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

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

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Background: 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. Results: 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. Conclusions: 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).

Original languageEnglish
Article number3
JournalBMC Molecular Biology
Volume19
Issue number1
DOIs
StatePublished - 14 Mar 2018

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Genes
Research Personnel
Cell Line
Epitopes
Gene Knock-In Techniques
Guide RNA
Kanamycin Kinase
Inverted Repeat Sequences
Mutation
Neomycin
Deoxyribonuclease I
Homologous Recombination
Gene Deletion
Immune System
Western Blotting
RNA
Polymerase Chain Reaction
DNA

Keywords

  • CRISPR Cas9
  • Custom donor vector design and construction
  • Mammalian cell lines

Cite this

Vazquez, Neftali ; Sanchez, Lilia ; Marks, Rebecca ; Martinez, Eduardo ; Fanniel, Victor ; Lopez, Alma ; Salinas, Andrea ; Flores, Itzel ; Hirschmann, Jesse ; Gilkerson, Robert ; Schuenzel, Erin ; Dearth, Robert ; Halaby, Reginald ; Innis-Whitehouse, Wendy ; Keniry, Megan. / A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone. In: BMC Molecular Biology. 2018 ; Vol. 19, No. 1.
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abstract = "Background: 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. Results: 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. Conclusions: 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).",
keywords = "CRISPR Cas9, Custom donor vector design and construction, Mammalian cell lines",
author = "Neftali Vazquez and Lilia Sanchez and Rebecca Marks and Eduardo Martinez and Victor Fanniel and Alma Lopez and Andrea Salinas and Itzel Flores and Jesse Hirschmann and Robert Gilkerson and Erin Schuenzel and Robert Dearth and Reginald Halaby and Wendy Innis-Whitehouse and Megan Keniry",
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Vazquez, N, Sanchez, L, Marks, R, Martinez, E, Fanniel, V, Lopez, A, Salinas, A, Flores, I, Hirschmann, J, Gilkerson, R, Schuenzel, E, Dearth, R, Halaby, R, Innis-Whitehouse, W & Keniry, M 2018, 'A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone', BMC Molecular Biology, vol. 19, no. 1, 3. https://doi.org/10.1186/s12867-018-0105-8

A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone. / Vazquez, Neftali; Sanchez, Lilia; Marks, Rebecca; Martinez, Eduardo; Fanniel, Victor; Lopez, Alma; Salinas, Andrea; Flores, Itzel; Hirschmann, Jesse; Gilkerson, Robert; Schuenzel, Erin; Dearth, Robert; Halaby, Reginald; Innis-Whitehouse, Wendy; Keniry, Megan.

In: BMC Molecular Biology, Vol. 19, No. 1, 3, 14.03.2018.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone

AU - Vazquez, Neftali

AU - Sanchez, Lilia

AU - Marks, Rebecca

AU - Martinez, Eduardo

AU - Fanniel, Victor

AU - Lopez, Alma

AU - Salinas, Andrea

AU - Flores, Itzel

AU - Hirschmann, Jesse

AU - Gilkerson, Robert

AU - Schuenzel, Erin

AU - Dearth, Robert

AU - Halaby, Reginald

AU - Innis-Whitehouse, Wendy

AU - Keniry, Megan

PY - 2018/3/14

Y1 - 2018/3/14

N2 - Background: 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. Results: 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. Conclusions: 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).

AB - Background: 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. Results: 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. Conclusions: 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).

KW - CRISPR Cas9

KW - Custom donor vector design and construction

KW - Mammalian cell lines

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U2 - 10.1186/s12867-018-0105-8

DO - 10.1186/s12867-018-0105-8

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