Biased and Unbiased Methods for the Detection of Off-Target Cleavage by CRISPR/Cas9: An Overview.
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 endonuclease (Cas9) derived from bacterial adaptive immune systems is a revolutionary tool used in both basic and applied science. It is a versatile system that enables the genome of different species to be modified by generating double strand breaks (DSBs) at specific locations. However, all of the CRISPR/Cas9 systems can also produce DSBs at off-target sites that differ substantially from on-target sites. The generation of DSBs in locations outside the intended site can produce mutations that need to be carefully monitored, especially when using these tools for therapeutic purposes. However, off-target analyses of the CRISPR/Cas9 system have been very challenging, particularly when performed directly in cells. In this manuscript, we review the different strategies developed to identify off-targets generated by CRISPR/cas9 systems and other specific nucleases (ZFNs, TALENs) in real target cells.
Genomic Medicine Department, GENYO-Centre for Genomics and Oncological Research Pfizer-Universidad de Granada-Junta de Andalucía, Avda de la Ilustración 114, 18007 Granada, Spain. francisco.martin@genyo.es.|LentiStem Biotech, GENYO, Avda de la Ilustración 114, 18007 Granada, Spain. francisco.martin@genyo.es.
Francisco
F
Martin
Genomic Medicine Department, GENYO-Centre for Genomics and Oncological Research Pfizer-Universidad de Granada-Junta de Andalucía, Avda de la Ilustración 114, 18007 Granada, Spain. sabina.sanchez@genyo.es.
Sabina
S
Sánchez-Hernández
Genomic Medicine Department, GENYO-Centre for Genomics and Oncological Research Pfizer-Universidad de Granada-Junta de Andalucía, Avda de la Ilustración 114, 18007 Granada, Spain. alejandra.gutierrez@genyo.es.
Alejandra
A
Gutiérrez-Guerrero
Genomic Medicine Department, GENYO-Centre for Genomics and Oncological Research Pfizer-Universidad de Granada-Junta de Andalucía, Avda de la Ilustración 114, 18007 Granada, Spain. javier.pinedo@genyo.es.
Javier
J
Pinedo-Gomez
Genomic Medicine Department, GENYO-Centre for Genomics and Oncological Research Pfizer-Universidad de Granada-Junta de Andalucía, Avda de la Ilustración 114, 18007 Granada, Spain. karim.benabdel@genyo.es.|LentiStem Biotech, GENYO, Avda de la Ilustración 114, 18007 Granada, Spain. karim.benabdel@genyo.es.
Karim
K
Benabdellah
D000818:Animals; D015982:Bias; D064113:CRISPR-Cas Systems; D000072669:Gene Editing / Q000379:methods* / Q000592:standards; D018390:Gene Targeting / Q000379:methods* / Q000592:standards; D006801:Humans
CHIPSeq; CRISPR/cas9; GUIDE-seq; IDLVs; LAM-HTGTS; biased; off-target sites; real target cells; specific nucleases; unbiased