Extended DNA-sensing GRNAS

摘要

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity and/or improving the specificity of RNA-programmable endonucleases, such as Cas9. For example, provided are guide RNAs (gRNAs) that are engineered to exist in an “on” or “off” state, which control the binding and hence cleavage activity of RNA-programmable endonucleases. Some aspects of this disclosure provide gRNAs that modulate the activity of an RNA-programmable endonuclease based on the presence or absence of an extended DNA (xDNA).

主权项

1. An extended DNA (xDNA)-sensing single-guide RNA (sgRNA) comprising:
(1) a domain that binds a Cas9 protein, comprising a sgRNA backbone sequence forming a first stem-loop structure; and (2) a DNA-targeting domain, comprising
(i) a guide region comprising a sequence of at least 10 contiguous nucleotides that is 100% complementary to a first region of a target DNA sequence;(ii) a guide block region comprising a sequence of at least 10 contiguous nucleotides that is 100% complementary to a nucleotide sequence of the guide region (i); and(iii) an xDNA sensor region that hybridizes to a second region of the target DNA,wherein the domain that binds a Cas9 protein (1) and the DNA-targeting domain (2) do not overlap, and wherein the guide region (i) and the guide block region (ii) do not overlap.