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

Abstract

BackgroundClustered 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.ResultsWe 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.ConclusionsHere 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).Electronic supplementary materialThe online version of this article (10.1186/s12867-018-0105-8) contains supplementary material, which is available to authorized users.

CRISPR-Cas9 Genome Editing in Human Cell Lines with Donor Vector Made by Gibson Assembly

“…Nuclear localization sequences were fused to hSpCas9 to ensure DNA access [19]. CRISPR heterologous systems are continually being refined to increase efficiency and reduce off-target effects in a vast array of biological systems from C. Elegans to humans [9].…”

“…The protocol highlighted in this chapter was published (in less detail) in Open Access BMC Molecular Biology; parts of this protocol are reproduced with permission from the initial journal [9]. This method employs a modified Cas9 nickase (Cas9D10A) purchased from Sigma: CRISPR Cas9D10A-GFP Nickase (catalog: CAS9D10AGFPP, Sigma, St. Louis, MO).…”

“…To select for FOXO3 disruption mutants, we prepared a donor vector containing the neomycin resistance gene via Gibson assembly using the commonly employed mammalian expression vector pcDNA3 as the backbone [9]. First, PCR was used to construct two "arms" containing FOXO3 chromosomal sequences.…”

“…CRISPR Cas9 and related mechanisms are endogenous adaptive immune responses found widely in bacteria and archaea to fend off invading DNAs, such as those from viruses [1][2][3][4][5][6][7][8]. Short segments of invading DNA are inserted into a specialized bacterial chromosomal locus containing repetitive DNA elements, the clustered regularly interspaced short palindromic repeats (CRISPR) [9][10][11][12][13][14][15]. The resulting transcribed RNA is processed into several short RNAs (crRNAs) that are incorporated into a riboprotein complex [11][12][13][14][15].The riboprotein complex then targets the foreign nucleic acids in a sequence specific manner due to the presence of the crRNAs.…”

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

“…The original article [1] contains three erroneous mentions of usage of a restriction enzyme— Bst Z17I—in the Methods section as displayed in the following sentences:[…] Therefore, FOXO3 gene fragments (PCR products) had pcDNA3 sequences on the ends that corresponded to upstream and downstream sequences of the utilized restriction sites ( Dra III for Arm1 and Bst Z17I for Arm2) in pcDNA3.[…] The intermediate vector (with FOXO3 Arm 1) was cut with Bst Z17I, which is on the other side of the neomycin resistance gene in the pcDNA3 plasmid compared to FOXO3 Arm 1.[…] FOXO3 Arm 2 was amplified with the primer pair specified in Table 1, producing a product that had sequences on each end that were identical to the sequences proximal to the Bst Z17I site in the intermediate FOXO3 Arm 1 vector.…”