Harvard College

Johnny Hao Hu

David R Liu

Alejandro Chavez

Weixin Tang

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for engineering Cas9 and Cas9 variants that have increased activity on target sequences that do not contain the canonical PAM sequence. In some embodiments, fusion proteins comprising such Cas9 variants and nucleic acid editing domains, e.g., deaminase domains, are provided.

Evolved Cas9 proteins for gene editing

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity and/or improving the specificity of RNA-programmable proteins, 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, in certain instances, cleavage activity of RNA-programmable proteins (e.g., RNA-programmable endonucleases). By incorporating ligand-responsive self-cleaving catalytic RNAs (aptazymes) into guide RNAs, a set of aptazyme-embedded guide RNAs was developed that enable small molecule-controlled nuclease-mediated genome editing and small molecule-controlled base editing, as well as small molecule-dependent transcriptional activation in mammalian cells.

Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation

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 mRNA-sensing gRNAs that modulate the activity of RNA-programmable endonucleases based on the presence or absence of a target mRNA. 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).

Switchable CAS9 nucleases and uses thereof

A nucleic acid construct is provided that encodes two or more or a plurality of spacer sequences separated by restriction endonuclease recognition site. A plurality of such nucleic acid sequences are provided as a library for making guide RNAs for use with CRISPR/Cas systems.

Methods of making guide RNA

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 endo-nucleases. Some aspects of this disclosure provide mRNA-sensing gRNAs that modulate the activity of RNA-programmable endo-nucleases based on the presence or absence of a target mRNA. 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).

Switchable Cas9 nucleases and uses thereof

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).

Extended DNA-sensing GRNAS

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 mRNA-sensing gRNAs that modulate the activity of RNA-programmable endonucleases based on the presence or absence of a target mRNA.

MRNA-sensing switchable gRNAs

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.

Growing community of inventors

Cambridge, MA, United States of America

Johnny Hao Hu