Although all engineered nucleases are designed to have single site specificity, as proteins with biochemical properties, there is the possibility that because off-target binding (determined by standard on- and off-rates), they might create DSBs at unintended genomic sites. Because DSBs have the potential to create genotoxicity, the measurement and minimization of potential off target breaks have been an important part of the genome editing field.[1]

There are multiple different methods to identify potential off target sites, including bioinformatic methods, in vitro biochemical methods, and cellular-based methods. Each of these methods has its own strengths and weaknesses, and currently the approach to evaluating the specificity of nucleases used for translational purposes is to use a combination of approaches. In contrast to knockdown approaches using short-hairpin RNAs (shRNAs), it is remarkable that there have been no reports of off-target effects confounding an experimental result, and thus, for research purposes, using bioinformatics to identify guides with low probability of off-target cutting based on sequence homology has been adequate. The gold standard for measuring potential off-target effects is to quantify the frequency of indels using amplicon deep sequencing from the cell population of interest. Currently the sensitivity of this approach is reliable only down to the 0.1% level.

Because of the importance of specificity, there have been several engineered variants of the Cas9 derived from Streptococcus pyogenes (the most used source for Cas9 for ex vivo editing) with increased specific ity.[2-6] The increased specificity has been obtained by targeting different biochemical properties of the Cas9 protein. However, some of these variants may have been overengineered and have lost on-target activity in the process of creating increased specificity. Care must then be used to identify which Cas9 is best designed for the specific project. Using these engineered specificity variants, there are now multiple examples of no measurable off-target indels being created using a given gRNA.

The potential for off-target breaks induced by a nuclease should also be put into context of the cell’s robust ability to repair the wide variety and large number of DNA insults it is challenged with each day. These include tens of DSBs at random sites and thousands if not tens of thousands of other lesions, most of which occur randomly in the genome, including in tumor suppressors and oncogenes. In contrast to lentiviral vectors, there has been no description of a well-designed engineered nuclease causing lesions in a tumor suppressor or oncogene as an off-target event.

References

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[1] Tsai SQ, Joung JK. Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases. Nat Rev Genet. 2016;17(5):300–312.

[2] Vakulskas CA, et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat Med. 2018;24(8):1216–1224.

[3] Slaymaker IM, et al. Rationally engineered Cas9 nucleases with improved specificity. Science. 2016;351(6268):84–88.

 [4] Kleinstiver BP, et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016;529(7587):490–495.

[5] Casini A, et al. A highly specific SpCas9 variant is identified by in vivo screening in yeast. Nat Biotechnol. 2018;36(3):265–271.

[6] Chen JS, et al. Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature. 2017;550(7676):407–410.

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مواضيع ذات صلة

A Broad Range of Genomic Changes by Homologous Recombination–Mediated Genome Editing

Early Transcription of Genes N and Cro

Lambda’s Relatives and Lambda Hybrids

The Genetic Structure of Lambda

The Physical Structure of Lambda

Other Attenuated Systems: Operons, Bacillus subtilis and HIV

Coupling Translation to Termination

trp Multiple Secondary Structures in trp Leader RNA

Early Explorations of the Hypersynthesis

The Serendipitous Discovery of trp Enzyme Hypersynthesis

Rapid Induction Capabilities of the trp Operon

The Aromatic Amino Acid Synthetic Pathway and its Regulation