Metabolite Damage and Damage-Control in a Minimal Genome

Analysis of the genes retained in the minimized Mycoplasma JCVI-Syn3A genome established that systems that repair or preempt metabolite damage are essential to life. Several genes with known metabolite damage repair or preemption functions were identified and experimentally validated, including 5-formyltetrahydrofolate cyclo-ligase, CoA disulfide reductase, and certain hydrolases. Furthermore, we discovered that an enigmatic YqeK hydrolase domain fused to NadD has a novel proofreading function in NAD synthesis and could double as a MutT-like sanitizing enzyme for the nucleotide pool. Finally, we combined metabolomics and cheminformatics approaches to extend the core metabolic map of JCVI-Syn3A to include promiscuous enzymatic reactions and spontaneous side reactions. This extension revealed that several key metabolite damage-control systems remain to be identified in JCVI-Syn3A, such as that for methylglyoxal.

Genome-scale top-down reduction of phages to generate viable minimal phage genomes

Reduction of tailed-phage genomes to generate viable minimal genome phages is important for expanding our understanding of phage biology, providing insights for phage synthetic biology. Many efforts have been made to minimize living cells, but such work remains a challenge for phages due to the extraordinary genomic diversity and lack of genome-scale editing techniques. Here, we developed a CRISPR/Cas9-based iterative phage genome reduction (CiPGr) approach to detect the nonessential gene set of phages and minimize phage genomes. By CiPGr, inactivated genes accumulated on the phage genome, and mutant progeny with robust growth gradually arose, eventually becoming predominant in the populations. CiPGr was applied to four distinct tailed phages (model phages T7 and T4; wild-type phages seszw and selz), resulting in mutants of these phages with deletion of 8–20% (3.3–33 kbp) sequences, and leading to minimal genomes. Metagenomic sequencing of the mutant phage populations generated showed that 46.7 to 65.4% of genes of these phages were removed. Loss of some genes (39.6%-50%) in the removable gene sets was likely severely detrimental to phage growth. This made the corresponding mutant progenies recede in the populations, leading to the failure of detection of these genes in the genomes of the isolated mutants. In summary, our results for these four distinct tailed phages demonstrated that CiPGr is a generic yet effective approach suitable for use in novel phages without prior knowledge.

Minimal genome-wide human CRISPR-Cas9 library

CRISPR guide RNA libraries have been iteratively improved to provide increasingly efficient reagents, although their large size is a barrier for many applications. We design an optimised minimal genome-wide human CRISPR-Cas9 library (MinLibCas9) by mining existing large-scale gene loss-of-function datasets, resulting in a greater than 42% reduction in size compared to other CRISPR-Cas9 libraries while preserving assay sensitivity and specificity. MinLibCas9 provides backward compatibility with existing datasets, increases the dynamic range of CRISPR-Cas9 screens and extends their application to complex models and assays.

Minimal genome-wide human CRISPR-Cas9 library

CRISPR guide-RNA libraries have been iteratively optimised to provide increasingly efficient reagents, although their large size is a barrier for many applications. We designed an optimised minimal genome-wide human CRISPR-Cas9 library (MinLibCas9), by mining existing large-scale gene loss-of-function datasets, resulting in a greater than 42% reduction in size compared to other libraries while preserving assay sensitivity and specificity. MinLibCas9 increases the dynamic range of CRISPR-Cas9 loss-of-function screens and extends their application to complex models and assays.