Compositions and methods for improved nucleases
AssigneeAstraZeneca AB
InventorANNA-LINA CAVALLO
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Abstract
The present disclosure provides recombinant Cas9 proteins with a faster degradation rate than wild-type Cas9. The present disclosure also provides recombinant Cas9 proteins and CRISPR-Cas systems with reduced off-target modifications. Also provided herein are methods of site-specific modification with reduced off-target modifications utilizing the recombinant Cas9 proteins of the present disclosure.
Claims57
A recombinant Cas9 protein comprising an engineered KFERQ motif or KFERQ-like motif.
A recombinant Cas9 protein comprising one or more amino acid modifications of a wild-type Cas9 protein that introduce a chaperone mediated autophagy (CMA) target motif or an endosomal microautophagy (eMI) target motif in the Cas9 protein, wherein the recombinant Cas9 protein degrades at least 20% faster in vivo than the wild-type Cas9 protein or a Cas9 protein that does not comprise the CMA or eMI target motif.
A recombinant Cas9 protein comprising one or more amino acid modifications in a wild-type Cas9 protein, wherein the recombinant Cas9 protein comprises a CMA target motif or an eMI target motif.
A recombinant Cas9 protein isolated from Streptococcus pyogenes (SpCas9) comprising an amino acid modification at one or more of position F185, A547, I548, T560, V561, D829, I830, L1087, S1088, P1199, K1200 of SEQ ID NO:1, or combinations thereof.
A recombinant Cas9 protein capable of binding to heat shock cognate protein of 70 kD (HSC70).
A recombinant Cas9 protein isolated from Streptococcus pyogenes (SpCas9), comprising an engineered KFERQ motif or KFERQ-like motif at amino acid position 185 of SEQ ID NO:1.
Dependencies are inferred from the claim text.