corresponding to the PAM sequence in the donor sequence. Such “PAM‐silent” mutations, once incorporated into the target site, will block re‐cleavage by the CRISPR nuclease. This has become a common practice for SNP/mutation generation to increase KI efficiencies especially when it is low (i.e. <5%). However, this would inevitably introduce a nucleotide mutation in the edited site, albeit as a silent codon change. This silent codon change may have an impact on mRNA (stability, secondary structure, or translation efficiency) and further confound the effect of the targeted nucleotide SNP/mutation on mRNA. It is therefore preferred not to adopt the PAM‐silent strategy, especially if the editing efficiencies are greater than 10%. If used, a QC assay at the mRNA level (i.e. RT‐qPCR) will need to be applied to assess the effect of the mutations (the on‐target nucleotide change plus PAM‐silent mutation) on mRNA among isogenic lines before functional assays are applied.
4.6 Summary
In this chapter, we reviewed important design considerations for CRISPR experiments and the major providers of reagents, and discussed ways of working with CROs for genome editing projects. Many of the considerations are equally applicable to projects carried out in‐house. Regardless of where the genome editing projects are to be conducted, we suggest applying the formula Model x Assay x Perturbation, as well as the triple constraint principle – quality, time, and cost – in designing and managing execution of each project. We hope that the guidelines discussed here will help researchers strike an appropriate balance of these principles to maximize scientific impact of this exciting technology within a given budget or resource.
Acknowledgments
We would like to thank Gerard Drewes and Michelle Kimberland for critical reading of the manuscript.
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