Golden Gate Assembly of CRISPR gRNA expression array for simultaneously targeting multiple genes

Abstract

The engineered CRISPR/Cas9 technology has developed as the most efficient and broadly used genome editing tool. However, simultaneously targeting multiple genes (or genomic loci) in the same individual cells using CRISPR/Cas9 remain one technical challenge. In this article, we have developed a Golden Gate Assembly method for the generation of CRISPR gRNA expression arrays, thus enabling simultaneous gene targeting. Using this method, the generation of CRISPR gRNA expression array can be accomplished in 2 weeks, and contains up to 30 gRNA expression cassettes. We demonstrated in the study that simultaneously targeting 10 genomic loci or simultaneously inhibition of multiple endogenous genes could be achieved using the multiplexed gRNA expression array vector in human cells. The complete set of plasmids is available through the non-profit plasmid repository Addgene.

Keywords: CRISPR; Cas9; Genome editing; Golden Gate Assembly; Simultaneous multiple gene inhibition; Simultaneous multiple gene knockout.

Fig. 1

Golden Gate Assembly of CRISPR gRNA expression array for simultaneously targeting of multiple genomic loci. Schematic illustration of Golden Gate Assembly of gRNA expression array using single modular, “array” vectors and the pMsgRNA-EGFP plasmid. Each gRNA guide oligonucleotide (T1 to T10, T11 to T20, or T21 to T30) is cloned into single modular plasmids (pMA1 to pMA10) based using the type IIS restriction enzyme BbsI correspondingly. The single pMA modular plasmid containing one gRNA expression cassette can be cut out and assembled into the “array” plasmids using the type IIS restriction enzyme BsaI, which will generate gRNA expression arrays carrying up to 10 cassettes. The gRNA expression arrays in the “array” plasmids can be further cut out and assembled into the pMsgRNA-EGFP plasmid using BsmBI, generating gRNA expression array containing 11–30 cassettes. GGC, Golden Gate Cloning; U6, U6 promoter for gRNA transcription; BB, BbsI restriction enzyme; Scr, SpCas9 gRNA scaffold sequences; EGFP, EGFP expression cassette driven by the CMV promoter

Fig. 2

PCR validation of a gRNA expression carrying 10 expression cassettes. a Schematic illustration of the universal PCR and guide specific PCR for screening of transformed E. coli colonies. F1 (U6-F) and R1 (Scr-R) are used for the universal PCR (Supplementary Table 1). Guide specific PCRs are using the pair sense and antisense gRNA oligonucleotides of the two adjacent gRNAs. b An example of the universal PCR results for a multiplexed gRNA array containing 10 expression cassettes. c An example of the 9 guide RNA specific PCRs for a multiplexed gRNA array containing 10 expression cassettes

Fig. 3

Functional validation of the M10 gRNA expression array using a dual-fluorescent reporter system (C-Check). Upper panel a C-Check reporter vector were generated for each genomic locus. For comparison, HEK293T cells were transfected with a C-Check plasmid, a SpCas9 expression plasmid, and equal molar amount of either a pair of gRNAs or the M10 gRNA, or an empty gRNA (control). Transfected cells were analyzed by flow cytometry 48 h after transfection. Lower panel representative dot and histogram plot of the flow cytometry analysis, EGFP intensity versus AsRED intensity. Bar plot of mean percentage efficiency (% GFP+/AsRED+ cells, n = 3) is presented to the right

Fig. 4

Simultaneous deletion of 5 genomic loci using gRNA expression array in human cells. a For each genomic locus, a pair of gRNAs is designed to mediate target deletion of a small DNA fragment, which facilitates the genotyping by PCR. The M10 gRNA expression array plasmid used in this study contain 10 gRNAs targeting 5 genomic loci. b PCR screening results of dual gRNAs or M10 gRNAs mediated targeted gene deletion in five genomic loci in HEK293T cells. Cells transfected with an empty gRNA plasmid were used as controls. The expected wild type (wt) and deleted (del) amplicons were indicated to the right. Transfections were performed in triplicates. “-”, water control for PCR

Fig. 5

Simultaneous inhibition of multiples genes using CRISPRi in human cells. a Inhibition of KLF4, C-MYC, SOX2 and OCT4 expression in HEK293T cells by 10 single gRNAs or the M10-CRISPRi gRNA. Relative mRNA expression was calculated as the mean (triplicates) percentage of basal expression level in cells transfected with an empty gRNA. Schematic illustration of the gRNA target site for each gene is shown on the top. Asterisk indicates statistically significant (p < 0.05 by ANOVA). b Correlation plot of the mean inhibition of gene expression (percentage of the basal expression level) and the basal expression level of each gene (mean delta Ct of the reference gene, beta-actin). Each plot represents mean of triplicates

Fig. 6

Schematic comparison of simultaneous delivery of many CRISPR gRNA into the same cells using co-transfection of many single gRNA plasmids or one multiplexed gRNA expression array, as well as the potential applications