Qiaxcel capillary electrophoresis instrument

Short 200–350 bp PCR products were amplified using Phusion Hot-start FLEX DNA polymerase. PCR products were purified using Ampure XP beads (Beckman Coulter Genomics) according to manufacturer’s instructions. Dual-indexed TruSeq Illumina deep sequencing libraries were prepared using a high-throughput library preparation system (Kapa Biosystems) on a Sciclone G3 liquid-handling workstation. Final adapter-ligated libraries were quantified using a Qiaxcel capillary electrophoresis instrument (Qiagen). 150 bp paired end sequencing was performed on an Illumina MiSeq Sequencer by the Dana-Farber Cancer Institute Molecular Biology Core.
MiSeq paired-end reads were mapped to human genome reference GChr37 using bwa^{31 (link)}. Reads with an average quality score >30 were analyzed for insertion or deletion mutations that overlapped the intended target or candidate off-target nuclease binding site. Mutation analyses were conducted using the Genome Analysis Toolkit (GATK) and Python.

Roughly 72 hr post-transfection, genomic DNA was extracted from U2OS cells using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter Genomics), and T7 endonuclease I (T7E1) assays were performed as previously described^{26 (link)}. Briefly, 600–800 nt amplicons surrounding on-target sites were amplified from ~100 ng of genomic DNA using Phusion Hot-Start Flex DNA Polymerase (New England Biolabs, NEB) using the primers listed in Supplementary Table 3. PCR products were visualized (using a QIAxcel capillary electrophoresis instrument, Qiagen), and purified (Agencourt Ampure XP cleanup, Beckman Coulter Genomics), Denaturation and annealing of ~200 ng of the PCR product was followed by digestion with T7EI (NEB). Digestion products were purified (Ampure) and quantified (QIAxcel) to approximate the mutagenesis frequencies induced by Cas9-sgRNA complexes.

T7E1 assays were performed as previously described^{33 (link)} to quantify Cas9-induced mutagenesis at endogenous loci in human cells. Approximately 72 hours post-transfection, genomic DNA was isolated using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter Genomics). Target loci were PCR-amplified from ~100 ng of genomic DNA using the primers listed in Supplementary Table 1. Following an Agencourt Ampure XP clean-up step (Beckman Coulter Genomics), ~200 ng purified PCR product was denatured and hybridized prior to digestion with T7E1 (New England Biolabs). Following a second clean-up step, mutagenesis frequencies were quantified using a Qiaxcel capillary electrophoresis instrument (Qiagen).

T7E1 assays were performed as previously described¹⁷ . Briefly, genomic DNA was isolated 72 hours post transfection using the Agencourt DNAdvance Genomic DNA Isolation kit (Beckman Coulter Genomics) according to the manufacturer’s instructions with a Sciclone G3 liquid-handling workstation (Caliper). PCR reactions to amplify genomic loci were performed using Phusion Hot-start Flex DNA polymerase (New England Biolabs). Samples were amplified using a two-step protocol (98 °C, 30 sec; (98 °C, 7 sec; 72 °C, 30 sec) × 35; 72 °C, 5 min) or a touchdown PCR protocol ((98 °C, 10 s; 72–62 °C, −1 °C/cycle, 15 s; 72 °C, 30 s) × 10 cycles, (98 °C, 10 s; 62 °C, 15 s; 72 °C, 30 s) × 25 cycles). 200 ng of purified PCR amplicons were denatured, hybridized and treated with T7 Endonuclease I (New England Biolabs). Mutation frequency was quantified using a Qiaxcel capillary electrophoresis instrument (Qiagen) as previously described¹⁷ .