Project description:The CRISPR-Cas9 system enables efficient sequence-specific mutagenesis for creating germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we established in vitro-assembled, fluorescent Cas9-sgRNA RNPs in stabilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. Sequence analysis of targeted loci in individual embryos reveals highly efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis reveals preliminary loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show efficient targeting of functional non-coding elements in gene-regulatory regions using saturating mutagenesis towards uncovering functional control elements in transgenic reporters and endogenous genes. Our results suggest that in vitro assembled, fluorescent Cas9-sgRNA RNPs provide a rapid reverse-genetics tool for direct and scalable loss-of-function studies beyond zebrafish applications.
Project description:Genotyping of RpoD mutants via amplicon sequencing from the following manuscript: \\"Systematic dissection of σ70 sequence diversity and function in bacteria\\" by Park and Wang (2020). Includes raw sequencing reads from samples from MAGE-seq single codon saturation mutagenesis and high-throughput fitness competition experiment as well as the RpoD ortholog mutants generated through recombineering and CRISPR selection.
Project description:Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. A major class of regulatory proteins is microbial allosteric transcription factors (aTFs), but aTF–inducer pairs are currently limited by those that naturally occur. Altering inducer specificity in these proteins is difficult because mutations that affect inducer binding may also disrupt allostery. Here, we engineer an aTF, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol or sucralose. We employ computational protein design, single-residue saturation mutagenesis, or random mutagenesis, along with multiplex assembly, and identify initial hits via a two-stage enrichment screen. Following activity maturation, we identify LacI variants with specificity to and induction by these new inducers comparable to that of wild-type LacI and its inducer, IPTG. The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits.