Homology-Directed Transgene-Free Gene Editing in Chlamydomonas reinhardtii

2020 ◽  
pp. 237-252
Author(s):  
Aron Ferenczi ◽  
Attila Molnar
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Gene Editing ◽  
Free Gene

scholarly journals DNA-free gene editing in plants: a brief overview

2020 ◽  
Vol 35 (1) ◽  
pp. 131-138
Author(s):  
Tsveta Tsanova ◽  
Lidia Stefanova ◽  
Lora Topalova ◽  
Atanas Atanasov ◽  
Ivelin Pantchev
Keyword(s):  
Gene Editing ◽  
Free Gene

scholarly journals Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application

2021 ◽  
Vol 3 ◽  
Author(s):  
Jin-Jun Yue ◽  
Jin-Ling Yuan ◽  
Fu-Hui Wu ◽  
Yu-Hsuan Yuan ◽  
Qiao-Wei Cheng ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Protoplast Regeneration ◽  
Self Incompatibility ◽  
Future Prospects ◽  
Juvenile Period ◽  
Free Gene ◽  
Protoplast Transfection ◽  
Cas Proteins

In the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system, protoplasts are not only useful for rapidly validating the mutagenesis efficiency of various RNA-guided endonucleases, promoters, sgRNA designs, or Cas proteins, but can also be a platform for DNA-free gene editing. To date, the latter approach has been applied to numerous crops, particularly those with complex genomes, a long juvenile period, a tendency for heterosis, and/or self-incompatibility. Protoplast regeneration is thus a key step in DNA-free gene editing. In this report, we review the history and some future prospects for protoplast technology, including protoplast transfection, transformation, fusion, regeneration, and current protoplast applications in CRISPR/Cas-based breeding.

scholarly journals Expanding the editable genome and CRISPR–Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking

2019 ◽  
Vol 48 (2) ◽  
pp. 974-995 ◽  
Author(s):  
Xiaoyu Chen ◽  
Francesca Tasca ◽  
Qian Wang ◽  
Jin Liu ◽  
Josephine M Janssen ◽  
...  
Keyword(s):  
Gene Editing ◽  
Cell Function ◽  
Chromosomal Rearrangements ◽  
Dna Breaks ◽  
Double Stranded Dna ◽  
Genome Wide ◽  
Donor And Acceptor ◽  
Free Gene ◽  
In Trans ◽  
Programmable Nucleases

Abstract Genome editing typically involves recombination between donor nucleic acids and acceptor genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR–Cas9). Yet, nucleases yield off-target mutations and, most pervasively, unpredictable target allele disruptions. Remarkably, to date, the untoward phenotypic consequences of disrupting allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness as a result of DSBs at allelic and non-allelic target sites and report that simultaneous single-stranded DNA break formation at donor and acceptor DNA by CRISPR–Cas9 nickases (in trans paired nicking) mostly overcomes such disruptive genotype-phenotype associations. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments into essential and multiple-copy genomic sites. As shown herein by genotyping assays and high-throughput genome-wide sequencing of DNA translocations, this is achieved while circumventing most allelic and non-allelic mutations and chromosomal rearrangements characteristic of nuclease-dependent procedures. Our work demonstrates that in trans paired nicking retains target protein dosages in gene-edited cell populations and expands gene editing to chromosomal tracts previously not possible to modify seamlessly due to their recurrence in the genome or essentiality for cell function.

scholarly journals Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and single-stranded DNA

2017 ◽  
Vol 114 (51) ◽  
pp. 13567-13572 ◽  
Author(s):  
Aron Ferenczi ◽  
Douglas Euan Pyott ◽  
Andromachi Xipnitou ◽  
Attila Molnar
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Gene Editing ◽  
Nuclear Gene ◽  
Single Step ◽  
Single Stranded Dna ◽  
Research Fields ◽  
Free Generation ◽  
Direct Delivery ◽  
Dna Editing ◽  
Endogenous Locus

The green alga Chlamydomonas reinhardtii is an invaluable reference organism to research fields including algal, plant, and ciliary biology. Accordingly, decades-long standing inefficiencies in targeted nuclear gene editing broadly hinder Chlamydomonas research. Here we report that single-step codelivery of CRISPR/Cpf1 ribonucleoproteins with single-stranded DNA repair templates results in precise and targeted DNA replacement with as much as ∼10% efficiency in C. reinhardtii. We demonstrate its use in transgene- and selection-free generation of sequence-specific mutations and epitope tagging at an endogenous locus. As the direct delivery of gene-editing reagents bypasses the use of transgenes, this method is potentially applicable to a wider range of species without the need to develop methods for stable transformation.

scholarly journals Fluorescent labeling of CRISPR/Cas9 RNP for gene knockout in HSPCs and iPSCs reveals an essential role for GADD45b in stress response

2019 ◽  
Vol 3 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Masoud Nasri ◽  
Perihan Mir ◽  
Benjamin Dannenmann ◽  
Diana Amend ◽  
Tessa Skroblyn ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Gene Editing ◽  
Gene Knockout ◽  
Cell Types ◽  
Fluorescent Labeling ◽  
Hematopoietic Stem ◽  
Guide Rna ◽  
Free Gene ◽  
Induced Pluripotent

Abstract CRISPR/Cas9-mediated gene editing of stem cells and primary cell types has several limitations for clinical applications. The direct delivery of ribonucleoprotein (RNP) complexes consisting of Cas9 nuclease and guide RNA (gRNA) has improved DNA- and virus-free gene modifications, but it does not enable the essential enrichment of the gene-edited cells. Here, we established a protocol for the fluorescent labeling and delivery of CRISPR/Cas9–gRNA RNP in primary human hematopoietic stem and progenitor cells (HSPCs) and induced pluripotent stem cells (iPSCs). As a proof of principle for genes with low-abundance transcripts and context-dependent inducible expression, we successfully deleted growth arrest and DNA-damage-inducible β (GADD45B). We found that GADD45B is indispensable for DNA damage protection and survival in stem cells. Thus, we describe an easy and efficient protocol of DNA-free gene editing of hard-to-target transcripts and enrichment of gene-modified cells that are generally difficult to transfect.

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