Repurposing CRISPR/Cas9 for in situ functional assays - PubMed (original) (raw)

Repurposing CRISPR/Cas9 for in situ functional assays

Abba Malina et al. Genes Dev. 2013.

Abstract

RNAi combined with next-generation sequencing has proven to be a powerful and cost-effective genetic screening platform in mammalian cells. Still, this technology has its limitations and is incompatible with in situ mutagenesis screens on a genome-wide scale. Using p53 as a proof-of-principle target, we readapted the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated 9) genome-editing system to demonstrate the feasibility of this methodology for targeted gene disruption positive selection assays. By using novel "all-in-one" lentiviral and retroviral delivery vectors heterologously expressing both a codon-optimized Cas9 and its synthetic guide RNA (sgRNA), we show robust selection for the CRISPR-modified Trp53 locus following drug treatment. Furthermore, by linking Cas9 expression to GFP fluorescence, we use an "all-in-one" system to track disrupted Trp53 in chemoresistant lymphomas in the Eμ-myc mouse model. Deep sequencing analysis of the tumor-derived endogenous Cas9-modified Trp53 locus revealed a wide spectrum of mutants that were enriched with seemingly limited off-target effects. Taken together, these results establish Cas9 genome editing as a powerful and practical approach for positive in situ genetic screens.

Keywords: CRISPR; Cas9; functional screening; genome editing; p53.

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Figures

Figure 1.

Figure 1.

Genome editing of a TLR locus in 293T cells using an engineered all-in-one type II CRISPR system. (A) Schematic diagram of LeGO-based lentivirus (pLC) constructs driving expression of Cas9 and sgRNAs. (B) Predicted secondary structure (

http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi

) of sgRNA showing alignment of trigger sequence with target and PAM. The first nucleotide of the trigger sequence is forcibly a G, since the sgRNA is expressed from the murine U6 promoter. (C) Schematic of TLR with the position and nucleotide sequence of the TLR trigger, PAM, and stop codon shown. (D) A genomically integrated TLR is efficiently targeted by pLC-TLR. Quantitation of 293T TLR cells transfected with the indicated Cas9/sgRNA expression constructs and, where indicated, in combination with Δ20 eGFP. (E) Immunoblot showing expression and subcellular localization of Cas9 from the experiment presented in D. (C) Cytoplasmic fraction; (M) membrane fraction; (N) nuclear fraction. Blots were probed with the antibodies indicated below each panel. (F) Lentiviral-mediated NHEJ and HDR in 293T TLR cells. Cells were infected with lentivirus expressing Cas9 and the corresponding sgRNA and analyzed by flow cytometry 6 d later. The Δ20 eGFP donor plasmid was introduced by transfection 1 d prior to transduction with the Cas9/sgRNA lentiviral construct.

Figure 2.

Figure 2.

Cas9-mediated editing of _Trp_53 in _Arf_−/− MEFs leads to Nutlin-3a resistance. (A) Schematic diagram of the pQ-based retroviral constructs driving expression of Cas9, GFP, and sgRNAs (pQCiG). (B) Flow cytometric analysis of _Arf_−/− and _p53_−/− MEFs transduced with QCiG-Rosa, QCiG-p53, or MLP-p53.1224 retroviruses, cultured 3 d later in the presence of vehicle or 10 μM Nutlin-3a for 24 h, and then allowed to recover for 4 d. (C) Colony formation assay of infected _Arf_−/− and _p53_−/− MEFs with QCiG-Rosa, QCiG-p53, or MLP-p53.1224. Five-thousand cells were seeded, exposed to 10 μM Nutlin-3a for 24 h, and allowed to recover for 12 d in the absence of drug, at which point they were stained with crystal violet. (D) SURVEYOR assay of DNA isolated from QCiG-p53- and QCiG-Rosa-infected _Arf_−/− MEFs exposed to 10 μM Nutlin-3a for 24 h and allowed to recover for 4 d. The arrowhead denotes the expected SURVEYOR cleavage products. (E) Immunoblot documenting Cas9 and p53 expression in QCiG- and MLP-infected MEFs. The asterisk denotes the position of a prominent p53 truncated product.

Figure 3.

Figure 3.

Cas9-mediated editing of _Trp_53 in _Arf_−/−Eμ-myc lymphomas is positively selected for following DXR treatment in vivo. (A) Schematic diagram of in vivo fitness assay. (B) Kaplan-Meier analysis of tumor-free survival of mice injected with Rosa26 or Trp53 Cas9 targeted _Arf_−/−Eμ-myc and _p53_−/−Eμ-myc lymphomas following treatment with DXR. (C) Detection of GFP in tumors arising from QCiG-p53-infected _Arf_−/−Eμ-myc lymphomas following exposure to DXR and analyzed 3 d later. White arrows denote GFP fluorescence in lymph nodes originating from the presence of QCiG-p53 in the resulting tumors. (D) FACS analysis of the indicated Cas9 targeted Eμ-myc lymphomas analyzed before injection into mice (input), from tumors arising in vivo (pre-DXR), and from tumors for which the host had received DXR treatment (post-DXR). (E) SURVEYOR assay of DNA from QCiG-p53- and QCiG-Rosa-infected _Arf_−/−Eμ-myc lymphomas isolated from mice prior to DXR treatment. (F) Immunoblot showing long-term Cas9, p53, and GFP expression in QCiG-Rosa and QCiG-p53 Arf_−/−_Eμ-Myc lymphomas in vivo. Samples are from three separate tumors isolated prior to (pre-DXR) or following (post-DXR) DXR treatment. In the case of post-DXR samples for QCiG-Rosa _Arf_−/−Eμ-myc lymphomas, tumors were harvested after relapse (∼10 d after post-DXR treatment). The asterisk highlights a truncated p53 protein arising in the Cas9-edited samples.

Figure 4.

Figure 4.

Analysis of indels at the Trp53 locus and at predicted off-target sites in _Arf_−/−MEFs and _Arf_−/−Eμ-myc tumors edited with Rosa26 and _Trp5_3 sgRNAs. (A) Total count and location of insertions and deletions in exon 7 of Trp53 in _Arf_−/−Eμ-myc cells prior to injection, post-implantation, and post-DXR treatment, respectively. The vertical dashed line represents the predicted Cas9 cleavage site. (B) Frequency of mutant reads obtained following sequencing of Trp53 exon 7 from the indicated cells and tumors. T-1, T-2, and T-3 represent three independent tumors. (C, top panel) Sequence alignment of the trigger site in the Trp53 and Trp53 pseudogene. Differences are highlighted in green. (Bottom panel) Pie charts illustrating the proportion of mutated sequence reads at Trp53 (left) and the Trp53 pseudogene (right) relative to wild-type sequences (wt; blue). DNA was isolated from samples of _Arf_−/−Eμ-myc lymphoma cells infected with QCiG-Rosa-infected (top), QCiG-p53-infected (middle), or QCiG-p53-infected cells that were exposed to 10 μM Nutlin-3a for 3 d followed by a 10-d recovery period (bottom). (D) Prediction of genomic sequences showing sequences complementary to the first 13 perfectly matched nucleotides 5′ to the PAM of the Trp53 trigger sequence with all possible combinations of PAM. The trigger sequence is shown in blue, PAM is in red, and flanking nucleotides are in black. The genomic location is shown at the right. (E) Percent mutant reads at the indicated genomic locus in _Rosa26_- and _Trp53_-modified _Arf_−/− MEFs. The total read count for each amplified region ranged from ∼11,000 to 15,000 (sample #8), ∼18,000 to 23,000 (sample #7), and ∼20,000 to 53,000 (all others). Read counts for locus #2 are absent, since the barcode that had been used in the preparation of that sample could not be deciphered from the output of reads.

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