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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1:

  Materials
  • 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, )
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., … Horvath, P. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science (New York, N.Y.), 315, 1709–1712. doi: 10.1126/science.1138140.
  Bell, C. C., Magor, G. W., Gillinder, K. R., & Perkins, A. C. (2014). A high‐throughput screening strategy for detecting CRISPR‐Cas9 induced mutations using next‐generation sequencing. BMC Genomics, 15, 1002. doi: 10.1186/1471‐2164‐15‐1002.
  Brinkman, E. K., Chen, T., Amendola, M., & Van Steensel, B. (2014). Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Research, 42, 1–8. doi: 10.1093/nar/gku936.
  Chang, C.‐T., Tsai, C.‐N., Tang, C. Y., Chen, C.‐H., Lian, J.‐H., Hu, C.‐Y., … Lee, Y. S. (2012). Mixed sequence reader: A program for analyzing DNA sequences with heterozygous base calling. The Scientific World Journal, 2012, 1–10. doi: 10.1100/2012/365104.
  Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., … Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science, 15, 819–823. doi: 10.1126/science.1231143.
  Dehairs, J., Talebi, A., Cherifi, Y., & Swinnen, J. V. (2016). CRISP‐ID: Decoding CRISPR mediated indels by Sanger sequencing. Scientific Reports, 6, 28973. doi: 10.1038/srep28973.
  Dmitriev, D. A., & Rakitov, R. A. (2008). Decoding of superimposed traces produced by direct sequencing of heterozygous indels. PLoS Computational Biology, 4, e1000113. doi: 10.1371/journal.pcbi.1000113.
  Doudna, J. A., & Charpentier, E. (2014). The new frontier of genome engineering with CRISPR‐Cas9. Science, 346, 1258096–1258096. doi: 10.1126/science.1258096.
  Goodwin, S., McPherson, J. D., & McCombie, W. R. (2016). Coming of age: Ten years of next‐generation sequencing technologies. Nature Reviews Genetics, 17, 333–351. doi: 10.1038/nrg.2016.49
  Güell, M., Yang, L., & Church, G. M. (2014). Genome editing assessment using CRISPR Genome Analyzer (CRISPR‐GA). Bioinformatics (Oxford, England), 30, 2968–2970. doi: 10.1093/bioinformatics/btu427.
  Hsu, P. D., Lander, E. S., & Zhang, F. (2014). Development and applications of CRISPR‐Cas9 for genome engineering. Cell, 157, 1262–1278. doi: 10.1016/j.cell.2014.05.010.
  Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual‐RNA‐guided DNA endonuclease in adaptive bacterial immunity. Science, 337, 816–822. doi: 10.1126/science.1225829.
  Liu, W., Xie, X., Ma, X., Li, J., Chen, J., & Liu, Y. G. (2015). DSDecode: A web‐based tool for decoding of sequencing chromatograms for genotyping of targeted mutations. Molecular Plant, 8, 1431–1433. doi: 10.1016/j.molp.2015.05.009.
  Ma, X., Chen, L., Zhu, Q., Chen, Y., & Liu, Y. G. (2015). Rapid decoding of sequence‐specific nuclease‐induced heterozygous and biallelic mutations by direct sequencing of PCR products. Molecular Plant, 8, 1285–1287. doi: 10.1016/j.molp.2015.02.012.
  Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., … Church, G. M. (2013). RNA‐guide human genome engineering via Cas9. Science, 339, 823–826. doi: 10.1126/science.1232033.
  Pinello, L., Canver, M. C., Hoban, M. D., Orkin, S. H., Kohn, D. B., Bauer, D. E., & Yuan, G.‐C. (2016). Analyzing CRISPR genome‐editing experiments with CRISPResso. Nature Biotechnology, 34, 695–697. doi: 10.1038/nbt.3583.
  Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013). Genome engineering using the CRISPR‐Cas9 system. Nature Protocols, 8, 2281–2308. doi: 10.1038/nprot.2013.143.
  Sander, J. D., & Joung, J. K. (2014). CRISPR‐Cas systems for editing, regulating and targeting genomes. Nature Biotechnology, 32, 347–355. doi: 10.1038/nbt.2842.
  Strauss, W. M. (2001). Preparation of genomic DNA from mammalian tissue. Current Protocols in Molecular Biology, 42, 2.2.1–2.2.3. doi: 10.1002/0471142727.mb0202s42.
  Wang, H., La Russa, M., & Qi, L. S. (2016). CRISPR/Cas9 in Genome Editing and Beyond. Annual Review of Biochemistry, 85, 227–264. doi: 10.1146/annurev‐biochem‐060815‐014607.
  Wiedenheft, B., Sternberg, S. H., & Doudna, J. A. (2012). RNA‐guided genetic silencing systems in bacteria and archaea. Nature, 482, 331–338. doi: 10.1038/nature10886.
  Yang, Z., Steentoft, C., Hauge, C., Hansen, L., Thomsen, A. L., Niola, F., … Bennett, E. P. (2015). Fast and sensitive detection of indels induced by precise gene targeting. Nucleic Acids Research, 43, e59. doi: 10.1093/nar/gkv126.
  Zhidkov, I., Cohen, R., Geifman, N., Mishmar, D., & Rubin, E. (2011). CHILD: A new tool for detecting low‐abundance insertions and deletions in standard sequence traces. Nucleic Acids Research, 39, e47. doi: 10.1093/nar/gkq1354.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library