Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system

Abstract

CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications, not only for single gene targeting, but also for multiple targeted mutagenesis. To make the most of the multiplexity of CRISPR/Cas9, we established a system for constructing all-in-one expression vectors containing multiple guide RNA expression cassettes and a Cas9 nuclease/nickase expression cassette. We further demonstrated successful examples of multiple targeting including chromosomal deletions in human cells using the all-in-one CRISPR/Cas9 vectors constructed with our novel system. Our system provides an efficient targeting strategy for multiplex genome/epigenome editing, simultaneous activation/repression of multiple genes, and beyond.

Figure 1. Schematic overview of the all-in-one CRISPR/Cas9 vector construction system for multiplex genome engineering.

Oligonucleotides corresponding to each target sequence are annealed and inserted into BpiI-digested pX330A or pX330S vectors (STEP 1). The constructed vectors harboring single gRNA expression cassettes are then assembled into an all-in-one vector harboring multiple gRNA cassettes using the Golden Gate assembly method (STEP 2). Amp, ampicillin; Spec, spectinomycin; U6, human U6 promoter; CBh, chicken beta-actin short promoter.

Figure 2. Multiplex genome editing with Cas9 nuclease and seven gRNAs.

(A) Schematic illustration of the all-in-one vector expressing seven gRNAs targeting seven different genomic loci and Cas9 nuclease. The blue and green bent arrows indicate the U6 and CBh promoters, respectively. (B) Genomic cleavage analysis of the seven genomic loci targeted with the all-in-one CRISPR/Cas9-nuclease vector. The products from untransfected control cells (C) and cells transfected with CRISPR/Cas9-nuclease vectors targeting seven (7) and single (1) loci were analyzed by agarose gel electrophoresis. The percentage of non-homologous end-joining (% NHEJ) was estimated using ImageJ software as previously described.

Figure 3. Multiplex genome editing with Cas9 nickase and six gRNAs.

(A) Schematic illustration of the all-in-one vector expressing six gRNAs targeting three different genomic loci and Cas9 nuclease. The black box indicates exon 14 of the human APC gene. The numbers from 2118 to 10804 represent the base positions in the APC gene transcript (NCBI reference sequence: NM_001127511.2). (B) The sizes of PCR products and cut images in each locus. (C) Genomic cleavage analysis of the three genomic loci targeted with the all-in-one CRISPR/Cas9-nickase vector. The products from untransfected control cells (C) and cells transfected with the CRISPR/Cas9-nickase vector expressing six gRNAs (6) were analyzed by agarose gel electrophoresis. The arrowheads indicate the approximate positions of the cleaved fragments. % NHEJ was estimated using ImageJ software as previously described. W, Wide-Range DNA Ladder (100–2,000 bp) (Takara Bio, Shiga, Japan).

Figure 4. Large deletions mediated by all-in-one CRISPR/Cas9 vectors.

(A) Schematic illustration of large deletion at the HPRT1 locus. The black boxes indicate exons. The blue lines and letters indicate PCR products. The zigzag lines indicate the HPRT1_A and HPRT1_B target sites. (B) Schematic illustration of large deletion at the APC locus. The black boxes indicate exons. The blue lines and letters indicate PCR products. The triangles indicate the off-5, off-8 and off-4 target sites. (C) Genomic PCR analysis of the HPRT1 and APC loci. The products from untransfected control cells (C) and cells transfected with the CRISPR/Cas9-nuclease vector expressing seven gRNAs (7) or the CRISPR/Cas9-nickase vector expressing six gRNAs (6) were analyzed by agarose gel electrophoresis. Red and blue asterisks indicate PCR products from un-deleted alleles, whereas yellow and green asterisks indicate PCR products from chromosomally deleted alleles. λ, λHindIII marker. W, Wide-Range DNA Ladder (100–2,000 bp) (Takara Bio). (D) Sequences of the PCR products from deleted alleles at the HPRT1 locus. The HPRT1_A and HPRT1_B target sites are indicated by red letters. PAM sites are indicated by black boxes. Deletions are indicated by dashes. (E) Sequences of the PCR products from deleted alleles at the APC locus. The off-5_L and off-4_R target sites are indicated by red letters. PAM sites are indicated by black boxes. Deletions are indicated by dashes.