Natural microbial sensing circuits can be rewired into new gene networks to build living sensors that detect and respond to disease-associated biomolecules. However, synthetic living sensors, once ingested, are cleared from the gastrointestinal (GI) tract within 48 h; retaining devices in the intestinal lumen is prone to intestinal blockage or device migration. To localize synthetic microbes and safely extend their residence in the GI tract for health monitoring and sustained drug release, an ingestible magnetic hydrogel carrier is developed to transport diagnostic microbes to specific intestinal sites. The magnetic living hydrogel is localized and retained by attaching a magnet to the abdominal skin, resisting the peristaltic waves in the intestine. The device retention is validated in a human intestinal phantom and an in vivo rodent model, showing that the ingestible hydrogel maintains the integrated living bacteria for up to seven days, which allows the detection of heme for GI bleeding in the harsh environment of the gut. The retention of microelectronics is also demonstrated by incorporating a temperature sensor into the magnetic hydrogel carrier.

Programmable and precise regulation of dCas9 functions in response to multiple molecular signals by using synthetic gene circuits will expand the application of the CRISPR-Cas technology. However, the application of CRISPR-Cas therapeutic circuits is still challenging due to the restrictive cargo size of existing viral delivery vehicles. Here, we construct logic AND circuits by integrating multiple split dCas9 domains, which is useful to reduce the size of synthetic circuits. In addition, we engineer sensory switches by exchanging split dCas9 domains, allowing differential regulations on one gene, or activating two different genes in response to cell-type specific microRNAs. Therefore, we provide a valuable split-dCas9 toolkit to engineer complex transcription controls, which may inspire new biomedical applications.

Although Cas9 nucleases are remarkably diverse in microorganisms, the range of genomic sequences targetable by a CRISPR/Cas9 system is restricted by the requirement of a short protospacer adjacent motif (PAM) at the target site. Here, we generate a group of chimeric Cas9 (cCas9) variants by replacing the key region in the PAM interaction (PI) domain of Staphylococcus aureus Cas9 (SaCas9) with the corresponding region in a panel of SaCas9 orthologs. By using a functional assay at target sites with different nucleotide recombinations at PAM position 3–6, we identify several cCas9 variants with expanded recognition capability at NNVRRN, NNVACT, NNVATG, NNVATT, NNVGCT, NNVGTG, and NNVGTT PAM sequences. In summary, we provide a panel of cCas9 variants accessible up to 1/4 of all the possible genomic targets in mammalian cells.

Nuclease dead Cas9 (dCas9) has been widely used for modulating gene expression by fusing with different activation or repression domains. However, delivery of the CRISPR/Cas system fused with various effector domains in a single adeno-associated virus (AAV) remains challenging due to the payload limit. Here, we engineered a set of downsized variants of Cas9 including Staphylococcus aureus Cas9 (SaCas9) that retained DNA binding activity by deleting conserved functional domains. We demonstrated that fusing FokI nuclease domain to the N-terminal of the minimal SaCas9 (mini-SaCas9) or to the middle of the split mini-SaCas9 can trigger efficient DNA cleavage. In addition, we constructed a set of compact transactivation domains based on the tripartite VPR activation domain and self-assembled arrays of split SpyTag:SpyCatch peptides, which are suitable for fusing to the mini-SaCas9. Lastly, we produced a single AAV containing the mini-SaCas9 fused with a downsized transactivation domain along with an optimized gRNA expression cassette, which showed efficient transactivation activity. Our results highlighted a practical approach to generate down-sized CRISPR/Cas9 and gene activation systems for in vivo applications.

Tumorigenesis is a complex process that is driven by a combination of networks of genes and environmental factors; however, efficient approaches to identifying functional networks that are perturbed by the process of tumorigenesis are lacking. In this study, we provide a comprehensive network-based strategy for the systematic discovery of functional synergistic modules that are causal determinants of inflammation-induced tumorigenesis. Our approach prioritizes candidate genes selected by integrating clinical-based and network-based genome-wide gene prediction methods and identifies functional synergistic modules based on combinatorial CRISPR-Cas9 screening. On the basis of candidate genes inferred de novo from experimental and computational methods to be involved in inflammation and cancer, we used an existing TGFβ1-induced cellular transformation model in colonic epithelial cells and a new combinatorial CRISPR-Cas9 screening strategy to construct an inflammation-induced differential genetic interaction network. The inflammation-induced differential genetic interaction network that we generated yielded functional insights into the genes and functional module combinations, and showed varied responses to the inflammation agents as well as active traditional Chinese medicine compounds. We identified opposing differential genetic interactions of inflammation-induced tumorigenesis: synergistic promotion and suppression. The synergistic promotion state was primarily caused by deletions in the immune and metabolism modules; the synergistic suppression state was primarily induced by deletions in the proliferation and immune modules or in the proliferation and metabolism modules. These results provide insight into possible early combinational targets and biomarkers for inflammation-induced tumorigenesis and highlight the synergistic effects that occur among immune, proliferation, and metabolism modules. In conclusion, this approach deepens the understanding of the underlying mechanisms that cause inflammation to potentially increase the cancer risk of colonic epithelial cells and accelerate the translation into novel functional modules or synergistic module combinations that modulate complex disease phenotypes.

The specificity of CRISPR/Cas9 genome editing is largely determined by the sequences of guide RNA (gRNA) and the targeted DNA, yet the sequence-dependent rules underlying off-target effects are not fully understood. To systematically explore the sequence determinants governing CRISPR/Cas9 specificity, here we describe a dual-target system to measure the relative cleavage rate between off- and on-target sequences (off-on ratios) of 1902 gRNAs on 13,314 synthetic target sequences, and reveal a set of sequence rules involving 2 factors in off-targeting: 1) a guide-intrinsic mismatch tolerance (GMT) independent of the mismatch context; 2) an “epistasis-like” combinatorial effect of multiple mismatches, which are associated with the free-energy landscape in R-loop formation and are explainable by a multi-state kinetic model. These sequence rules lead to the development of MOFF, a model-based predictor of Cas9-mediated off-target effects. Moreover, the “epistasis-like” combinatorial effect suggests a strategy of allele-specific genome editing using mismatched guides. With the aid of MOFF prediction, this strategy significantly improves the selectivity and expands the application domain of Cas9-based allele-specific editing, as tested in a high-throughput allele-editing screen on 18 cancer hotspot mutations.

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level did not only promote naïve pluripotent gene expression but also enhanced cell survival and clonogenicity, and it enabled integration of hESCs with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.