s):  
Emma Haapaniemi ◽  
Sandeep Botla ◽  
Jenna Persson ◽  
Bernhard Schmierer ◽  
Jussi Taipale
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Homologous Recombination ◽  
Cell Cycle Arrest ◽  
Genome Editing ◽  
Dna Damage Response ◽  
Normal Cells ◽  
Cycle Arrest ◽  
Damage Response

AbstractWe report here that genome editing by CRISPR/Cas9 induces a p53-mediated DNA damage response and cell cycle arrest. Transient inhibition of p53 prevents this response, and increases the rate of homologous recombination more than five-fold. This provides a way to improve precision genome editing of normal cells, but warrants caution in using CRISPR for human therapies until the mechanism of the activation of p53 is elucidated.

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