Guide RNA Molecules Research Articles

A Guide to Computational Tools and Design Strategies for Genome Editing Experiments in Zebrafish Using CRISPR/Cas9.

The development of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology for mainstream biotechnological use based on its discovery as an adaptive immune mechanism in bacteria has dramatically improved the ability of molecular biologists to modify genomes of model organisms. The zebrafish is highly amenable to applications of CRISPR/Cas9 for mutation generation and a variety of DNA insertions. Cas9 protein in complex with a guide RNA molecule recognizes where to cut the homologous DNA based on a short stretch of DNA termed the protospacer-adjacent motif (PAM). Rapid and efficient identification of target sites immediately preceding PAM sites, quantification of genomic occurrences of similar (off target) sites and predictions of cutting efficiency are some of the features where computational tools play critical roles in CRISPR/Cas9 applications. Given the rapid advent and development of this technology, it can be a challenge for researchers to remain up to date with all of the important technological developments in this field. We have contributed to the armamentarium of CRISPR/Cas9 bioinformatics tools and trained other researchers in the use of appropriate computational programs to develop suitable experimental strategies. Here we provide an in-depth guide on how to use CRISPR/Cas9 and other relevant computational tools at each step of a host of genome editing experimental strategies. We also provide detailed conceptual outlines of the steps involved in the design and execution of CRISPR/Cas9-based experimental strategies, such as generation of frameshift mutations, larger chromosomal deletions and inversions, homology-independent insertion of gene cassettes and homology-based knock-in of defined point mutations and larger gene constructs.

CRISPR Content Correlates with the Pathogenic Potential of Escherichia coli.

Guide RNA molecules (crRNA) produced from clustered regularly interspaced short palindromic repeat (CRISPR) arrays, altogether with effector proteins (Cas) encoded by cognate cas (CRISPR associated) genes, mount an interference mechanism (CRISPR-Cas) that limits acquisition of foreign DNA in Bacteria and Archaea. The specificity of this action is provided by the repeat intervening spacer carried in the crRNA, which upon hybridization with complementary sequences enables their degradation by a Cas endonuclease. Moreover, CRISPR arrays are dynamic landscapes that may gain new spacers from infecting elements or lose them for example during genome replication. Thus, the spacer content of a strain determines the diversity of sequences that can be targeted by the corresponding CRISPR-Cas system reflecting its functionality. Most Escherichia coli strains possess either type I-E or I-F CRISPR-Cas systems. To evaluate their impact on the pathogenicity of the species, we inferred the pathotype and pathogenic potential of 126 strains of this and other closely related species and analyzed their repeat content. Our results revealed a negative correlation between the number of I-E CRISPR units in this system and the presence of pathogenicity traits: the median number of repeats was 2.5-fold higher for commensal isolates (with 29.5 units, range 0–53) than for pathogenic ones (12.0, range 0–42). Moreover, the higher the number of virulence factors within a strain, the lower the repeat content. Additionally, pathogenic strains of distinct ecological niches (i.e., intestinal or extraintestinal) differ in repeat counts. Altogether, these findings support an evolutionary connection between CRISPR and pathogenicity in E. coli.

RNA-guided gene activation by CRISPR-Cas9–based transcription factors

Technologies for engineering synthetic transcription factors have enabled many advances in medicine and science. In contrast to existing methods based on engineering of new DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Co-expression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.

CRISPR Immunity Relies on the Consecutive Binding and Degradation of Negatively Supercoiled Invader DNA by Cascade and Cas3

The prokaryotic CRISPR/Cas immune system is based on genomic loci that contain incorporated sequence tags from viruses and plasmids. Using small guide RNA molecules, these sequences act as a memory to reject returning invaders. Both the Cascade ribonucleoprotein complex and the Cas3 nuclease/helicase are required for CRISPR interference in Escherichia coli, but it is unknown how natural target DNA molecules are recognized and neutralized by their combined action. Here we show that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a protospacer-adjacent motif (PAM). PAM recognition by Cascade exclusively involves the crRNA-complementary DNA strand. After Cascade-mediated R loop formation, the Cse1 subunit recruits Cas3, which catalyzes nicking of target DNA through its HD-nuclease domain. The target isthen progressively unwound and cleaved by the joint ATP-dependent helicase activity and Mg(2+)-dependent HD-nuclease activity of Cas3, leading to complete target DNA degradation and invader neutralization.

Widespread Promiscuous Genetic Information Transfer From DNA to RNA

### Widespread RNA and DNA Sequence Differences in the Human Transcriptome Li et al Science . 2011;333:53–58. New genomic studies show that the transfer of genetic information from DNA to RNA within humans is not very faithful. If true, frequent, or purposeful, then the implications for human health and disease are significant, but the burden of proof required is significant. Commentary: Molecular biology and genetics has a “central dogma.” First postulated by Francis Crick1,2 in 1958, shortly after the discovery of the double helix, it states that the molecular transfer or flow of genetic information occurs only as DNA→RNA→ protein. This argument is intrinsic to our understanding of genotypes, phenotypes, and human disease. The central “dogma,” a word whose precise meaning Crick acknowledged he did not precisely know3 and that could have been given the less glamorous moniker “hypothesis,” is frequently misunderstood as also implying high accuracy or even inflexibility.2 We now know that genetic information flow can be sometimes corrupted and can also occur in other directions in particular biological systems. A recent study by Li and colleagues,4 from the laboratory of Vivian Cheung at the University of Pennsylvania, now demonstrates apparent widespread DNA→RNA sequence differences in humans. The experiments they conducted involved sequencing reverse-transcribed RNA from cultured B cells isolated from 27 different humans and comparing them with their host genome sequences. Surprisingly, after appropriate quality controls, they discovered numerous base positions at which the RNA sequence was different from the DNA sequence in multiple molecules, a phenomenon they refer to as RDD (RNA-DNA difference). Specifically, they identified 28766 RDDs in the 27 individuals at 10210 exonic sites within 4741 genes. Each individual had high-quality RNA-based sequence data at 9.8 million sites: so, RDD was detected at a rate of 1.1×10−4 per exonic site (base) across human genes. Interestingly, they observed all 12 types …

Structural insights into mRNA recognition from a PIWI domain-siRNA guide complex.

RNA interference and related RNA silencing phenomena use short antisense guide RNA molecules to repress the expression of target genes. Argonaute proteins, containing amino-terminal PAZ (for PIWI/Argonaute/Zwille) domains and carboxy-terminal PIWI domains, are core components of these mechanisms. Here we show the crystal structure of a Piwi protein from Archaeoglobus fulgidus (AfPiwi) in complex with a small interfering RNA (siRNA)-like duplex, which mimics the 5' end of a guide RNA strand bound to an overhanging target messenger RNA. The structure contains a highly conserved metal-binding site that anchors the 5' nucleotide of the guide RNA. The first base pair of the duplex is unwound, separating the 5' nucleotide of the guide from the complementary nucleotide on the target strand, which exits with the 3' overhang through a short channel. The remaining base-paired nucleotides assume an A-form helix, accommodated within a channel in the PIWI domain, which can be extended to place the scissile phosphate of the target strand adjacent to the putative slicer catalytic site. This study provides insights into mechanisms of target mRNA recognition and cleavage by an Argonaute-siRNA guide complex.

The structure and replication of kinetoplast DNA.

Kinetoplast DNA (kDNA), the mitochondrial DNA of flagellated protozoa of the order Kinetoplastida, is unique in its structure, function and mode of replication. It consists of few dozen maxicircles, encoding typical mitochondrial proteins and ribosomal RNA, and several thousands minicircles, encoding guide RNA molecules that function in the editing of maxicircles mRNA transcripts. kDNA minicircles and maxicircles in the parasitic species of the family Trypanosomatidae are topologically linked, forming a two dimensional fishnet-type DNA catenane. Studies of early branching free-living and parasitic species of the Bodonidae family revealed various other forms of this remarkable DNA structure and suggested the evolution of kDNA from unlinked DNA circles and covalently-linked concatamers into a giant topological catenane. The replication of kDNA occurs during nuclear S phase and includes the duplication of free detached minicircles and catenated maxicircle and the generation of two progeny kDNA networks that segregate upon cell division. Recent reports of sequence elements and specific proteins that regulate the periodic expression of replication proteins advanced our understanding of the mechanisms that regulate the temporal link between mitochondrial and nuclear DNA synthesis in trypanosomatids. Studies on kDNA replication enzymes and binding proteins revealed their remarkable organization in clusters at defined sites flanking the kDNA disk, in correlation with the progress in the cell cycle and the process of kDNA replication. In this review I describe the recent advances in the study of kDNA and discuss some of the major challenges in deciphering the structure, replication and segregation of this remarkable DNA structure.

Guide RNA molecules not engaged in RNA editing form ribonucleoprotein complexes free of mRNA

Mitochondrial pre-mRNAs in kinetoplastid organisms undergo uridine additions and deletions after transcription, a phenomenon termed kRNA editing. The reaction involves small, mitochondrial DNA transcripts, so called guide RNAs which provide the editing information via base pairing to the pre-mRNAs and furthermore may act as the U-nucleotide donors. Guide RNAs are not maintained as free molecules within the mitochondrial organelle, instead form several high molecular weight ribonucleoprotein complexes. Here we report the identification of two new gRNA containing RNP complexes, 8S and 15S in size, that only assemble with upstream gRNA molecules which require editing of their cognate pre-mRNA before they can base pair. The two complexes do not contain pre-mRNA molecules and the 8S RNP can be assembled in vitro. It contains two polypeptides under these conditions with apparent molecular weights of 90 and 21 kDa that can be cross-linked to the gRNA molecule. Our observation suggests the existence of structurally simple gRNA/protein complexes that might function as building blocks for the assembly of a high molecular weight editing machinery.

The formation of mitochondrial ribonucleoproteincomplexes involving guide RNA molecules in Trypanosoma brucei.

Transcripts from mitochondrial (maxicircle) genes of kinetoplastid organisms undergo RNA editing characterized by a series of reactions that insert and delete uridine nucleotides within the sequence of the pre-mRNAs. Guide RNAs, which complement fully edited mRNAs, provide the information for the edited sequence by an unknown mechanism. We report here that guide RNA molecules associate with other mitochondrial components to form four specific, stable ribonucleoprotein complexes. The complexes form very rapidly at a low monovalent cation concentration, and their formation is blocked by heparin or pretreatment of the mitochondrial lysate with SDS. ATP hydrolysis is not required but slightly stimulates complex association up to concentrations of 5 mM. The results are suggestive of a sequential assembly of the ribonucleoprotein complexes, and their possible involvement during the kinetoplastid RNA editing is discussed.

RNA editing as a source of genetic variation.

Kinetoplastid RNA editing alters mitochondrial RNA transcripts by addition and deletion of uridine residues, producing open reading frames that may be twice as long as the original RNA. Although the COIII gene encoding cytochrome c oxidase subunit III in Trypanosoma brucei is edited along its entire length, the presumably homologous genes in two related trypanosomes, Leishmania tarentolae and Crithidia fasciculata, are only modestly edited at their 5' ends. We used a comparative approach to investigate the evolution of an edited gene and to determine how well editing creates conserved protein sequences. As RNA editing probably involves the pairing of several guide RNA molecules with the messenger RNA, we expected the edited proteins to be resistant to evolutionary change. Here we report that RNA editing is extensive in the mitochondria of four species of the insect parasite Herpetomonas, which is possibly an evolutionary precursor of T. brucei and L. tarentolae, and the discovery that RNA editing is a novel source of frameshift mutations over evolutionary time. The edited proteins accumulate mutations nearly twice as rapidly as the unedited versions.

Partially edited mRNAs for cytochrome b and subunit III of cytochrome oxidase from leishmania tarentolae mitochondria: RNA editing intermediates

Partially edited mRNAs were selected by the polymerase chain reaction and sequenced. In the case of cytochrome b, 102 out of 106 clones displayed patterns of editing that were consistent with a strictly progressive 3′ to 5′ editing process, as predicted by the guide RNA model of RNA editing. In the case of cytochrome oxidase subunit III (COIII), 177 out of 304 clones displayed strictly progressive 3′ to 5′ patterns of editing. However, the remaining 127 COIII clones displayed unexpected patterns in which upstream editing preceded downstream editing, uridines were inserted at sites not normally edited, and purine residues were deleted. We suggest that many of these RNAs are produced by normal 3′ to 5′ editing of the COIII mRNA with incorrect guide RNA molecules.