Sayer, A. | Search bioRxiv

bioRxiv2026

Comparative Single-Cell Profiling of CRISPR Knockout and Interference Defines Modality-Specific Strengths in Functional Genomics

Pooled CRISPR screens coupled with single-cell RNA sequencing enable high-throughput functional interrogation of gene regulatory networks, yet systematic comparisons of CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) remain limited. We established a single-cell CRISPRko workflow and benchmarked it against CRISPRi using 87 sgRNAs targeting 29 unfolded protein response genes. CRISPRko generated transcriptional phenotypes are highly concordant with CRISPRi, and induced comparable pathway-level responses. While CRISPRi allows direct assessment of target gene repression, CRISPRko provides an effective complementary approach for complete loss-of-function studies, expanding the toolkit for single-cell functional genomics.

genomics↗

bioRxiv2026

Cellular basis of B12 uptake and remodelling in microalgae revealed using a novel bioassay

O_LIVitamin B12, an essential micronutrient for many microalgae and humans, is synthesised only by certain prokaryotes. B12 is a complex tetrapyrrole that can exist in many forms (vitamers), some more bioavailable than others. Some microalgae are able to interconvert, or remodel, different B12 vitamers. As microalgae are important primary producers, it is crucial to understand how diverse microalgae acquire, utilise, and remodel this micronutrient. C_LIO_LIThrough the development of a novel algal bioassay for B12 quantification that distinguishes between B12 vitamers with different lower axial ligands, and the generation of targeted knock-out lines, we characterised the role of proteins involved in algal B12 uptake and remodelling. C_LIO_LIWe found that the previously characterised protein CoBalamin-Acquisition protein 1 (CBA1) is also necessary for the acquisition of pseudocobalamin, a less bioavailable form of B12. In addition, we provide the first experimental evidence that COBT is required for Chlamydomonas reinhardtii to remodel B12. C_LIO_LIWe apply the algal B12 bioassay to show that the edible alga Chlorella vulgaris can accumulate pseudocobalamin but is unable to remodel it, highlighting the need for thorough investigation of the metabolic requirements and capabilities of microalgae, especially given the growing interest in microalgae-based food additives. C_LI

microbiology↗

bioRxiv2022

Exploring the onset of B12-based mutualisms using a recently evolved Chlamydomonas auxotroph and B12-producing bacteria

Cobalamin (vitamin B12), is a cofactor for crucial metabolic reactions in multiple eukaryotic taxa, including major primary producers such as algae, and yet only prokaryotes can produce it. Many bacteria can colonise the algal phycosphere, forming stable communities that gain preferential access to exudates and in return provide compounds, such as B12. Extended coexistence can then drive gene loss, leading to greater algal-bacterial interdependence. In this study, we investigate how a recently evolved B12-dependent strain of Chlamydomonas reinhardtii, metE7, forms a mutualism with certain bacteria, including the rhizobium Mesorhizobium loti and even a strain of the gut bacterium E. coli engineered to produce cobalamin. Although metE7 was supported by B12 producers, its growth in co-culture was slower than the B12-independent wild-type, suggesting that high bacterial B12 provision may be necessary to favour B12 auxotrophs and their evolution. Moreover, we found that an E. coli strain that releases more B12 makes a better mutualistic partner, and although this trait may be more costly in isolation, greater B12 release provided an advantage in co-cultures. We hypothesise that, given the right conditions, bacteria that release more B12 may be selected for, particularly if they form close interactions with B12-dependent algae. Originality-Significance statementMicroalgae are fundamental to the global carbon cycle, and yet despite being photosynthetic they often rely on other organisms for micronutrients. One of these micronutrients is vitamin B12 (cobalamin), which they receive from bacteria. Many environmental studies support the widespread role of B12 in algal-bacterial mutualisms, so here we wished to investigate how these mutualisms may arise evolutionarily by using an experimentally evolved B12-dependent alga and various B12-producers. A B12-producing rhizobium, Mesorhizobium loti, could stably support the B12-dependent Chlamydomonas reinhardtii metE7 strain, and vice versa, but nutrient supplementation increased growth of both species further. metE7 could also be supported by E. coli strains engineered to produce B12, and engineering a strain to release a higher proportion of B12 led to better algal growth, which increased bacterial growth in turn. We suggest that as B12-based mutualisms develop, increased B12 release may be selected for and therefore lead to more productive symbioses.

microbiology↗