Krisp: A Python package to aid in the design of CRISPR and amplification-based diagnostic assays from whole genome sequencing data.

Recent pandemics like COVID-19 highlighted the importance of rapidly developing diagnostics to detect evolving pathogens. CRISPR-Cas technology has recently been used to develop diagnostic assays for sequence-specific recognition of DNA or RNA. These assays have similar sensitivity to the gold standard qPCR but can be deployed as easy to use and inexpensive test strips. However, the discovery of diagnostic regions of a genome flanked by conserved regions where primers can be designed requires extensive bioinformatic analyses of genome sequences. We developed the Python package krisp to aid in the discovery of primers and diagnostic sequences that differentiate groups of samples from each other, using either unaligned genome sequences or a variant call format (VCF) file as input. Krisp has been optimized to handle large datasets by using efficient algorithms that run in near linear time, use minimal RAM, and leverage parallel processing when available. The validity of krisp results has been demonstrated in the laboratory with the successful design of a CRISPR diagnostic assay to distinguish the sudden oak death pathogen Phytophthora ramorum from closely related Phytophthora species. Krisp is released open source under a permissive license with all the documentation needed to quickly design CRISPR-Cas diagnostic assays.

Can the RNA World Still Function without Cytidine?

Most scenarios for the origin of life assume that RNA played a key role in both catalysis and information storage. The A, U, G, and C nucleobases in modern RNA all participate in secondary structure formation and replication. However, the rapid deamination of C to U and the absence of C in meteorite samples suggest that prebiotic RNA may have been deficient in cytosine. Here, we assess the ability of RNA sequences formed from a three-letter AUG alphabet to perform both structural and genetic roles in comparison to sequences formed from the AUGC alphabet. Despite forming less thermodynamically stable helices, the AUG alphabet can find a broad range of structures and thus appears sufficient for catalysis in the RNA World. However, in the AUG case, longer sequences are required to form structures with an equivalent complexity. Replication in the AUG alphabet requires GU pairing. Sequence fidelity in the AUG alphabet is low whenever G's are present in the sequence. We find that AUG sequences evolve to AU sequences if GU pairing is rare, and to RU sequences if GU pairing is common (R denotes A or G). It is not possible to conserve a G at a specific site in either case. These problems do not rule out the possibility of an RNA World based on AUG, but they show that it wouldbe significantly more difficult than with a four-base alphabet.

Developmental Systems Drift and the Drivers of Sex Chromosome Evolution.

Phenotypic invariance-the outcome of purifying selection-is a hallmark of biological importance. However, invariant phenotypes might be controlled by diverged genetic systems in different species. Here, we explore how an important and invariant phenotype-the development of sexually differentiated individuals-is controlled in over two dozen species in the frog family Pipidae. We uncovered evidence in different species for 1) an ancestral W chromosome that is not found in many females and is found in some males, 2) independent losses and 3) autosomal segregation of this W chromosome, 4) changes in male versus female heterogamy, and 5) substantial variation among species in recombination suppression on sex chromosomes. We further provide evidence of, and evolutionary context for, the origins of at least seven distinct systems for regulating sex determination among three closely related genera. These systems are distinct in their genomic locations, evolutionary origins, and/or male versus female heterogamy. Our findings demonstrate that the developmental control of sexual differentiation changed via loss, sidelining, and empowerment of a mechanistically influential gene, and offer insights into novel factors that impinge on the diverse evolutionary fates of sex chromosomes.

Rolling-circle and strand-displacement mechanisms for non-enzymatic RNA replication at the time of the origin of life.

It is likely that RNA replication began non-enzymatically, and that polymerases were later selected to speed up the process. We consider replication mechanisms in modern viruses and ask which of these is possible non-enzymatically, using mathematical models and experimental data found in the literature to estimate rates of RNA synthesis and replication. Replication via alternating plus and minus strands is found in some single-stranded RNA viruses. However, if this occurred non-enzymatically it would lead to double-stranded RNA that would not separate. With some form of environmental cycling, such as temperature, salinity, or pH cycling, double-stranded RNA can be melted to form single-stranded RNA, although re-annealing of existing strands would then occur much faster than synthesis of new strands. We show that re-annealing blocks this form of replication at a very low concentration of strands. Other kinds of viruses synthesize linear double strands from single strands and then make new single strands from double strands via strand-displacement. This does not require environmental cycling and is not blocked by re-annealing. However, under non-enzymatic conditions, if strand-displacement occurs from a linear template, we expect the incomplete new strand to be almost always displaced by the tail end of the old strand through toehold-mediated displacement. A third kind of replication in viruses and viroids is rolling-circle replication which occurs via strand-displacement on a circular template. Rolling-circle replication does not require environmental cycling and is not prevented by toehold-mediated displacement. Rolling-circle replication is therefore expected to occur non-enzymatically and is a likely starting point for the evolution of polymerase-catalysed replication.

Error thresholds for RNA replication in the presence of both point mutations and premature termination errors.

We consider a spatial model of replication in the RNA World in which polymerase ribozymes use neighbouring strands as templates. Point mutation errors create parasites that have the same replication rate as the polymerase. We have shown previously that spatial clustering allows survival of the polymerases as long as the error rate is below a critical error threshold. Here, we additionally consider errors where a polymerase prematurely terminates replication before reaching the end of the template, creating shorter parasites that are replicated faster than the functional polymerase. In well-known experiments where Qß RNA is replicated by an RNA polymerase protein, the virus RNA is rapidly replaced by very short non-functional sequences. If the same thing were to occur when the polymerase is a ribozyme, this would mean that termination errors could potentially destroy the RNA World. In this paper, we show that this is not the case in the RNA replication model studied here. When there is continued generation of parasites of all lengths by termination errors, the system can survive up to a finite error threshold, due to the formation of travelling wave patterns; hence termination errors are important, but they do not lead to the inevitable destruction of the RNA World by short parasites. The simplest assumption is that parasite replication rate is inversely proportional to the strand length. In this worst-case scenario, the error threshold for termination errors is much lower than for point mutations. We also consider a more realistic model in which the time for replication of a strand is the sum of a time for binding of the polymerase, and a time for polymerization. When the binding step is considered, termination errors are less serious than in the worst case. In the limit where the binding time is dominant, replication rates are equal for all lengths, and the error threshold for termination is the same as for point mutations.

Survival of RNA Replicators is much Easier in Protocells than in Surface-Based, Spatial Systems.

In RNA-World scenarios for the origin of life, replication is catalyzed by polymerase ribozymes. Replicating RNA systems are subject to invasion by non-functional parasitic strands. It is well-known that there are two ways to avoid the destruction of the system by parasites: spatial clustering in models with limited diffusion, or group selection in protocells. Here, we compare computational models of replication in spatial models and protocells as closely as possible in order to determine the relative importance of these mechanisms in the RNA World. For the survival of the polymerases, the replication rate must be greater than a minimum threshold value, kmin, and the mutation rate in replication must be less than a maximum value, Mmax, which is known as the error threshold. For the protocell models, we find that kmin is substantially lower and Mmax is substantially higher than for the equivalent spatial models; thus, the survival of polymerases is much easier in protocells than on surfaces. The results depend on the maximum number of strands permitted in one protocell or one lattice site in the spatial model, and on whether replication is limited by the supply of monomers or the population size of protocells. The substantial advantages that are seen in the protocell models relative to the spatial models are robust to changing these details. Thus, cooperative polymerases with limited accuracy would have found it much easier to operate inside lipid compartments, and this suggests that protocells may have been a very early step in the development of life. We consider cases where parasites have an equal replication rate to polymerases, and cases where parasites multiply twice as fast as polymerases. The advantage of protocell models over spatial models is increased when the parasites multiply faster.

Constraining the Time Interval for the Origin of Life on Earth.

Estimates of the time at which life arose on Earth make use of two types of evidence. First, astrophysical and geophysical studies provide a timescale for the formation of Earth and the Moon, for large impact events on early Earth, and for the cooling of the early magma ocean. From this evidence, we can deduce a habitability boundary, which is the earliest point at which Earth became habitable. Second, biosignatures in geological samples, including microfossils, stromatolites, and chemical isotope ratios, provide evidence for when life was actually present. From these observations we can deduce a biosignature boundary, which is the earliest point at which there is clear evidence that life existed. Studies with molecular phylogenetics and records of the changing level of oxygen in the atmosphere give additional information that helps to determine the biosignature boundary. Here, we review the data from a wide range of disciplines to summarize current information on the timings of these two boundaries. The habitability boundary could be as early as 4.5 Ga, the earliest possible estimate of the time at which Earth had a stable crust and hydrosphere, or as late as 3.9 Ga, the end of the period of heavy meteorite bombardment. The lack of consensus on whether there was a late heavy meteorite bombardment that was significant enough to prevent life is the largest uncertainty in estimating the time of the habitability boundary. The biosignature boundary is more closely constrained. Evidence from carbon isotope ratios and stromatolite fossils both point to a time close to 3.7 Ga. Life must have emerged in the interval between these two boundaries. The time taken for life to appear could, therefore, be within 200 Myr or as long as 800 Myr. Key Words: Origin of life-Astrobiology-Habitability-Biosignatures-Geochemistry-Early Earth. Astrobiology 18, 343-364.

The Role of Templating in the Emergence of RNA from the Prebiotic Chemical Mixture.

Biological RNA is a uniform polymer in three senses: it uses nucleotides of a single chirality; it uses only ribose sugars and four nucleobases rather than a mixture of other sugars and bases; and it uses only 3'-5' bonds rather than a mixture of different bond types. We suppose that prebiotic chemistry would generate a diverse mixture of potential monomers, and that random polymerization would generate non-uniform strands of mixed chirality, monomer composition, and bond type. We ask what factors lead to the emergence of RNA from this mixture. We show that template-directed replication can lead to the emergence of all the uniform properties of RNA by the same mechanism. We study a computational model in which nucleotides react via polymerization, hydrolysis, and template-directed ligation. Uniform strands act as templates for ligation of shorter oligomers of the same type, whereas mixed strands do not act as templates. The three uniform properties emerge naturally when the ligation rate is high. If there is an exact symmetry, as with the chase of chirality, the uniform property arises via a symmetry-breaking phase transition. If there is no exact symmetry, as with monomer selection and backbone regioselectivity, the uniform property emerges gradually as the rate of template-directed ligation is increased.