Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells

Authors:
Luhan Yang, Dennis Grishin, Gang Wang, John Aach, Cheng-Zhong Zhang, Raj Chari, Jason Homsy, Xuyu Cai, Yue Zhao, Jian-Bing Fan, Christine Seidman, Jonathan Seidman, William Pu, George Church
In:
Source: Nat Commun.
Publication Date: (2014)
Issue: 5: 5507
Research Area:
Basic Research
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Platform:
4D-Nucleofector™ X-Unit
Experiment
Genome editing of hiPSCs PGP1 iPSCs were acquired from Personal Genome Project15. Cells were maintained on Matrigel (BD Biosciences)-coated plates in mTeSR1 medium (Stemcell Technologies) at 37?°C and 5% CO2 in a humidified incubator. Cultures were passaged every 5–7 days with TrypLE Express (Invitrogen). Genome editing on iPSCs using PiggyBac-inserted Cas9 was described before16. Briefly, PiggyBac carrying inducible Cas9 was integrated into the genome aided by transposase to generate Cas9-PGP1 iPSCs. After establishing the cell line, we transfected the cells with gRNA constructs through nucleofection and added 1?µg?ml-1 doxycycline to the media to induce Cas9 expression. Single cells were expanded into clones and on-target effects were validated through Sanger sequencing following the previously described protocol Site-specific deep sequencing Cas9-PGP1 iPSCs were nucleofected using P3 Primary Cell 4D-Nucleofector X Kit (Lonza). A total of 5 × 105 cells were harvested using TrypLE Express (Invitrogen) and resuspended in 20?µl nucleofection mixture containing 16.4?µl of P3 Nucleofector solution, 3.6?µl supplement, 1?µg of gRNA construct (and 1?µg Cas9 construct for transient transfection of Cas9). The nucleofection reactions were then conducted using the CB150 program. Thereafter the cells were plated on Matrigel-coated plates in mTeSR1 medium supplemented with ROCK inhibitor (Calbiochem Y-27632) and 1?µg?ml-1 DOX. After 48?h the cells were harvested and the genomic DNA was extracted using ZyGEM prepGEM extraction kit. Primers used in the deep sequencing can be found in Supplementary Table 3. The libraries were purified with QIAquick PCR Purification Kit (Qiagen) and sequenced on the MiSeq platform. Cas9 activity was measured through indel detection and relative Cas9 cutting efficiencies were calculated by normalising the off-target cutting efficiency to on-target efficiency.
Abstract
CRISPR/Cas9 has demonstrated a high-efficiency in site-specific gene targeting. However, potential off-target effects of the Cas9 nuclease represent a major safety concern for any therapeutic application. Here, we knock out the Tafazzin gene by CRISPR/Cas9 in human-induced pluripotent stem cells with 54% efficiency. We combine whole-genome sequencing and deep-targeted sequencing to characterise the off-target effects of Cas9 editing. Whole-genome sequencing of Cas9-modified hiPSC clones detects neither gross genomic alterations nor elevated mutation rates. Deep sequencing of in silico predicted off-target sites in a population of Cas9-treated cells further confirms high specificity of Cas9. However, we identify a single high-efficiency off-target site that is generated by a common germline single-nucleotide variant (SNV) in our experiment. Based on in silico analysis, we estimate a likelihood of SNVs creating off-target sites in a human genome to be ~1.5-8.5%, depending on the genome and site-selection method, but also note that mutations might be generated at these sites only at low rates and may not have functional consequences. Our study demonstrates the feasibility of highly specific clonal ex vivo gene editing using CRISPR/Cas9 and highlights the value of whole-genome sequencing before personalised CRISPR design.