A small cassette enables conditional gene inactivation by CRISPR/Cas9

Abstract

The availability of CRISPR/Cas9 technology has enabled the rapid establishment of gene knockouts in many cell types and even whole organisms. However, conditional inactivation of essential genes remains a challenge. We devised an approach named DECAI (DEgradation based on Cre-regulated- Artificial Intron). It utilizes a small cassette of just 201 nucleotides that is inserted into the coding exon of a target gene using CRISPR/Cas9 technology and homology-directed repair. As its sequence is derived from an artificial intron, the cassette is removed by the splicing machinery and thus leaves no trace in the "off-state". Upon activation with Cre recombinase ("on-state"), the intron is crippled and the target gene is disrupted by a series of stop codons. We exemplify the utility of this approach on several non-essential and essential human genes. Clones bearing the conditional knockout cassette are recovered at frequencies above 5% and cassette function can be traced at the genomic DNA and the mRNA level. Importantly, cassette activation leads to loss of gene expression as judged by flow cytometry, Western blot or immunofluorescence. Altogether, this highlights the broad utility of the approach for conditional gene inactivation and suggests that this tool could be used to study the loss-of-function phenotypes of essential genes.

Figure 1

Approach for conditional gene inactivation using artificial introns. (A) For the purpose of conditional gene inactivation, an artificial intron is introduced into a coding exon of a gene. In the artificial intron (marked in blue), the branch point (in yellow) is flanked by two parallel loxP sites (black triangles). Under steady-state conditions, the intron gets removed by splicing and leaves the mRNA of the target gene intact. (B) Upon recombination with Cre, the branch point is excised and the intron is inactivated. Consequently, the intron is no longer removed and the ribosome reads through the intronic sequence, running into one of three stop codons that were added to the artificial intron cassette. (C) DNA sequence of the artificial intron that was used for most of the experiments presented in this manuscript. Important features are highlighted: splice donor in green, loxP sites in grey, stop codons in three reading frames in red, branch point in yellow, polypyrimidine tract is underlined and splice acceptor in blue.

Figure 2

Identification of the artificial intron configuration that enables conditional gene inactivation. (A) HAP1 cells were transfected with NanoLuc without intron (“No intron”) or one harbouring the intron without further modification (“Intron”). In addition, a set of cassette variants (Var 1–5) were tested in which loxP sites positioning relative to the branch point varied. Note that we included two designs for each cassette, one in which the cassette was intact (“no Cre”) and one in which the sequence between the loxP sites had been removed (“Cre”). NanoLuc levels were measured 24 h post transfection using the Nano-Glo Dual assay. (B) Variant 4 identified in Fig. 2A was placed into various sequence contexts within the NanoLuc gene, reflecting one of four possible insertion sites: CAG-G, AAG-G, CAG-A and AAG-A (where the hyphen denotes the site where the intron got inserted). For each of these, two sites were evaluated. HEK293T cells were transiently transfected with these various constructs and NanoLuc luciferase activity was measured using the NanoGlo Dual assay 48 h hours after transfection.

Figure 3

Cre-recombination leads to branch point excision. (A) Genomic DNA was isolated from cells transfected with Cre (for CD46) or cells transduced with Cre-ERT2 and treated with 4-hydroxitamoxifen (4-OHT; for METTL16). Samples were genotyped using PCR primers specific for CD46 or METTL16. (B) mRNA was isolated from cells expressing Cre, reverse transcribed using oligo (dT) and analysed with primers specific for the CD46 cDNA.

Figure 4

Artificial intron cassette activation leads to conditional gene inactivation. (A and B) HAP1 cells bearing the artificial intron in CD46 were transfected with Cre recombinase. For Western blotting, cells were analysed using a CD46-specific antibody. For flow cytometry, cells were stained with a CD46-specific antibody. (C) METTL16-DECAI cells were transduced with Cre-ERT2, treated with 4-OHT as indicated and stained with Crystal Violet.

Figure 5

Generation of Oct4-conditional knockout human iPS cells. (A) Wild-type cells (+/+) or cells bearing the artificial intron in exon 1 of Oct4 (Oct4-DECAI #1 and Oct4-DECAI #2, created with two independent gRNAs) were transfected with Cre-ERT2 recombinase and 4-OHT or left untreated. Cells were fixed in 4% PFA and stained for Oct4 and SSEA4 using specific antibodies. DAPI was used for nuclear staining. Images were acquired with an Olympus IX83 microscope (10X magnification, 100 µm scale bars). For bright field images see Supplementary Figure 5. (B) Flow cytometry analysis of the cells before (No Cre) and after Cre treatment (+Cre) stained with Oct4 and SSEA4 specific antibodies. (C) qRT-PCR analysis was performed before and after induction of Cre recombination. Expression levels of Oct4, two additional pluripotency markers (Nanog and Sox2), and two differentiation markers (Cdx2 and Eomes) are shown. qRT-PCR values are normalized to GAPDH and shown as relative to the highest value of that target across all samples.