Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector

Abstract

Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.

Figure 1.

Validation of sgRNA and lentiviral Cas9 expression constructs. (A) Constructs encoding unique Pol III promoters expressing sgRNAs targeting the AAVS1 locus or a construct containing the hU6 promoter immediately followed by poly-thymidine to terminate expression (‘Empty vector’) were transfected into HEK293T cells. End-point RT-PCR was used to probe for expression of each indicated promoter/sgRNA construct 2 days post-transfection. –RT: no reverse transcription control. (B) HEK293T cells were transfected with expression vectors encoding the AAVS1-targeted zinc finger nuclease or Cas9-T2A-GFP and the indicated promoter/sgRNA expression cassettes and assessed for gene modification levels 3 days post-transfection using the Surveyor assay. (C) HEK293T cells were transduced with a single lentiviral vector expressing four sgRNAs targeting the IL1RN promoter. End-point RT-PCR was used to probe for expression of each indicated promoter/sgRNA construct at 10 days post-transduction. –RT: no reverse transcription control. (D) HEK293T cells were transduced with lentiviral constructs encoding the indicated Cas9-T2A-GFP constructs without sgRNAs and assessed for Cas9 expression by western blot 7 days post-transduction by probing for a FLAG epitope tag on the N-terminus of the Cas9 protein.

Figure 2.

Golden Gate assembly of single lentiviral vectors encoding CRISPR/Cas9 and multiple sgRNA expression cassettes. In the first step, individual sgRNAs are cloned into separate expression vectors and sequence verified. Fragments containing each sgRNA expression cassette are then transferred into a Cas9-expressing lentivirus in step 2 by Golden Gate assembly as shown. Black triangles shown in the lentiviral plasmid represent loxP sites flanking the entire sgRNA and Cas9 expression cassette.

Figure 3.

Delivery of a multiplex CRISPR/Cas9 system with a single lentivirus. (A) Four sgRNAs targeting distinct genomic loci were cloned into a lentiviral vector expressing the active Cas9 nuclease. (B) HEK293T cells and primary human dermal fibroblasts were transduced with lentivirus expressing the indicated sgRNAs and assayed for nuclease activity using the Surveyor assay. HEK293T cells were assayed 7 days post-transduction. The human fibroblasts were assayed 10 days post-transduction. (C) Summary of gene editing events at four targeted loci in 10 single cell-derived clonal populations of HEK293T cells transduced with a single lentiviral Cas9 vector. Surveyor analysis for each individual clone is shown in Supplementary Figure S8.

Figure 4.

Transient gene activation in HEK293T cells stably expressing dCas9^VP64. HEK293T cells were transduced with lentivirus to stably express dCas9^VP64 and were subsequently transfected with plasmid expressing the indicated sgRNA combinations. By varying the number of sgRNAs delivered, the level of endogenous gene activation was tunable for both the (A) IL1RN and (B) HBG1 loci at 3 days post-transfection (*P < 0.05 versus no sgRNAs). Peak levels of (C) IL1RN and (D) HBG1 were observed 3–6 days post-transfection and the level of activation returned to background levels between days 15 and 20.

Figure 5.

Stable gene activation by dCas9^VP64 in HEK293T cells using a single multiplex lentiviral vector. HEK293T cells were transduced with lentivirus to stably express dCas9^VP64 and the indicated combinations of sgRNAs. Activation of the endogenous (A) IL1RN and (B) HBG1 loci at 7 days post-transduction were tunable by varying the number of sgRNAs delivered (*P < 0.05 versus no sgRNAs). Peak levels of endogenous (C) IL1RN and (D) HBG1 were observed 6 days post-transduction and the level of activation was sustained through day 21.

Figure 6.

Stable gene activation by dCas9^VP64 in primary human fibroblasts using a single multiplex lentiviral vector. Primary human dermal fibroblasts were transduced with lentivirus to stably co-express dCas9^VP64 and four sgRNAs targeted to the IL1RN promoter. Peak levels of endogenous IL1RN were observed 14 days post-transduction and the level of activation was sustained through day 21.