Streptococcus thermophilus CRISPR1 Cas9 (St1Cas9)

Characteristics

Encoded by the CRISPR1 locus of S. thermophilus, distinct from the CRISPR3-encoded St3Cas9 of the same organism, which recognizes a different NGGNG PAM and is a larger protein. St1Cas9 recognizes a 5'-NNAGAAW PAM immediately 3' of the target. At matched on-target activity, it produces significantly lower off-target mutagenesis than SpCas9 at the human PRKDC and CARD11 loci. Its compact size lets St1Cas9 and its guide RNA be encoded together in a single AAV vector, unlike SpCas9.

Applications: Used for knockout editing at endogenous human loci (PRKDC, CARD11) with lower off-target mutagenesis than SpCas9 at matched on-target activity. Delivered as a single AAV8 vector encoding St1Cas9 and its guide RNA to disrupt Hpd in neonatal mice, rescuing lethality in a hereditary tyrosinemia model. Also used as the nuclease scaffold for St1Cas9-based cytosine and adenine base editors (St1BE4max, St1ABEmax).

Limitations: The NNAGAAW PAM is more restrictive than SpCas9's NGG, reducing the number of targetable sites. St1Cas9 is distinct from the CRISPR3-encoded St3Cas9 of the same organism, which recognizes an NGGNG PAM and is a larger protein; the two are not interchangeable. Reaching high editing efficiency in human cells required added NLS sequences and sgRNA scaffold optimization.

References

1. Cong et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science - Cong 2013 SpCas9
2. Müller et al. (2016). Streptococcus thermophilus CRISPR-Cas9 systems enable specific editing of the human genome. Mol Ther - Müller 2016 St1Cas9
3. Agudelo et al. (2020). Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9. Genome Res - Agudelo 2020 St1Cas9