Is it helpful to use HDR templates that introduce silent mutations in the PAM sequence or sgRNA seeding region?
Is it helpful to use HDR templates that introduce silent mutations in the PAM sequence or sgRNA seeding region?
Cas9-sgRNA RNPs can re-cut a given genomic target following HDR if the editing outcome does not involve alteration of the PAM or sgRNA seeding region in the protospacer sequence, such that the RNP complex can still bind to the genomic target. Given this, a common strategy for increasing the percentage of successful HDR in coding regions is the incorporation of silent mutations in HDR templates that disrupt Cas9-sgRNA RNP binding without altering corresponding amino acid sequences.
If your PAM sequence is in a coding region and it can be silently mutated, it is advisable to do so to maximize the precision of genome editing and prevent subsequent Cas9 re-cutting and indel creation following HDR (Paquet et al. 2016). In seeking to mutate the PAM to disrupt RNP binding, please take into account other canonical PAM sequences (different from NGG) that can also be recognized less effectively by SpCas9 (i.e., NAG or NGA) (Zhang et al. 2014; Hsu et al. 2013) or Cas12a (i.e., TCTA, TCCA, or CCCA) (Yamano et al. 2017).
As mentioned above, the protospacer sequence can also be mutated. However, since some guide RNAs may tolerate a mismatch in the binding sequence, we recommend changing several bases as close to the PAM as possible in the seeding region of the sgRNA (Paquet et al. 2016; Kwart et al. 2017).
References
Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–32 (2013).
Kwart, D., Paquet, D., Teo, S. & Tessier-Lavigne, M. Precise and efficient scarless genome editing in stem cells using CORRECT. Nat. Protoc. 12, 329–354 (2017).
Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016).
Yamano, T. et al. Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1. Mol. Cell 67, 633–645.e3 (2017).
Zhang, Y. et al. Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells. Sci. Rep. 4, doi: 10.1038/srep05405 (2014).