Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors

The utility of CRISPR-Cas9 and TALENs for genome editing may be compromised by their off-target activity. We show that integrase-defective lentiviral vectors (IDLVs) can detect such off-target cleavage with a frequency as low as 1%. In the case of Cas9, we find frequent off-target sites with a one-base bulge or up to 13 mismatches between the single guide RNA (sgRNA) and its genomic target, which refines sgRNA design.

[1]  Stephan Wolf,et al.  Genome-wide high-throughput integrome analyses by nrLAM-PCR and next-generation sequencing , 2010, Nature Protocols.

[2]  David R. Liu,et al.  Revealing Off-Target Cleavage Specificities of Zinc Finger Nucleases by In Vitro Selection , 2011, Nature Methods.

[3]  Jeffrey C. Miller,et al.  An unbiased genome-wide analysis of zinc-finger nuclease specificity , 2011, Nature Biotechnology.

[4]  Volker Brendel,et al.  TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction , 2012, Nucleic Acids Res..

[5]  Eli J. Fine,et al.  DNA targeting specificity of RNA-guided Cas9 nucleases , 2013, Nature Biotechnology.

[6]  David R. Liu,et al.  High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity , 2013, Nature Biotechnology.

[7]  Christopher M. Vockley,et al.  RNA-guided gene activation by CRISPR-Cas9-based transcription factors , 2013, Nature Methods.

[8]  David A. Scott,et al.  Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity , 2013, Cell.

[9]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[10]  Christof von Kalle,et al.  TALEN-based gene correction for epidermolysis bullosa. , 2013, Molecular therapy : the journal of the American Society of Gene Therapy.

[11]  Bo Zhang,et al.  Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish , 2013, Nucleic acids research.

[12]  G. Church,et al.  CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering , 2013, Nature Biotechnology.

[13]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[14]  J. Keith Joung,et al.  High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells , 2013, Nature Biotechnology.

[15]  Xiaoling Wang,et al.  Precise Gene Modification Mediated by TALEN and Single-Stranded Oligodeoxynucleotides in Human Cells , 2014, PloS one.

[16]  E. Lander,et al.  Genetic Screens in Human Cells Using the CRISPR-Cas9 System , 2013, Science.

[17]  Neville E. Sanjana,et al.  Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells , 2014, Science.

[18]  Gang Bao,et al.  CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences , 2014, Nucleic acids research.

[19]  Jin-Soo Kim,et al.  Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases , 2014, Genome research.

[20]  Jiajie Zhang,et al.  PEAR: a fast and accurate Illumina Paired-End reAd mergeR , 2013, Bioinform..