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A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone

Overview of attention for article published in BMC Molecular Biology, March 2018
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Title
A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone
Published in
BMC Molecular Biology, March 2018
DOI 10.1186/s12867-018-0105-8
Pubmed ID
Authors

Neftali Vazquez, Lilia Sanchez, Rebecca Marks, Eduardo Martinez, Victor Fanniel, Alma Lopez, Andrea Salinas, Itzel Flores, Jesse Hirschmann, Robert Gilkerson, Erin Schuenzel, Robert Dearth, Reginald Halaby, Wendy Innis-Whitehouse, Megan Keniry

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone. We describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9 = CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines. Here we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications).

Twitter Demographics

The data shown below were collected from the profiles of 4 tweeters who shared this research output. Click here to find out more about how the information was compiled.

Mendeley readers

The data shown below were compiled from readership statistics for 53 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 53 100%

Demographic breakdown

Readers by professional status Count As %
Student > Master 11 21%
Student > Bachelor 10 19%
Student > Ph. D. Student 8 15%
Researcher 4 8%
Other 2 4%
Other 3 6%
Unknown 15 28%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 24 45%
Agricultural and Biological Sciences 7 13%
Medicine and Dentistry 2 4%
Pharmacology, Toxicology and Pharmaceutical Science 1 2%
Immunology and Microbiology 1 2%
Other 3 6%
Unknown 15 28%

Attention Score in Context

This research output has an Altmetric Attention Score of 2. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 13 September 2018.
All research outputs
#7,605,706
of 13,511,578 outputs
Outputs from BMC Molecular Biology
#129
of 267 outputs
Outputs of similar age
#132,882
of 272,337 outputs
Outputs of similar age from BMC Molecular Biology
#1
of 1 outputs
Altmetric has tracked 13,511,578 research outputs across all sources so far. This one is in the 42nd percentile – i.e., 42% of other outputs scored the same or lower than it.
So far Altmetric has tracked 267 research outputs from this source. They receive a mean Attention Score of 2.6. This one has gotten more attention than average, scoring higher than 51% of its peers.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 272,337 tracked outputs that were published within six weeks on either side of this one in any source. This one is in the 49th percentile – i.e., 49% of its contemporaries scored the same or lower than it.
We're also able to compare this research output to 1 others from the same source and published within six weeks on either side of this one. This one has scored higher than all of them