scholarly journals Marker-free coselection for CRISPR-driven genome editing in human cells

2017 ◽  
Vol 14 (6) ◽  
pp. 615-620 ◽  
Author(s):  
Daniel Agudelo ◽  
Alexis Duringer ◽  
Lusiné Bozoyan ◽  
Caroline C Huard ◽  
Sophie Carter ◽  
...  
Keyword(s):  
Genome Editing ◽  
Human Cells ◽  
Marker Free

CRISPR-Cpf1-mediated genome editing and gene regulation in human cells

2019 ◽  
Vol 37 (1) ◽  
pp. 21-27 ◽  
Author(s):  
Tianwen Li ◽  
Linwen Zhu ◽  
Bingxiu Xiao ◽  
Zhaohui Gong ◽  
Qi Liao ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Genome Editing ◽  
Human Cells

scholarly journals Efficient target cleavage by Type V Cas12a effector programmed with split CRISPR RNA

2020 ◽  
Author(s):  
Regina Tkach ◽  
Natalia Nikitchina ◽  
Nikita Shebanov ◽  
Vladimir Mekler ◽  
Egor Ulashchik ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Target Recognition ◽  
Human Cells ◽  
Stable Complex ◽  
Slight Effect ◽  
Target Dna ◽  
Type V

ABSTRACTCRISPR RNAs (crRNAs) directing target DNA cleavage by type V-A Cas12a nucleases consist of repeat-derived 5’-scaffold moiety and 3’-spacer moiety. We demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by Cas12a ortholog from Acidaminococcus sp (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer part only, while crRNAs split into two individual moieties (scaffold and spacer RNAs) catalyzed highly specific and efficient cleavage of target DNA. Our data also indicate that AsCas12a combined with split crRNA forms a stable complex with the target. These observations were also confirmed in lysates of human cells expressing AsCas12a. The ability of the AsCas12a nuclease to be programmed with split crRNAs opens new lines of inquiry into the mechanisms of target recognition and cleavage and will further facilitate genome editing techniques based on Cas12a nucleases.

Rapid Control of Genome Editing in Human Cells by Chemical-Inducible CRISPR-Cas Systems

2018 ◽  
pp. 267-288 ◽  
Author(s):  
Kaiwen Ivy Liu ◽  
Muhammad Nadzim Bin Ramli ◽  
Norfala-Aliah Binte Sutrisnoh ◽  
Meng How Tan
Keyword(s):  
Genome Editing ◽  
Human Cells ◽  
Rapid Control

scholarly journals CRISPR-Cas9 genome editing in human cells works via the Fanconi Anemia pathway

2017 ◽  
Author(s):  
Chris D Richardson ◽  
Katelynn R Kazane ◽  
Sharon J Feng ◽  
Nicholas L Bray ◽  
Axel J Schäfer ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Fanconi Anemia ◽  
High Efficiency ◽  
Genetic Diseases ◽  
Dermal Fibroblasts ◽  
Human Cells ◽  
Individual Gene ◽  
Break Site ◽  
Repair Pathways

AbstractCRISPR-Cas9 genome editing creates targeted double strand breaks (DSBs) in eukaryotic cells that are processed by cellular DNA repair pathways. Co-administration of single stranded oligonucleotide donor DNA (ssODN) during editing can result in high-efficiency (>20%) incorporation of ssODN sequences into the break site. This process is commonly referred to as homology directed repair (HDR) and here referred to as single stranded template repair (SSTR) to distinguish it from repair using a double stranded DNA donor (dsDonor). The high efficacy of SSTR makes it a promising avenue for the treatment of genetic diseases1,2, but the genetic basis of SSTR editing is still unclear, leaving its use a mostly empiric process. To determine the pathways underlying SSTR in human cells, we developed a coupled knockdown-editing screening system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. Unexpectedly, we found that SSTR requires multiple components of the Fanconi Anemia (FA) repair pathway, but does not require Rad51-mediated homologous recombination, distinguishing SSTR from repair using dsDonors. Knockdown of FA genes impacts SSTR without altering break repair by non-homologous end joining (NHEJ) in multiple human cell lines and in neonatal dermal fibroblasts. Our results establish an unanticipated and central role for the FA pathway in templated repair from single stranded DNA by human cells. Therapeutic genome editing has been proposed to treat genetic disorders caused by deficiencies in DNA repair, including Fanconi Anemia. Our data imply that patient genotype and/or transcriptome profoundly impact the effectiveness of gene editing treatments and that adjuvant treatments to bias cells towards FA repair pathways could have considerable therapeutic value.

scholarly journals Efficient Targeted Gene Insertion in Maize using Agrobacterium-Mediated Delivery

2020 ◽  
Author(s):  
Sergei Svitashev ◽  
Dave Peterson ◽  
Pierjuigi Barone ◽  
Brian Lenderts ◽  
Chris Schwartz ◽  
...  
Keyword(s):  
Genome Editing ◽  
Strand Break ◽  
Plant Genome ◽  
Specific Gene ◽  
Crop Species ◽  
Gene Insertion ◽  
Maize Transformation ◽  
Dna Double Strand Break ◽  
Genomic Location ◽  
Marker Free

Abstract CRISPR-Cas is a powerful DNA double strand break technology with wide-ranging applications in plant genome modification. However, the efficiency of genome editing depends on various factors including plant genetic transformation processes and types of modifications desired. Agrobacterium infection is the preferred method of transformation and delivery of editing components into the plant cell. While this method has been successfully used to generate gene knock-outs in multiple crops, precise nucleotide replacement and especially gene insertion into a pre-defined genomic location remain highly challenging. Here we report an efficient, heritable, selectable marker-free site-specific gene insertion in maize using Agrobacterium-mediated delivery. Advancements in maize transformation and new vector design enabled targeted insertion with frequencies as high as 8–10%. Importantly, these advancements allowed not only an improvement of the frequency but also of the quality of generated events. These results further enable the application of genome editing for trait product development in a wide variety of crop species amenable to Agrobacterium-mediated transformation.

scholarly journals Unintended inhibition of protein function using GFP nanobodies in human cells

2019 ◽  
Author(s):  
Cansu Küey ◽  
Gabrielle Larocque ◽  
Nicholas I. Clarke ◽  
Stephen J. Royle
Keyword(s):  
Developmental Biology ◽  
Genome Editing ◽  
Cell Lines ◽  
Protein Function ◽  
Human Cells ◽  
Popular Approach ◽  
Inactive State ◽  
Dynamin 2 ◽  
Gfp Tag

Tagging a protein-of-interest with GFP using genome editing is a popular approach to study protein function in cell and developmental biology. To avoid re-engineering cell lines or organisms in order to introduce additional tags, functionalized nanobodies that bind GFP can be used to extend the functionality of the GFP tag. We developed functionalized nanobodies, which we termed “dongles”, that could add, for example, an FKBP tag to a GFP-tagged protein-of-interest; enabling knocksideways experiments in GFP knock-in cell lines. The power of knocksideways is that it allows investigators to rapidly switch the protein from an active to an inactive state. We show that dongles allow for effective knocksideways of GFP-tagged proteins in genome-edited human cells. However, we discovered that nanobody binding to dynamin-2-GFP caused inhibition of dynamin function prior to knocksideways. While this limitation might be specific to the protein studied, it was significant enough to convince us not to pursue development of dongle technology further.

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