Figure 3: Development of LV-EF1α-SpCas9-eGFP and LV-GMF-sgRNAs:

a) Selection of GMF specific sgRNAs: The panel A shows the DNA sequence of mouse GMF exon 2 flanked by the introns. The GMF amino acid sequence is indicated by single letter codes. The GMF sgRNA1 sequence which is slightly different than the GMF sgRNA used in the AAV (due to the differences between SaCas9 and SpCas9 PAM sequences) is also located in the anticlockwise orientation on the lower strand is depicted by the blue arrow flanked by the two vertical bars. b) The panel B shows the GMF sgRNA deisgn of GMF sgRNA2 and GMF sgRNA3 both of which are located in the GMF coding sequence in the exon 3. c) Generation of LV-EF1α-SpCas9-eGFP-Neo and LV-GMF-sgRNAs: A set of 5 different VSV enveloped pseudotyped third generation lentiviral vectors were developed. In the lentiviral vector LV-SpCas9-eGFP-Neo, the EF1α promoter drives the expression of CRISPR-Cas9 and a SV40 promoter drives the expression of eGFP reporter and Neo resistance genes. The lentiviral vector LV-GMF-sgRNA 1, 2 and 3 express GMF sgRNAs under the control of the U6 promoter while the SV40 promoter drives the expression of mCherry reporter and Puromycin resistance marker. In the control lentiviral vector LV-SCRAM-sgRNA a scrambled sgRNA that does not target any known gene was used as a negative gene editing control virus. d) Generation of stable CRISPR-Cas9 expressing cell line: BV2 cells were transduced with the lentiviral vector LV-EF1α-SpCas9-eGFP-Neo to generate a stable cell line using neomycin selection. Co-transduction of BV2-CRISPR-Cas9 cells with LV-GMF-sgRNA 1 shows the presence of mCherry expressing cells and the merged image shows the presence of a small subset of cells showing both eGFP and mCherry expression. Puromycin selection was used to generate stable BV2-CRISPR-Cas9 and GMF-sgRNA expressing cells.