CRISPR/Cas9-mediated targeted knock-in of large constructs using nocodazole and RNase HII

Abstract

On-target integration of large cassettes via homology-directed repair (HDR) has several applications. However, the HDR-mediated targeted knock-in suffered from low efficiency. In this study, we made several large plasmids (12.1-13.4 kb) which included the CRISPR/Cas9 system along with a puromycin transgene as part of the large DNA donor (5.3-7.1 kb insertion cassettes) and used them to evaluate their targeted integration efficiency into a transgenic murine embryonic fibroblast (MEF) cell line carrying a single copy of a Venus transgene. We established a detection assay by which HDR events could be discriminated from the error-prone non-homologous end-joining (NHEJ) events. Improving the plasmid quality could considerably leverage the cell toxicity impediment of large plasmids. The use of the TILD (targeted integration with linearized dsDNA) cassettes did not improve the HDR rate compared to the circular plasmids. However, the direct inclusion of nocodazole into the electroporation solution significantly improved the HDR rate. Also, simultaneous delivery of RNase HII and the donor plasmids into the electroporated cells considerably improved the HDR events. In conclusion, the results of this study showed that using cell synchronization reagents in the electroporation medium can efficiently induce HDR rate in the mammalian genome.

Figure 1

Schematic presentation of the gRNAs and large plasmids. (A) The location of three gRNAs on the CAGGS promoter and one gRNA on the 5´coding part of the Venus transgene. Forward and reverse primers for amplification of Venus are depicted in blue colour. (B) The genomic location of Venus transgene and its homology arms with SGD constructs as well as the insertion cassettes. Compared to the pSGD52, pSGD73 and pSGD74 constructs, the gRNA was translocated after the Cas9 and puromycin genes in pSGD69, pSGD75, and pSGD77. The location of a primer complementary to the puromycin sequence is depicted with a red box. This primer was used combined with the Venus Rev primer for detection of the HDR-mediated SGD insertion into the Venus transgene. pSGD 52 and 62 contained gRNA-252, pSGD73, 74, and 77 contained gRNA-72, pSGD 69 contained gRNA-69, and pSGD75 contained both gRNA-72 and + 121. The figure was created in Microsoft PowerPoint 2016.

Figure 2

HDR-mediated Venus knock-out using the SGD52 construct. The MEF cells carrying a single copy of Venus were electrotransfected with either pX459 plasmid expressing gRNA-252 and Cas9, or the SGD52 vector expressing gRNA-252 and Cas9 and puromycin plus an HDR cassette. The pSGD52-electrotransfected cells were selected against puromycin for 105 days. In parallel, cells from the pX459-252 and the non-transfected cells (positive control), expressing a single copy of Venus, were also cultured for 105 days. (A) Results of FACS analysis. The Venus expression pattern of PK and pX459/ pSGD52-electroporated cells are shown in red color. The MEF cells with no Venus transgene was considered as the negative control and was depicted in blue color. (B) Fluorescence microscopy and (C) PCR. Two sets of primers were used to amplify the Venus transgene and the Puromycin-Venus. Specific primers for Venus could detect only the wild type copy of Venus transgene under the CAGGS promoter in non-transfected cells, but not the truncated Venus using the SGD cassette. The presence of the HDR-mediated SGD cassette was verified using specific primers covering the part of the puromycin transgene and the proximal part of the truncated Venus. The SGD cells are electrotransfected cells with pSGD plasmids.

Figure 3

HDR-mediated Venus knock-out using various SGD constructs. The MEF cells carrying a single copy of Venus transgene were electrotransfected with pSGD plasmids. The pSGD75 plasmid which carried gRNA + 121 and induced NHEJ-mediated Venus knock-out was used as the transfection control. The electrotransfected cells were selected with puromycin for 105 days. (A) Results of FACS analysis. The Venus expression pattern of either PK or pSGD-electrotransfected cells are shown in red color. The MEF cells with no Venus transgene were considered as the negative control and was depicted in blue color. (B) and (C) End-point PCR using two sets of primer pairs on supernatants and genomic DNA of SGD groups, respectively. After 105 days, pSGD-electroporated cells were DNaseI-treated. A fraction of supernatants was used for PCR to assess plasmid carry-over from the initial step of electroporation. Genomic DNA was extracted from cell pellets and used for PCR. Two sets of primers were used to amplify the puromycin-Venus which was detectable only in the HDR events although with different lengths of amplicons. Moreover, Cas9 primers were used for detection of the Cas9 transgene. This primer set produced a same band of PCR product in all SGD groups. (C) Digital PCR assays specific for detection of Venus and Cas9 sequences. Two sets of primer–probe assays were used to detect the copy number of Venus and Cas9 genes and the rate of Cas9 copies to Venus copies was calculated.

Figure 4

Evaluation of DNA quality on the cell survival rate following electroporation. (A) Twenty micrograms of SGD plasmids were used for electrotransfection either in 10 µl (2 µg/µl) or 20 µl (1 µg/µl) of the electroporation medium (250 µl). (B) The effect of ethanol washing of anion exchange column-prepared DNA was assessed on the cell survival rate using different large plasmids.

Figure 5

Comparison of TILD versus circular DNA donors for HDR induction. The pSGD plasmids carrying the CRISPR system and the DNA donor which had BamH1 restriction sequence at both 5′ and 3′ ends. The constructs were digested with BamHI enzyme to release the TILD fragments and co-transfected with the corresponding gRNA-encoding plasmid into MEF cells carrying the Venus transgene. The Venus knockout rate was compared between TILD and circular plasmids. The same process was used for the pSGD75 which induced both HDR and NHEJ knockout. This plasmid was considered as the positive control for transfection rate.

Figure 6

Effect of RNase HII on the HDR rate. MEF cells were electrotransfected with pSGD plasmids carrying both the CRISPR system and DNA donor. We included different concentration of RNase HII into the electroporation media. The pSGD75 plasmid caused both NHEJ- and HDR-induced Venus knock-out.

Figure 7

Effect of nocodazole on the HDR rate. MEF cells were electrotransfected with pSGD plasmids carrying both the CRISPR system and DNA donor in the presence of nocodazole into the electroporation media (70 ng/µl final concentration). The pSGD75 plasmid associated with both NHEJ- and HDR-induced Venus knock-out. (A) the HDR-mediated Venus knock-out rate using SGD cassettes. (B) Fluorescence microscopy of MEF cells electroporated with the pSGD plasmids.