CRISPR-Cas systems: Challenges and future prospects.

The advancement gained over the past couple of decades in clustered regularly interspaced short palindromic repeats and CRISPR associated proteins (CRISPR-Cas) systems have revolutionized the field of synthetic biology, therapeutics, diagnostics and metabolic engineering. The technique has enabled the process of genome editing to be very precise, rapid, cost-effective and highly efficient which were the downfalls for the previously debuted zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) technologies. However, despite its great potential, challenges including off-target activity, method of delivery, ethical and regulatory issues still remain unresolved for the CRISPR-Cas systems.

Critical Roles of Translation Initiation and RNA Uridylation in Endogenous Retroviral Expression and Neural Differentiation in Pluripotent Stem Cells.

Previous studies have suggested that the loss of the translation initiation factor eIF4G1 homolog NAT1 induces excessive self-renewability of naive pluripotent stem cells (PSCs); yet the role of NAT1 in the self-renewal and differentiation of primed PSCs is still unclear. Here, we generate a conditional knockout of NAT1 in primed PSCs and use the cells for the functional analyses of NAT1. Our results show that NAT1 is required for the self-renewal and neural differentiation of primed PSCs.

Continuous genetic recording with self-targeting CRISPR-Cas in human cells.

The ability to record molecular events in vivo would enable monitoring of signaling dynamics within cellular niches and critical factors that orchestrate cellular behavior. We present a self-contained analog memory device for longitudinal recording of molecular stimuli into DNA mutations in human cells. This device consists of a self-targeting guide RNA (stgRNA) that repeatedly directs Streptococcus pyogenes Cas9 nuclease activity toward the DNA that encodes the stgRNA, enabling localized, continuous DNA mutagenesis as a function of stgRNA expression.

Foundations and Emerging Paradigms for Computing in Living Cells.

Genetic circuits, composed of complex networks of interacting molecular machines, enable living systems to sense their dynamic environments, perform computation on the inputs, and formulate appropriate outputs. By rewiring and expanding these circuits with novel parts and modules, synthetic biologists have adapted living systems into vibrant substrates for engineering. Diverse paradigms have emerged for designing, modeling, constructing, and characterizing such artificial genetic systems.

Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM.

The orchestrated action of genes controls complex biological phenotypes, yet the systematic discovery of gene and drug combinations that modulate these phenotypes in human cells is labor intensive and challenging to scale. Here, we created a platform for the massively parallel screening of barcoded combinatorial gene perturbations in human cells and translated these hits into effective drug combinations. This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of combinatorial genetics en masse (CombiGEM) to rapidly assemble barcoded combinatorial genetic libraries that can be tracked with high-throughput sequencing.

Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells.

RNA-based regulation and CRISPR/Cas transcription factors (CRISPR-TFs) have the potential to be integrated for the tunable modulation of gene networks. A major limitation of this methodology is that guide RNAs (gRNAs) for CRISPR-TFs can only be expressed from RNA polymerase III promoters in human cells, limiting their use for conditional gene regulation. We present new strategies that enable expression of functional gRNAs from RNA polymerase II promoters and multiplexed production of proteins and gRNAs from a single transcript in human cells.

Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas.

Transcriptional regulation is central to the complex behavior of natural biological systems and synthetic gene circuits. Platforms for the scalable, tunable, and simple modulation of transcription would enable new abilities to study natural systems and implement artificial capabilities in living cells. Previous approaches to synthetic transcriptional regulation have relied on engineering DNA-binding proteins, which necessitate multistep processes for construction and optimization of function.