Preparation of CRISPR/Cas9 and gRNA plasmids
Human genome engineering has been transformed by the introduction of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system found in most bacteria and archaea. Type II CRISPR/Cas systems have been engineered to induce RNA-guided genome editing in human cells, where small RNAs function together with Cas9 nucleases for sequence-specific cleavage of target sequences. Here we describe the protocol for Cas9-mediated human genome engineering, including construct building and transfection methods necessary for delivering Cas9 and guide RNA (gRNA) into human-induced pluripotent stem cells (hiPSCs) and HEK293 cells. Following genome editing, we also describe methods to assess genome editing effi- ciency using next-generation sequencing and isolate monoclonal hiPSCs with the desired modifications for downstream applications. Keywords: genome engineering • CRISPR • human stem cells
Steps to reproduce
1. Obtain plasmid from Addgene. 2. Use a sterile pipet tip or toothpick to scrape the bacterial stock from the Addgene bacterial stab, and streak it onto an LB agar plate containing 100 μg/ml ampicillin. Incubate plate at 37°C for 10 hr or overnight. 3. Once colonies are formed, pick a single colony from the plate to inoculate 200 ml of LB liquid medium containing 100 μg/ml ampicillin. Grow overnight at 37°C with shaking at 200 rpm. 4. Isolate plasmid DNA using a plasmid Maxiprep kit. Use Nanodrop microspectrophotometer to measure DNA concentration. Resuspend DNA at 1 μg/μl in water. Use this product for transfection. 5. Using sequence analysis software, identify all 22-bp regions within 50 bp of the intended genomic target in the form of 5′-N19-NGG-3′. 6. For each candidate sequence, query for alternate binding sites in the reference genome. Because of the higher tolerance of mismatches in the first 7 bp of the target sequence, search the reference genome for the last 13 bp of the target sequence with the NGG protospacer adjacent motif (S13NGG). Use NCBI BLASTN or other online software to choose the one with minimal off-target sites at region of interest. Finalize the design of the customized gRNA expression fragment (455 bp) by including the selected target sequence (N19) in the gRNA expression fragment below. 7. Synthesize the final gRNA expression fragment (455 bp) as a standard gBlock without any 5′ modifications from gene synthesis companies. 8. Resuspend the gBlock (delivered at 200 ng) in 20 μl of water for a final concentration of 10 ng/μl. 9. Pipet 1 μl gBlock, 1 μl pCRII-Blunt-TOPO vector, and 4 μl salt solution (from PCR-Blunt II-Topo kit) in a 1.5-ml microcentrifuge tube, mixing gently. Incubate at room temperature for at least 5 min. 10. To transform 5 μl of product into Top10 Chemically Competent E. coli cells, thaw one aliquot of Top10 cells in ice for 10 min, add 5 μl of the TOPO cloning reaction from the previous step, and incubate on ice for 30 min. Heat-shock the cells at 42°C, then return to ice for 2 min. Add 250 μl of room temperature SOC medium (from PCR-Blunt II-Topo kit) and incubate at 37°C with shaking for 1 hr. 11. Spread 100 μl of the transformation mixture using sterilized glass beads onto a prewarmed LB agar plate containing 50 μg/ml kanamycin by gently swirling the plate or, alternatively, using an inoculating loop for spreading. Incubate overnight at 37°C. 12. After incubation, pick 5 colonies for Sanger sequencing using the M13 Forward and M13 Reverse universal sequencing primers. 13. After identifying the colonies with the correct sequence, grow a maxiprep culture of the correct transformant by inoculating 200 ml of LB medium containing 50 μg/ml kanamycin with 100 μl of the original culture (step 11). Grow overnight at 37°C with shaking at 200 rpm. 14. Isolate plasmid DNA using a plasmid maxiprep kit. Resuspend plasmid DNA at 1 μg/ml in water. Use this product for transfection.