Illumina MiSeq paired end sequencing; Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish (Run 9)
Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish (Run 9)
Sample 5_p
For detailed protocols refer to Gagnon et al., 2014, Methods section with details in Supplementary Protocols. Here we listed shortened version as we can't upload images. There may be a more up-to-date version of this protocol on this website – http://labs.mcb.harvard.edu/schier/index.htmlTarget site selection for the initial screenBriefly, target sites were selected in exons across the genome that matched the sequence GG-N19-GG, GA-N19-GG, or AG-N19-GG. These sites were checked for uniqueness in RefSeq protein coding regions using Bowtie and the initially defined specificity rules, which suggested an intolerance for mismatches in the 3′ twelve bases of the target site.Cas9 cloning and protein expressionFor in vivo expression of Cas9, the Cas9 open reading frame was amplified from hCas9 and cloned into the pCS2 vector to generate pCS2-Cas9. For in vitro expression of Cas9, the hCas9 open reading frame cloned into pET-28b to generate pET-28b-Cas9-His. Cas9 protein was expressed in E. coli Rosetta cells (Novagen) using the auto-induction method, growing for 12 hours at 37°C, followed by 24 hour expression at 18°C. Purification was performed using his-tag resin (G-Biosciences). Buffers were 20 mM Tris pH 8, 30 mM Imidazole, 500 mM NaCl for washes and 20 mM Tris pH 8, 500 mM Imidazole, 500 mM NaCl for elutions. Fractions were dialyzed into 20 mM Tris, 200 mM KCl, 10 mM MgCl2 and single-use aliquots were frozen in liquid nitrogen and stored at −80°C. Plasmids are available from Addgene at http://www.addgene.org/Alex_Schier/.sgRNA template generation and transcriptionTo generate templates for sgRNA transcription, gene-specific oligonucleotides containing the T7 (5′-TAATACGACTCACTATA-3′) or SP6 (5′- ATTTAGGTGACACTATA-3′) promoter sequence, the 20 base target site without the PAM, and a complementary region were annealed to a constant oligonucleotide encoding the reverse-complement of the tracrRNA tail (Supplementary Figure S1 and Table S1). The ssDNA overhangs were filled in with T4 DNA polymerase (NEB), and the resulting sgRNA template were purified using Qiaquick columns (Qiagen). sgRNAs were transcribed using Megascript kits (Ambion). All sgRNAs were then DNase treated and precipitated with ammonium actetate/ethanol. Cas9 mRNA was transcribed from linearized template DNA using mMachine SP6 kit (Ambion), DNase treated, and precipitated with lithium chloride. RNA concentration was quantified using Nanodrop spectrophotometer and diluted and aliquoted as a 500 ng/ul 20× stock (sgRNAs) or 600 ng/ul 2× stock (Cas9 mRNA). Fish husbandry and microinjectionFor the initial screen, zebrafish TLAB strain zygotes were collected and injected through the chorion with a mix of 25 pg sgRNA, 300 pg Cas9 mRNA, and phenol red dye in a single mix. Embryos were grown to 24–30hpf and genomic DNA extracted from pools of 8–10 embryos using the HotSHOT method. For comparison between Cas9 mRNA and protein, higher levels of sgRNA were co-injected (200–300 pg). Cas9/sgRNA complex was formed by incubating protein with sgRNA at room temperature for 5 minutes before injection. Determination of somatic mutagenesis ratesTo determine indel percentage using sequencing, a fusion PCR method was used to attach Illumina P5 sequencing adapters and barcodes to amplicons designed to surround the target site. Amplicons were quantified by visualization on agarose gel before being pooled at roughly equal molar ratios. Pools of amplicons were gel extracted and sequenced with MiSeq Personal Sequencer (Illumina), 150 bp paired-end sequencing.