The recently discovered CRISPR/Cas9 endonuclease system, comprised of a guide RNA for the recognition of a DNA target and the Cas9 nuclease protein for binding and processing the target, has been extensively studied and has been widely applied in genome editing, synthetic biology, and transcriptional modulation in cells and animals. Toward more precise genomic modification and further expansion of the CRISPR/Cas9 system as a spatiotemporally controlled gene regulatory system, several approaches of conditional activation of Cas9 function using small molecules and light have recently been developed. These methods have led to improvements in the genome editing specificity of the CRISPR/Cas9 system and enabled its activation with temporal and spatial precision.

Das CRISPR/Cas9-Endonukleasesystem, das aus einer Guide-RNA zur Erkennung einer Zielsequenz der DNA und dem Cas9-Nukleaseprotein zur Bindung und Prozessierung der Ziel-DNA besteht, wurde intensiv erforscht und findet bereits breite Anwendung im Gen-Editing, der Synthesebiologie und der transkriptionellen Modulation in Zellen und Tieren. Mit dem Ziel einer präziseren Genmodifizierung und der Weiterentwicklung des CRISPR/Cas9-Systems zu einem räumlich-zeitlich kontrollierbaren Genregulationssystem wurden kürzlich mehrere Ansätze zur konditionalen Aktivierung der Cas9-Funktion durch niedermolekulare Verbindungen (“kleine Moleküle”) oder Licht entwickelt. Diese Methoden führten zu einer verbesserten Spezifität des CRISPR/Cas9-Systems im Gen-Editing und ermöglichten dessen Aktivierung mit zeitlicher und räumlicher Präzision.

A method has been developed to produce and integrate single-stranded DNA into genomic locations in bacteria in response to exogenous signals. The system functions similarly to a cellular tape recorder by writing information into DNA and reading it at a later time. Much like other cellular memory platforms, its operation is based on DNA recombinase function. However, the scalability and recording capacity have been improved over previous designs. In addition, memory storage was reversible and could be recorded in response to analogue inputs, such as light exposure. This modular memory writing system is an important addition to the genomic editing toolbox available for synthetic biology.

DNA logic gates are devices composed entirely of DNA that perform Boolean logic operations on one or more oligonucleotide inputs. Typical outputs of DNA logic gates are oligonucleotides or fluorescent signals. Direct activation of protein function has not been engineered as an output of a DNA-based computational circuit. Explicit control of protein activation enables the immediate triggering of enzyme function and could yield DNA computation outputs that are otherwise difficult to generate. By using zinc-finger proteins, AND, OR, and NOR logic gates were created that respond to short oligonucleotide inputs and lead to the activation or deactivation of a split-luciferase enzyme. The gate designs are simple and modular, thus enabling integration with larger multigate circuits, and the modular structure gives flexibility in the choice of protein output. The gates were also modified with translator circuits to provide protein activation in response to microRNA inputs as potential cellular cancer markers.

Spectrally differentiated caged morpholino oligonucleotides (cMOs) and wavelength-selective illumination have been used to sequentially inactivate organismal gene function. The efficacy of these reverse-genetic chemical probes has been demonstrated in zebrafish embryos, and these reagents have been employed to examine the mechanisms of mesoderm patterning.

We report the genetic incorporation of caged cysteine and caged homocysteine into proteins in bacterial and mammalian cells. The genetic code of these cells was expanded with an engineered pyrrolysine tRNA/tRNA synthetase pair that accepts both light-activatable amino acids as substrates. Incorporation was validated by reporter assays, western blots, and mass spectrometry, and differences in incorporation efficiency were explained by molecular modeling of synthetase–amino acid interactions. As a proof-of-principle application, the genetic replacement of an active-site cysteine residue with a caged cysteine residue in Renilla luciferase led to a complete loss of enzyme activity; however, upon brief exposure to UV light, a >150-fold increase in enzymatic activity was observed, thus showcasing the applicability of the caged cysteine in live human cells. A simultaneously conducted genetic replacement with homocysteine yielded an enzyme with greatly reduced activity, thereby demonstrating the precise probing of a protein active site. These discoveries provide a new tool for the optochemical control of protein function in mammalian cells and expand the set of genetically encoded unnatural amino acids.

Spectrally differentiated caged morpholino oligonucleotides (cMOs) and wavelength-selective illumination have been used to sequentially inactivate organismal gene function. The efficacy of these reverse-genetic chemical probes has been demonstrated in zebrafish embryos, and these reagents have been employed to examine the mechanisms of mesoderm patterning.

DNA logic gates are devices composed entirely of DNA that perform Boolean logic operations on one or more oligonucleotide inputs. Typical outputs of DNA logic gates are oligonucleotides or fluorescent signals. Direct activation of protein function has not been engineered as an output of a DNA-based computational circuit. Explicit control of protein activation enables the immediate triggering of enzyme function and could yield DNA computation outputs that are otherwise difficult to generate. By using zinc-finger proteins, AND, OR, and NOR logic gates were created that respond to short oligonucleotide inputs and lead to the activation or deactivation of a split-luciferase enzyme. The gate designs are simple and modular, thus enabling integration with larger multigate circuits, and the modular structure gives flexibility in the choice of protein output. The gates were also modified with translator circuits to provide protein activation in response to microRNA inputs as potential cellular cancer markers.