CRISPR

The CRISPR/Cas system evolved in nature as a means for bacteria to protect themselves from invading viruses and bacteriophages by inserting pieces of their DNA into the host genome. This allowed the adaptive immune system to respond accordingly on a subsequent infection. It was discovered in Streptococcus pyogenes and later found across many other species. Repeated sequences The discovery of clustered DNA repeats took place independently in three parts of the world. The first description of what would later be called CRISPR is Ishino, et al. in 1987. They accidentally cloned part of a CRISPR sequence together with the iap gene (isozyme conversion of alkaline phosphatase) from Escherichia coli. The organization of the repeats surprised them, as clustered repeated sequences are more typically arranged consecutively, without interspersing sequences. and used this property to design a typing method called spoligotyping, which remains in use. Mojica studied the function of repeats in the archaeal genera Haloferax and Haloarcula. Mojica's supervisor surmised that the clustered repeats had a role in segregating replicated DNA into daughter cells during cell division, because plasmids and chromosomes with identical repeat arrays could not coexist in Haloferax volcanii. They noted the transcription of the interrupted repeats for the first time; CRISPR's first full characterization. By 2000, Mojica and his students, after an automated search of published genomes, identified interrupted repeats in 20 species of microbes as belonging to the same family. Because those sequences were interspaced, Mojica initially called these sequences "short regularly spaced repeats" (SRSR). In 2001, Mojica and Jansen, who were searching for additional interrupted repeats, proposed the acronym CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to encompass the numerous acronyms then in use. In 2002, Tang, et al. showed evidence that CRISPR repeat regions in Archaeoglobus fulgidus were transcribed into long RNA molecules subsequently processed into unit-length small RNAs, plus some longer forms of 2, 3, or more spacer-repeat units. In 2005, Barrangou discovered that S thermophilus, after iterative phage infection challenges, develops increased phage resistance due to the incorporation of additional CRISPR spacer sequences. CRISPR-associated systems A major advance came with Jansen's observation that the prokaryote repeat cluster was accompanied by four homologous genes, CRISPR-associated systems, Cas 1–4. The Cas proteins showed helicase and nuclease motifs, suggesting a role in the dynamic structure of the CRISPR loci. However, CRISPR's function remained enigmatic. In 2005, three independent research groups showed that some CRISPR spacers are derived from phage DNA and extrachromosomal DNA such as plasmids. In effect, the spacers are fragments of DNA gathered from viruses that previously attacked the cell. The source of the spacers was a sign that the CRISPR-cas system could have a role in adaptive immunity in bacteria. All three studies proposing this idea were initially rejected by high-profile journals, but eventually appeared elsewhere. The first publication Experimental work by several groups revealed the basic mechanisms of CRISPR-Cas immunity. In 2007, the first experimental evidence that CRISPR was part of the adaptive immune system was published. Cascade, crRNA and a helicase/nuclease (Cas3) were required to provide a bacterial host with immunity against infection by a DNA virus. By designing an anti-virus CRISPR, they demonstrated that two orientations of the crRNA (sense/antisense) provided immunity, indicating that the crRNA guides were targeting dsDNA. That year Marraffini and Sontheimer confirmed that a CRISPR sequence of S. epidermidis targeted DNA and not RNA to prevent conjugation. This finding was at odds with the proposed RNA-interference-like mechanism of CRISPR-Cas immunity, although a CRISPR-Cas system that targets foreign RNA was later found in Pyrococcus furiosus. In 2012, Jinek et al. fused crRNA and tracrRNA into a single-guide RNA, simplifying Cas9 targeting. Šikšnys, et al., reported that Cas9 from S. thermophilus can target specific DNA by altering crRNA. In 2013, Cong, et al., as well as Mali, et al., applied CRISPR-Cas9 to edit human cell cultures. In 2015, Liang, et al., edited human tripronuclear zygotes, achieving successful cleavage in 28 of 54 embryos. Cas9 and Cas9 with the position of DNA cleavage shown relative to their PAM sequences in a zoom-in A simpler CRISPR system from S. pyogenes uses Cas9, an endonuclease functioning with two small RNAs—crRNA and tracrRNA—to form a four-component complex. In 2012, Doudna and Charpentier simplified this into a two-component system by fusing the RNAs into a single-guide RNA, enabling Cas9 to target and cut specific DNA sequences—a breakthrough that earned them the 2020 Nobel Prize in Chemistry. Parallel work showed the S. thermophilus Cas9 could be similarly reprogrammed by altering the crRNA sequence. Cas12a Cas12a, a Class II Type V CRISPR-associated nuclease, was characterized in 2015 and was formerly known as Cpf1. This collateral cleavage property is exploited in the development of various diagnostic technologies. == Locus structure ==

Repeats and spacers The CRISPR array is made up of an AT-rich leader sequence followed by short repeats that are separated by unique spacers. CRISPR repeats typically range in size from 28 to 37 base pairs (bps), though there can be as few as 23 bp and as many as 55 bp. Some show dyad symmetry, implying the formation of a secondary structure such as a stem-loop ('hairpin') in the RNA, while others are designed to be unstructured. The size of spacers in different CRISPR arrays is typically 32 to 38 bp (range 21 to 72 bp). CRISPR-Cas systems fall into two classes. Class 1 systems use a complex of multiple Cas proteins to degrade foreign nucleic acids. Class 2 systems use a single large Cas protein for the same purpose. Class 1 is divided into types I, III, and IV; class 2 is divided into types II, V, and VI. The 6 system types are divided into 33 subtypes. Each type and most subtypes are characterized by a "signature gene" found almost exclusively in the category. Classification is also based on the complement of cas genes that are present. Most CRISPR-Cas systems have a Cas1 protein. The phylogeny of Cas1 proteins generally agrees with the classification system, but exceptions exist due to module shuffling. The sporadic distribution of the CRISPR-Cas subtypes suggests that the CRISPR-Cas system is subject to horizontal gene transfer during microbial evolution. == Mechanism ==

and Cas2 and cleavage of a protospacer. (2) The protospacer is ligated to the direct repeat adjacent to the leader sequence and (3) single strand extension repairs the CRISPR and duplicates the direct repeat. The crRNA processing and interference stages occur differently in each of the three major CRISPR systems. (4) The primary CRISPR transcript is cleaved by cas genes to produce crRNAs. (5) In type I systems Cas6e/Cas6f cleave at the junction of ssRNA and dsRNA formed by hairpin loops in the direct repeat. Type II systems use a trans-activating (tracr) RNA to form dsRNA, which is cleaved by Cas9 and RNaseIII. Type III systems use a Cas6 homolog that does not require hairpin loops in the direct repeat for cleavage. (6) In type II and type III systems secondary trimming is performed at either the 5' or 3' end to produce mature crRNAs. (7) Mature crRNAs associate with Cas proteins to form interference complexes. (8) In type I and type II systems, interactions between the protein and PAM sequence are required for degradation of invading DNA. Type III systems do not require a PAM for successful degradation and in type III-A systems basepairing occurs between the crRNA and mRNA rather than the DNA, targeted by type III-B systems. CRISPR-Cas immunity is a natural process of bacteria and archaea. CRISPR-Cas prevents bacteriophage infection, conjugation and natural transformation by degrading foreign nucleic acids that enter the cell. Spacer acquisition When a microbe is invaded by a bacteriophage, the first stage of the immune response is to capture phage DNA and insert it into a CRISPR locus in the form of a spacer. Cas1 and Cas2 are found in both types of CRISPR-Cas immune systems, which indicates that they are involved in spacer acquisition. Mutation studies confirmed this hypothesis, showing that removal of Cas1 or Cas2 stopped spacer acquisition, without affecting CRISPR immune response. Multiple Cas1 proteins have been characterised and their structures resolved. Cas1 proteins have diverse amino acid sequences. However, their crystal structures are similar and all purified Cas1 proteins are metal-dependent nucleases/integrases that bind to DNA in a sequence-independent manner. or (double strand) dsDNA- specific endoribonuclease activity. In the I-E system of E. coli, Cas1 and Cas2 form a complex where a Cas2 dimer bridges two Cas1 dimers. In this complex, Cas2 performs a non-enzymatic scaffolding role, New spacers are usually added at the beginning of the CRISPR next to the leader sequence creating a chronological record of viral infections. In E. coli a histone like protein called integration host factor (IHF), which binds to the leader sequence, is responsible for the accuracy of this integration. IHF also enhances integration efficiency in the type I-F system of Pectobacterium atrosepticum, but in other systems, different host factors may be required Protospacer adjacent motifs (PAM) Bioinformatic analysis of regions of phage genomes that were excised as spacers (termed protospacers) revealed that they were not randomly selected but instead were found adjacent to short (3–5 bp) DNA sequences termed protospacer adjacent motifs (PAM). Analysis of CRISPR-Cas systems showed PAMs to be important for type I and type II, but not type III systems during acquisition. In type I and type II systems, protospacers are excised at positions adjacent to a PAM sequence, with the other end of the spacer cut using a ruler mechanism, thus maintaining the regularity of the spacer size in the CRISPR array. The conservation of the PAM sequence differs between CRISPR-Cas systems and appears to be evolutionarily linked to Cas1 and the leader sequence. New spacers are added to a CRISPR array in a directional manner, but not exclusively, adjacent This suggests that the spacer acquisition machinery generates single-stranded overhangs in the second-to-last position of the direct repeat and in the PAM during spacer insertion. However, not all CRISPR-Cas systems appear to share this mechanism as PAMs in other organisms do not show the same level of conservation in the final position. created by the pairing of identical repeats that flank the crRNA. These Cas proteins cleave the longer transcript at the edge of the paired region, leaving a single crRNA along with a small remnant of the paired repeat region. Type III systems also use Cas6, however, their repeats do not produce stem-loops. Cleavage instead occurs by the longer transcript wrapping around the Cas6 to allow cleavage just upstream of the repeat sequence. Type II systems lack the Cas6 gene and instead utilize RNaseIII for cleavage. Functional type II systems encode an extra small RNA that is complementary to the repeat sequence, known as a trans-activating crRNA (tracrRNA). The type I-E complex (commonly referred to as Cascade) requires five Cas proteins bound to a single crRNA. Interference During the interference stage in type I systems, the PAM sequence is recognized on the crRNA-complementary strand and is required along with crRNA annealing. In type I systems correct base pairing between the crRNA and the protospacer signals a conformational change in Cascade that recruits Cas3 for DNA degradation. Type II systems rely on a single multifunctional protein, Cas9, for the interference step. The type III systems analysed from S. solfataricus and P. furiosus both target the mRNA of phages rather than phage DNA genome, The DNA cleavage was shown to be transcription dependent. The mechanism for distinguishing self from foreign DNA during interference is built into the crRNAs and is therefore likely common to all three systems. Throughout the distinctive maturation process of each major type, all crRNAs contain a spacer sequence and some portion of the repeat at one or both ends. It is the partial repeat sequence that prevents the CRISPR-Cas system from targeting the chromosome as base pairing beyond the spacer sequence signals self and prevents DNA cleavage. RNA-guided CRISPR enzymes are classified as type V restriction enzymes. == Evolution ==

The cas genes in the adaptor and effector modules of the CRISPR-Cas system are believed to have evolved from two different ancestral modules. A transposon-like element called casposon encoding the Cas1-like integrase and potentially other components of the adaptation module was inserted next to the ancestral effector module, which likely functioned as an independent innate immune system. The highly conserved cas1 and cas2 genes of the adaptor module evolved from the ancestral module while a variety of class 1 effector cas genes evolved from the ancestral effector module. The evolution of these various class 1 effector module cas genes was guided by various mechanisms, such as duplication events. On the other hand, each type of class 2 effector module arose from subsequent independent insertions of mobile genetic elements. These mobile genetic elements took the place of the multiple gene effector modules to create single gene effector modules that produce large proteins which perform all the necessary tasks of the effector module. The non-random evolution of these spacer regions has been found to be highly dependent on the environment and the particular foreign mobile genetic elements it contains. CRISPR-Cas can immunize bacteria against certain phages and thus halt transmission. For this reason, Koonin described CRISPR-Cas as a Lamarckian inheritance mechanism. However, this was disputed by a critic who noted, "We should remember [Lamarck] for the good he contributed to science, not for things that resemble his theory only superficially. Indeed, thinking of CRISPR and other phenomena as Lamarckian only obscures the simple and elegant way evolution really works". But as more recent studies have been conducted, it has become apparent that the acquired spacer regions of CRISPR-Cas systems are indeed a form of Lamarckian evolution because they are genetic mutations that are acquired and then passed on. On the other hand, the evolution of the Cas gene machinery that facilitates the system evolves through classic Darwinian evolution. The basic model of CRISPR evolution is newly incorporated spacers driving phages to mutate their genomes to avoid the bacterial immune response, creating diversity in both the phage and host populations. To resist a phage infection, the sequence of the CRISPR spacer must correspond perfectly to the sequence of the target phage gene. Phages can continue to infect their hosts' given point mutations in the spacer. Metagenomic analysis of two acid-mine-drainage biofilms showed that one of the analyzed CRISPRs contained extensive deletions and spacer additions versus the other biofilm, suggesting a higher phage activity/prevalence in one community than the other. Although most were body-site specific, some within a body site are widely shared among individuals. One of these loci originated from streptococcal species and contained ≈15,000 spacers, 50% of which were unique. Similar to the targeted studies of the oral cavity, some showed little evolution over time. During the same interval, the phage developed single-nucleotide polymorphisms that became fixed in the population, suggesting that targeting had prevented phage replication absent these mutations. The chemostat and observational studies suggest many nuances to CRISPR and phage (co)evolution. == Identification ==

CRISPRs are widely distributed among bacteria and archaea Analysis of CRISPRs in metagenomic data is more challenging, as CRISPR loci do not typically assemble, due to their repetitive nature or through strain variation, which confuses assembly algorithms. Where many reference genomes are available, polymerase chain reaction (PCR) can be used to amplify CRISPR arrays and analyse spacer content. However, this approach yields information only for specifically targeted CRISPRs and for organisms with sufficient representation in public databases to design reliable polymerase PCR primers. Degenerate repeat-specific primers can be used to amplify CRISPR spacers directly from environmental samples; amplicons containing two or three spacers can be then computationally assembled to reconstruct long CRISPR arrays. or by using direct repeat sequences in partially assembled CRISPR arrays from contigs (overlapping DNA segments that together represent a consensus region of DNA) as a hook for identifying direct repeats in individual reads. == Use by phages ==

Another way for bacteria to defend against phage infection is by having chromosomal islands. A subtype of chromosomal islands called phage-inducible chromosomal island (PICI) is excised from a bacterial chromosome upon phage infection and can inhibit phage replication. PICIs are induced, excised, replicated, and finally packaged into small capsids by certain staphylococcal temperate phages. PICIs use several mechanisms to block phage reproduction. In the first mechanism, PICI-encoded Ppi differentially blocks phage maturation by binding or interacting specifically with phage TerS, hence blocking phage TerS/TerL complex formation responsible for phage DNA packaging. In the second mechanism PICI CpmAB redirects the phage capsid morphogenetic protein to make 95% of SaPI-sized capsid and phage DNA can package only 1/3rd of their genome in these small capsids and hence become nonviable phage. The third mechanism involves two proteins, PtiA and PtiB, that target the LtrC, which is responsible for the production of virion and lysis proteins. This interference mechanism is modulated by a modulatory protein, PtiM, binds to one of the interference-mediating proteins, PtiA, and hence achieves the required level of interference. One study showed that lytic ICP1 phage, which specifically targets Vibrio cholerae serogroup O1, has acquired a CRISPR-Cas system that targets a V. cholera PICI-like element. The system has 2 CRISPR loci and 9 Cas genes. It seems to be homologous to the I-F system found in Yersinia pestis. Moreover, like the bacterial CRISPR-Cas system, ICP1 CRISPR-Cas can acquire new sequences, which allows phage and host to co-evolve. Certain archaeal viruses were shown to carry mini-CRISPR arrays containing one or two spacers. It has been shown that spacers within the virus-borne CRISPR arrays target other viruses and plasmids, suggesting that mini-CRISPR arrays represent a mechanism of heterotypic superinfection exclusion and participate in interviral conflicts. == Applications ==

CRISPR gene editing is a revolutionary technology that allows for precise, targeted modifications to the DNA of living organisms. Developed from a natural defense mechanism found in bacteria, CRISPR-Cas9 is the most commonly used system. Gene editing with CRISPR-Cas9 involves a Cas9 nuclease and an engineered guide RNA, which come together to allow for the precise "cutting" of one or both strands of DNA at specific locations within the genome. It makes use of the cell's natural DNA repair systems, including non-homologous end joining, homology-directed repair, or mismatch repair, to modify, insert, or delete genetic material at these specific cut sites. This technology has transformed fields such as genetics, medicine, and agriculture, offering potential treatments for genetic disorders, advancements in crop engineering, and research into the fundamental workings of life. However, its ethical implications and potential unintended consequences have sparked significant debate. == Experiences with CRISPR==

In February of 2025 the first case of CRISPR gene editing therapy on a child was documented. It was used to treat Severe Carbamoyl Phosphate Synthetase 1 Deficiency. It was administered At Children's Hospital of Philadelphia and is currently successful in its use to counteract the disease. == See also ==