CRISPR‐Cas9‐Edited Site Sequencing (CRES‐Seq): An Efficient and High‐Throughput Method for the Selection of CRISPR‐Cas9‐Edited Clones

Yaligara Veeranagouda1, Delphine Debono‐Lagneaux1, Hamida Fournet1, Gilbert Thill1, Michel Didier1

1 Molecular Biology and Genomics, Translational Sciences, Sanofi R&D, Chilly‐Mazarin
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 31.14
DOI:  10.1002/cpmb.53
Online Posting Date:  January, 2018
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The emergence of clustered regularly interspaced short palindromic repeats–Cas9 (CRISPR‐Cas9) gene editing systems has enabled the creation of specific mutants at low cost, in a short time and with high efficiency, in eukaryotic cells. Since a CRISPR‐Cas9 system typically creates an array of mutations in targeted sites, a successful gene editing project requires careful selection of edited clones. This process can be very challenging, especially when working with multiallelic genes and/or polyploid cells (such as cancer and plants cells). Here we described a next‐generation sequencing method called CRISPR‐Cas9 Edited Site Sequencing (CRES‐Seq) for the efficient and high‐throughput screening of CRISPR‐Cas9‐edited clones. CRES‐Seq facilitates the precise genotyping up to 96 CRISPR‐Cas9‐edited sites (CRES) in a single MiniSeq (Illumina) run with an approximate sequencing cost of $6/clone. CRES‐Seq is particularly useful when multiple genes are simultaneously targeted by CRISPR‐Cas9, and also for screening of clones generated from multiallelic genes/polyploid cells. © 2018 by John Wiley & Sons, Inc.

Keywords: CRISPR; Cas9; CRISPR‐Cas9; CRISPR clone; gene editing; NGS; multiallelic genes; NGS; polyploidy

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • Cells from CRISPR‐Cas9‐edited clones, or genomic DNA from CRISPR clones
  • Lysis buffer (see recipe)
  • NEBNext High‐Fidelity 2× PCR Master Mix (New England Biolabs, cat no. M0541S)
  • 10 µM FA‐TSFp primer (Table 31.14.1)
  • 10 µM RA‐TSRp (Table 31.14.1)
  • 10 µM i5‐D501‐508 primer (Table 31.14.2)
  • 10 µM i7‐D701‐12 primer (Table 31.14.2)
  • DNA Clean & Concentrator 25 kit (Zymo Research, cat. no. D4033) or comparable PCR cleaning and concentration kits from other suppliers (e.g., PureLink Pro 96 PCR Purification Kit; for simultaneous purification of 96 samples; Invitrogen, cat. no. K310096A)
  • Agilent high‐sensitivity DNA kit (Agilent Technologies, cat no. 5067‐4626)
  • Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, cat. no. Q32851)
  • PhiX Control V3 library (Illumina)
  • PhiX Control Kit v3 (Illumina, cat. no. FC‐110‐3001)
  • Thermal cycler
  • 0.2‐ml PCR tubes
  • 1.5‐ml and 0.2‐ml nuclease‐free microcentrifuge tubes
  • NanoDrop 2000 or UV‐vis spectrophotometer
  • Benchtop centrifuge and microcentrifuge (≥13,000 × g )
  • Vortexer/multi‐vial vortex shaker
  • Agilent 2100 Bioanalyzer system (Agilent Technologies)
  • Agilent 2100 chip priming station and IKA vortex mixer (Agilent Technologies)
  • Qubit 2.0 (or above) fluorometer (Thermo Fisher Scientific)
  • Illumina sequencer (any MiniSeq, Miseq, or NextSeq500 system) and appropriate sequencing kits with 150 or 300 cycles
  • Additional reagents and equipment for preparation of genomic DNA (Strauss, )
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Literature Cited

Literature Cited
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