A Comparison of Techniques to Evaluate the Effectiveness of Genome Editing

The number of methods to assess the efficiency of genome editing is increasing very quickly. Recent techniques tend to exploit recent advances in laboratory practice (e.g., next-generation sequencing, computational analysis). Methods for genome-wide screening developed can simultaneously determine the specificity of genome editing tools and identify the presence of off-targets. While these methods are costly and time-consuming, they are indispensable when genome editing is planned to be used in vivo. The trend in in vitro research applications is to develop techniques that simplify as much as possible the first step of testing the efficiency of a newly designed genome editing tool. Genome editing using engineered nucleases (meganucleases, zinc finger nucleases, transcription activator-like effector nucleases) has created many recent breakthroughs. Prescreening for efficiency and specificity is a critical step prior to using any newly designed genome editing tool for experimental purposes. The current standard screening methods of evaluation are based on DNA sequencing or use mismatch-sensitive endonucleases. They can be time-consuming and costly or lack reproducibility. Here, we review and critically compare standard techniques with those more recently developed in terms of reliability, time, cost, and ease of use. Genome editing using engineered nucleases (meganucleases, zinc finger nucleases, transcription activator-like effector nucleases) has created many recent breakthroughs. Prescreening for efficiency and specificity is a critical step prior to using any newly designed genome editing tool for experimental purposes. The current standard screening methods of evaluation are based on DNA sequencing or use mismatch-sensitive endonucleases. They can be time-consuming and costly or lack reproducibility. Here, we review and critically compare standard techniques with those more recently developed in terms of reliability, time, cost, and ease of use. a frequent feature in many malignancies, chromosomal translocations are the result of an exchange of chromosomal fragments between nonhomologous chromosomes. To occur, they require DSBs to be created in each chromosome involved. the simultaneous breakage of the two DNA strands in close proximity within a given DNA sequence. DSB is physiological when occurring in certain cells such as maturing T or B lymphocytes; but, DSB can have pathological consequences if occurring at an abnormal rate or if it is not properly repaired. the targeted modification of a DNA sequence in living cells. Used to add, remove, replace, or modify existing DNA sequences, it can also induce specific chromosomal rearrangements or modify gene expression. one of the two main pathways for repair of DNA DSBs. HR requires the availability of a template DNA (e.g., a sister chromatid produced during DNA replication). insertion or deletion of a certain number of nucleotides within a given DNA sequence. a non-Sanger-based technology for DNA sequencing that allows for rapid sequencing of the whole genome. one of the two main pathways for repair of DNA DSBs. NHEJ is an error-prone process that can take place at any time during a cell cycle. unwanted genomic regions targeted during the genome editing procedure. short (approximately 20 nucleotide) RNA sequences that drive the CRISPR/Cas9 system to the targeted DNA sequence.