CRISPR-Cas Inhibits Natural Transformation Through Altruistic Group Defense and Self-Sacrifice

SummaryCRISPR-Cas systems present an evolutionary tradeoff: does defense against phages and other parasitic DNA also prevent cells from acquiring potentially helpful new genes? Genomic analyses of this conundrum have arrived at often contradictory conclusions. Meanwhile, experimental studies have focused mainly on phages, conjugation, or artificial transformation, but less work has examined natural competence, a major driver of evolution and antibiotic resistance. Here, we use Acinetobacter baylyi, which combines high natural competence with a functional CRISPR-Cas system, to experimentally probe the interactions between CRISPR-Cas and natural competence. In these bacteria, the endogenous CRISPR array largely allows natural transformation by targeted DNA. However, CRISPR-Cas then kills the newly autoimmune cells in a form of programmed cell death. CRISPR-Cas often allows self-targeting cells to form colonies, albeit with fitness costs. Thus CRISPR-Cas appears to block natural transformation in a process more akin to altruistic group defense than an individual immune system.

Enhanced Cas12a multi-gene regulation using a CRISPR array separator

The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple guide RNA (gRNAs). Here, we report that Cas12a array performance is hypersensitive to the GC content of gRNA spacers, as high-GC spacers can impair activity of the downstream gRNA. We analyze naturally occurring CRISPR arrays and observe that natural repeats always contain an AT-rich fragment that separates gRNAs, which we term a CRISPR separator. Inspired by this observation, we design short, AT-rich synthetic separators (synSeparators) that successfully remove the disruptive effects between gRNAs. We further demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously underexplored feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.

CRISPR-Cas System: The Powerful Modulator of Accessory Genomes in Prokaryotes

Being frequently exposed to foreign nucleic acids, bacteria and archaea have developed an ingenious adaptive defense system, called CRISPR-Cas. The system is composed of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) array, together with CRISPR (<i>cas</i>)-associated genes. This system consists of a complex machinery that integrates fragments of foreign nucleic acids from viruses and mobile genetic elements (MGEs), into CRISPR arrays. The inserted segments (spacers) are transcribed and then used by cas proteins as guide RNAs for recognition and inactivation of the targets. Different types and families of CRISPR-Cas systems consist of distinct adaptation and effector modules with evolutionary trajectories, partially independent. The origin of the effector modules and the mechanism of spacer integration/deletion is far less clear. A review of the most recent data regarding the structure, ecology, and evolution of CRISPR-Cas systems and their role in the modulation of accessory genomes in prokaryotes is proposed in this article. The CRISPR-Cas system&apos;s impact on the physiology and ecology of prokaryotes, modulation of horizontal gene transfer events, is also discussed here. This system gained popularity after it was proposed as a tool for plant and animal embryo editing, in cancer therapy, as antimicrobial against pathogenic bacteria, and even for combating the novel coronavirus – SARS-CoV-2; thus, the newest and promising applications are reviewed as well.

CRISPR-Cas: a brief overview

CRISPR-Cas is an adaptive immunity in prokaryotes against infections by viruses and plasmids. CRISPR array recognizes foreign sequences of the invaders and Cas destroys them. Using this system seems possible to find the unwanted sequences in the genome and to destroy or to change them with the suitable ones. This system might not only protect ourselves from the future infections but also correct congenital abnormalities which may predispose to carcinogenesis or some congenital diseases.

Pruning and Tending Immune Memories: Spacer Dynamics in the CRISPR Array

CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes) is a type of prokaryotic immune system that is unique in its ability to provide sequence-specific adaptive protection, which can be updated in response to new threats. CRISPR-Cas does this by storing fragments of DNA from invading genetic elements in an array interspersed with short repeats. The CRISPR array can be continuously updated through integration of new DNA fragments (termed spacers) at one end, but over time existing spacers become obsolete. To optimize immunity, spacer uptake, residency, and loss must be regulated. This mini-review summarizes what is known about how spacers are organized, maintained, and lost from CRISPR arrays.

Searching for fat tails in CRISPR-Cas systems: Data analysis and mathematical modeling

Understanding CRISPR-Cas systems—the adaptive defence mechanism that about half of bacterial species and most of archaea use to neutralise viral attacks—is important for explaining the biodiversity observed in the microbial world as well as for editing animal and plant genomes effectively. The CRISPR-Cas system learns from previous viral infections and integrates small pieces from phage genomes called spacers into the microbial genome. The resulting library of spacers collected in CRISPR arrays is then compared with the DNA of potential invaders. One of the most intriguing and least well understood questions about CRISPR-Cas systems is the distribution of spacers across the microbial population. Here, using empirical data, we show that the global distribution of spacer numbers in CRISPR arrays across multiple biomes worldwide typically exhibits scale-invariant power law behaviour, and the standard deviation is greater than the sample mean. We develop a mathematical model of spacer loss and acquisition dynamics which fits observed data from almost four thousand metagenomes well. In analogy to the classical ‘rich-get-richer’ mechanism of power law emergence, the rate of spacer acquisition is proportional to the CRISPR array size, which allows a small proportion of CRISPRs within the population to possess a significant number of spacers. Our study provides an alternative explanation for the rarity of all-resistant super microbes in nature and why proliferation of phages can be highly successful despite the effectiveness of CRISPR-Cas systems.

Ectopic Spacer Acquisition in Streptococcus thermophilus CRISPR3 Array

Streptococcus thermophilus relies heavily on two type II-A CRISPR-Cas systems, CRISPR1 and CRISPR3, to resist siphophage infections. One hallmark of these systems is the integration of a new spacer at the 5′ end of the CRISPR arrays following phage infection. However, we have previously shown that ectopic acquisition of spacers can occur within the CRISPR1 array. Here, we present evidence of the acquisition of new spacers within the array of CRISPR3 of S. thermophilus. The analysis of randomly selected bacteriophage-insensitive mutants of the strain Uy01 obtained after phage infection, as well as the comparison with other S. thermophilus strains with similar CRISPR3 content, showed that a specific spacer within the array could be responsible for misguiding the adaptation complex. These results also indicate that while the vast majority of new spacers are added at the 5′ end of the CRISPR array, ectopic spacer acquisition is a common feature of both CRISPR1 and CRISPR3 systems in S. thermophilus, and it can still provide phage resistance. Ectopic spacer acquisition also appears to have occurred naturally in some strains of Streptococcus pyogenes, suggesting that it is a general phenomenon, at least in type II-A systems.