CRISPR-associated transposons

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CRISPR-associated transposons or CASTs are mobile genetic elements (MGEs) that have evolved to make use of minimal CRISPR systems for RNA-guided transposition of their DNA. [1] Unlike traditional CRISPR systems that contain interference mechanisms to degrade targeted DNA, CASTs lack proteins and/or protein domains responsible for DNA cleavage. [2] Specialized transposon machinery, similar to that of the well characterized Tn7 transposon, complexes with the CRISPR RNA (crRNA) and associated Cas proteins for transposition. [1] CAST systems have been characterized in a wide range of bacteria and make use of variable CRISPR configurations including Type I-F, Type I-B, Type I-C, Type I-D, Type I-E, Type IV, and Type V-K. [1] [2] [3] [4] MGEs remain an important part of genetic exchange by horizontal gene transfer and CASTs have been implicated in the exchange of antibiotic resistance and antiviral defense mechanisms, as well as genes involved in central carbon metabolism. [5] [6] These systems show promise for genetic engineering due to their programmability, PAM flexibility, and ability to insert directly into the host genome without double strand breaks requiring activation of host repair mechanisms. [3] [7]   They also lack Cas1 and Cas2 proteins and so rely on other more complete CRISPR systems for spacer acquisition in trans. [2] [3]

Contents

Natural structure and function

CRISPR-associated transposons are similar to the Tn7 transposon which functions with a cut and paste mechanism. [1] It contains a heteromeric transposase consisting of TnsA and TnsB proteins, and a regulator protein TnsC. [1] Structural analysis has shown binding of the TnsB protein and sequence specific motifs on the ends of the transposon which allows for excision and mobility. [8] Targeting for integration is done by the TnsD or TnsE proteins which preferentially target safe sites within the host chromosome or mobile elements (plasmids or bacteriophages), respectively. [1] TnsE is not found in CASTs  but a TnsD homolog, TniQ, is present and functions to bridge the gap between the transposase and CRISPR-Cas. [9] Multiple CRISPR types have been found to associate with transposons with two of the most studied being Type I-F, which makes use of a multi-subunit effector (Cascade), and Type V-K, which makes use of a single Cas12k effector. [3] [7] In both cases, Tn7 transposons have evolved to make use of these effectors to create R loops for site-specific integration. [3] While TnsA is present in Type I-F systems, it is notably absent in Type V-K systems which showed higher off-target integrations during initial characterization. [3] [10]

Type IF-3

A Type IF-3 CAST (Tn6677) was initially identified in Vibrio Cholerae and has been extensively studied. [7] This system contains proteins TnsA, TnsB, and TnsC that complex with Cas6, Cas7, and a Cas5-Cas8 fusion through interactions with TniQ. [9] Initial integration steps include TniQ-Cascade binding at the target site and TnsA and TnsB excision of the transposon, which is followed by TnsC binding to TniQ and transposase binding to TnsC. [9] There can be off-targeting prior to this final step, but TnsB and TnsC binding leads to a final proofreading step to maintain a high on-target percentage. [11] Tn6677 integration has been validated at near 100% on-target efficiency at site specific locations in multiple points in the host genome. [7] Other systems have also been characterized and validated in this class with varying ranges of efficiency, and include orthogonal systems for multiplexed insertions up to 10kb. [6] [12]

A unique characteristic of Type IF-3 systems is the presence of self-targeting guide RNA that are used to target the host chromosome. These systems have privatized the corresponding spacers through the use of atypical crRNA that prevent endogenous Type 1F systems from using the guides and their interference mechanisms to degrade the host. [13] Another privatization mechanism is the use of mismatch tolerance allowing only CAST systems to target locations in the genome without an exact match to the spacer. [13]

Type V-K

A Type V-K system was originally characterized from a cyanobacteria, Scytonema hofmanni, and contains a single Cas effector, Cas12k, that functions with a tracrRNA. [3] This system functions similarly to Tn7 but does not have a TnsA protein which can result in off-targeting and chimera formation during over-expression. [10] The Cas12k and tracrRNA complex bind to the target site and TnsC is polymerized directly adjacent prior to TniQ attachment and TnsB recognition and integration. [14] While these systems use traditional tracrRNA characteristic of Type II CRISPR systems, they can also target with short crRNA located adjacent to the transposon end. [15] Type V-K spacers preferentially target locations near tRNA genes, but other sites have been observed in these short crRNA guides which have been acquired by non-traditional means. [15]  

Applications in genetic engineering

CRISPR-associated transposons have been harnessed for in vitro and in vivo gene editing at different targets, in different hosts, and with different payloads. All CAST components of the Tn6677 system from Vibrio cholerae have been combined into a single plasmid and confirmed to deliver up to 10kb transposons at near 100% efficiency. [16] This has also been shown in a community context with conjugative delivery of suicide vectors to provide antibiotic resistance or enhanced metabolic function to only a single microbe. [17] Much of the initial characterization of these systems has been done in E. coli, but functionality has been confirmed in beta- and gammaproteobacteria with high efficiency, and in alphaproteobacteria at somewhat lower efficiency. [18] A single plasmid Tn677 has also been shown to function in human HEK293T cells showing potential therapeutic use in the future. [19]

Related Research Articles

<span class="mw-page-title-main">Transposable element</span> Semiparasitic DNA sequence

A transposable element (TE), also transposon, or jumping gene, is a type of mobile genetic element, a nucleic acid sequence in DNA that can change its position within a genome, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size.

Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.

A transposase is any of a class of enzymes capable of binding to the end of a transposon and catalysing its movement to another part of a genome, typically by a cut-and-paste mechanism or a replicative mechanism, in a process known as transposition. The word "transposase" was first coined by the individuals who cloned the enzyme required for transposition of the Tn3 transposon. The existence of transposons was postulated in the late 1940s by Barbara McClintock, who was studying the inheritance of maize, but the actual molecular basis for transposition was described by later groups. McClintock discovered that some segments of chromosomes changed their position, jumping between different loci or from one chromosome to another. The repositioning of these transposons allowed other genes for pigment to be expressed. Transposition in maize causes changes in color; however, in other organisms, such as bacteria, it can cause antibiotic resistance. Transposition is also important in creating genetic diversity within species and generating adaptability to changing living conditions.

<span class="mw-page-title-main">Insertion (genetics)</span> Type of mutation

In genetics, an insertion is the addition of one or more nucleotide base pairs into a DNA sequence. This can often happen in microsatellite regions due to the DNA polymerase slipping. Insertions can be anywhere in size from one base pair incorrectly inserted into a DNA sequence to a section of one chromosome inserted into another. The mechanism of the smallest single base insertion mutations is believed to be through base-pair separation between the template and primer strands followed by non-neighbor base stacking, which can occur locally within the DNA polymerase active site. On a chromosome level, an insertion refers to the insertion of a larger sequence into a chromosome. This can happen due to unequal crossover during meiosis.

<span class="mw-page-title-main">CRISPR</span> Family of DNA sequence found in prokaryotic organisms

CRISPR is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. Each sequence within an individual prokaryotic cell is derived from a DNA fragment of a bacteriophage that had previously infected the prokaryote or one of its ancestors. These sequences are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral defense system of prokaryotes and provide a form of heritable, acquired immunity. CRISPR is found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.

Guide RNA (gRNA) or single guide RNA (sgRNA) is a short sequence of RNA that functions as a guide for the Cas9-endonuclease or other Cas-proteins that cut the double-stranded DNA and thereby can be used for gene editing. In bacteria and archaea, gRNAs are a part of the CRISPR-Cas system that serves as an adaptive immune defense that protects the organism from viruses. Here the short gRNAs serve as detectors of foreign DNA and direct the Cas-enzymes that degrades the foreign nucleic acid.

<span class="mw-page-title-main">Mobile genetic elements</span> DNA sequence whose position in the genome is variable

Mobile genetic elements (MGEs), sometimes called selfish genetic elements, are a type of genetic material that can move around within a genome, or that can be transferred from one species or replicon to another. MGEs are found in all organisms. In humans, approximately 50% of the genome are thought to be MGEs. MGEs play a distinct role in evolution. Gene duplication events can also happen through the mechanism of MGEs. MGEs can also cause mutations in protein coding regions, which alters the protein functions. These mechanisms can also rearrange genes in the host genome generating variation. These mechanism can increase fitness by gaining new or additional functions. An example of MGEs in evolutionary context are that virulence factors and antibiotic resistance genes of MGEs can be transported to share genetic code with neighboring bacteria. However, MGEs can also decrease fitness by introducing disease-causing alleles or mutations. The set of MGEs in an organism is called a mobilome, which is composed of a large number of plasmids, transposons and viruses.

Transposon mutagenesis, or transposition mutagenesis, is a biological process that allows genes to be transferred to a host organism's chromosome, interrupting or modifying the function of an extant gene on the chromosome and causing mutation. Transposon mutagenesis is much more effective than chemical mutagenesis, with a higher mutation frequency and a lower chance of killing the organism. Other advantages include being able to induce single hit mutations, being able to incorporate selectable markers in strain construction, and being able to recover genes after mutagenesis. Disadvantages include the low frequency of transposition in living systems, and the inaccuracy of most transposition systems.

In molecular biology, trans-activating CRISPR RNA (tracrRNA) is a small trans-encoded RNA. It was first discovered by Emmanuelle Charpentier in her study of the human pathogen Streptococcus pyogenes, a type of bacteria that causes harm to humanity. In bacteria and archaea, CRISPR-Cas constitute an RNA-mediated defense system that protects against viruses and plasmids. This defensive pathway has three steps. First, a copy of the invading nucleic acid is integrated into the CRISPR locus. Next, CRISPR RNAs (crRNAs) are transcribed from this CRISPR locus. The crRNAs are then incorporated into effector complexes, where the crRNA guides the complex to the invading nucleic acid and the Cas proteins degrade this nucleic acid. There are several CRISPR system subtypes.

<span class="mw-page-title-main">Cas9</span> Microbial protein found in Streptococcus pyogenes M1 GAS

Cas9 is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic engineering applications. Its main function is to cut DNA and thereby alter a cell's genome. The CRISPR-Cas9 genome editing technique was a significant contributor to the Nobel Prize in Chemistry in 2020 being awarded to Emmanuelle Charpentier and Jennifer Doudna.

<span class="mw-page-title-main">CRISPR interference</span> Genetic perturbation technique

CRISPR interference (CRISPRi) is a genetic perturbation technique that allows for sequence-specific repression of gene expression in prokaryotic and eukaryotic cells. It was first developed by Stanley Qi and colleagues in the laboratories of Wendell Lim, Adam Arkin, Jonathan Weissman, and Jennifer Doudna. Sequence-specific activation of gene expression refers to CRISPR activation (CRISPRa).

<span class="mw-page-title-main">Epigenome editing</span>

Epigenome editing or epigenome engineering is a type of genetic engineering in which the epigenome is modified at specific sites using engineered molecules targeted to those sites. Whereas gene editing involves changing the actual DNA sequence itself, epigenetic editing involves modifying and presenting DNA sequences to proteins and other DNA binding factors that influence DNA function. By "editing” epigenomic features in this manner, researchers can determine the exact biological role of an epigenetic modification at the site in question.

A protospacer adjacent motif (PAM) is a 2–6-base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. The PAM is a component of the invading virus or plasmid, but is not found in the bacterial host genome and hence is not a component of the bacterial CRISPR locus. Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence. PAM is an essential targeting component which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by the CRISPR-associated nuclease.

<span class="mw-page-title-main">Conservative transposition</span>

Transposition is the process by which a specific genetic sequence, known as a transposon, is moved from one location of the genome to another. Simple, or conservative transposition, is a non-replicative mode of transposition. That is, in conservative transposition the transposon is completely removed from the genome and reintegrated into a new, non-homologous locus, the same genetic sequence is conserved throughout the entire process. The site in which the transposon is reintegrated into the genome is called the target site. A target site can be in the same chromosome as the transposon or within a different chromosome. Conservative transposition uses the "cut-and-paste" mechanism driven by the catalytic activity of the enzyme transposase. Transposase acts like DNA scissors; it is an enzyme that cuts through double-stranded DNA to remove the transposon, then transfers and pastes it into a target site.

<span class="mw-page-title-main">Cas12a</span> DNA-editing technology

Cas12a is an RNA-guided endonuclease that forms an essential component of the CRISPR systems found in some bacteria and archaea. In its natural context, Cas12a targets and destroys the genetic material of viruses and other foreign mobile genetic elements, thereby protecting the host cell from infection. Like other Cas enzymes, Cas12a binds to a "guide" RNA which targets it to a DNA sequence in a specific and programmable matter. In the host organism, the crRNA contains a constant region that is recognized by the Cas12a protein and a "spacer" region that is complementary to a piece of foreign nucleic acid that previously infected the cell.

CRISPR activation (CRISPRa) is a gene regulation technique that utilizes an engineered form of the CRISPR-Cas9 system to enhance the expression of specific genes without altering the underlying DNA sequence. Unlike traditional CRISPR-Cas9, which introduces double-strand breaks to edit genes, CRISPRa employs a modified, catalytically inactive Cas9 (dCas9) fused with transcriptional activators to target promoter or enhancer regions, thereby boosting gene transcription. This method allows for precise control of gene expression, making it a valuable tool for studying gene function, creating gene regulatory networks, and developing potential therapeutic interventions for a variety of diseases.

<span class="mw-page-title-main">CRISPR gene editing</span> Gene editing method

CRISPR gene editing (CRISPR, pronounced "crisper", refers to "clustered regularly interspaced short palindromic repeats") is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. It is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added in vivo.

<span class="mw-page-title-main">Anti-CRISPR</span> Group of proteins found in phages

Anti-CRISPR is a group of proteins found in phages, that inhibit the normal activity of CRISPR-Cas, the immune system of certain bacteria. CRISPR consists of genomic sequences that can be found in prokaryotic organisms, that come from bacteriophages that infected the bacteria beforehand, and are used to defend the cell from further viral attacks. Anti-CRISPR results from an evolutionary process occurred in phages in order to avoid having their genomes destroyed by the prokaryotic cells that they will infect.

Prime editing is a 'search-and-replace' genome editing technology in molecular biology by which the genome of living organisms may be modified. The technology directly writes new genetic information into a targeted DNA site. It uses a fusion protein, consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA), capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides. It mediates targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates.

<span class="mw-page-title-main">CRISPR RNA</span> RNA transcript from the CRISPR locus

CRISPR RNA or crRNA is a RNA transcript from the CRISPR locus. CRISPR-Cas is an adaptive immune system found in bacteria and archaea to protect against mobile genetic elements, like viruses, plasmids, and transposons. The CRISPR locus contains a series of repeats interspaced with unique spacers. These unique spacers can be acquired from MGEs.

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