The CRISPR-Cas system: its origin, functions and applications in biotechnology: A Review

The CRISPR-Cas system is a genome editing system seen in prokaryotic immune system. Bacteria and archaea protect itself against invading viruses and plasmid by targeting RNA or DNA of the invading element predominantly using this gene-editing tool. The CRISPR- Cas defense mechanism is carried out in three stages; adaptation stage where the spacers are inserted into the CRISPR locus, the expression stage where crRNA is formed by transcription of the CRISPR loci and the interference stage where the invading element is destroyed by the crRNA and cas proteins. The CRISPR-cas has been involved in many other functions apart from the immune defense they include; DNA repair, regulation of virulence, genome evolution, inhibit biofilm formation etc. The application of CRISPR-Cas system include genome engineering, agriculture to efficiently target and mutate plants, improve crop yield and crop resistance, in medicine to eradicate genetic diseases. However, ethical considerations are a major setback of CRISPR-Cas application especially in medicine. CRISPR-Cas has been used in variety of species including cultured human cell, rice, drosophila and mice.

Characterization of 67 Confirmed Clustered Regularly Interspaced Short Palindromic Repeats Loci in 52 Strains of Staphylococci

Staphylococcus aureus (S. aureus), which is one of the most important species of Staphylococci, poses a great threat to public health. Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) are an adaptive immune platform to combat foreign mobile genetic elements (MGEs) such as plasmids and phages. The aim of this study is to describe the distribution and structure of CRISPR-Cas system in S. aureus, and to explore the relationship between CRISPR and horizontal gene transfer (HGT). Here, we analyzed 67 confirmed CRISPR loci and 15 companion Cas proteins in 52 strains of Staphylococci with bioinformatics methods. Comparing with the orphan CRISPR loci in Staphylococci, the strains harboring complete CRISPR-Cas systems contained multiple CRISPR loci, direct repeat sequences (DR) forming stable RNA secondary structures with lower minimum free energy (MFE), and variable spacers with detectable protospacers. In S. aureus, unlike the orphan CRISPRs away from Staphylococcal cassette chromosome mec (SCCmec), the complete CRISPR-Cas systems were in J1 region of SCCmec. In addition, we found a conserved motif 5′-TTCTCGT-3′ that may protect their downstream sequences from DNA interference. In general, orphan CRISPR locus in S. aureus differed greatly from the structural characteristics of the CRISPR-Cas system. Collectively, our results provided new insight into the diversity and characterization of the CRISPR-Cas system in S. aureus.

Antimicrobial Resistance and CRISPR Typing Among Salmonella Isolates From Poultry Farms in China

Although knowledge of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has been applied in many research areas, comprehensive studies of this system in Salmonella, particularly in analysis of antibiotic resistance, have not been reported. In this work, 75 Salmonella isolates obtained from broilers or broilers products were characterized to determine their antimicrobial susceptibilities, antibiotic resistance gene profiles, and CRISPR array diversities, and genotyping was explored. In total, 80.00% (60/75) of the strains were multidrug resistant, and the main pattern observed in the isolates was CN-AZM-AMP-AMC-CAZ-CIP-ATM-TE-SXT-FOS-C. The resistance genes of streptomycin (aadA), phenicol (floR-like and catB3-like), β-lactams (blaTEM, blaOXA, and blaCTX), tetracycline [tet(A)-like], and sulfonamides (sul1 and sul2) appeared at higher frequencies among the corresponding resistant isolates. Subsequently, we analyzed the CRISPR arrays and found 517 unique spacer sequences and 31 unique direct repeat sequences. Based on the CRISPR spacer sequences, we developed a novel typing method, CRISPR locus three spacer sequences typing (CLTSST), to help identify sources of Salmonella outbreaks especially correlated with epidemiological data. Compared with multi-locus sequence typing (MLST), conventional CRISPR typing (CCT), and CRISPR locus spacer pair typing (CLSPT), discrimination using CLTSST was weaker than that using CCT but stronger than that using MLST and CLSPT. In addition, we also found that there were no close correlations between CRISPR loci and antibiotics but had close correlations between CRISPR loci and antibiotic resistance genes in Salmonella isolates.

Application of Genome Editing Technologies for Disease Treatment: Review

The improvement of particularly versatile genome-modifying advancements has outfitted experts with the ability to rapidly and monetarily bring sequence-specific changes into the genomes of a wide scope of cell types and organisms. The CRISPR framework was first found as a protection system in Escherichia coli against infections. Short portions of unfamiliar DNA are coordinated inside the CRISPR locus and translated into CRISPR RNA (crRNA), which at that point toughen to trans-activating crRNA (tracrRNA) to coordinate sequence specific debasement of pathogenic DNA by the Cas9 protein. Many studies have now revealed insight into the primary premise of DNA recognition by Cas9, showing that the heteroduplex shaped by the gRNA and its complementary strand of DNA is housed in a positively charged groove between the two nuclease areas (RuvC and HNH) inside the Cas9 protein, and that PAM recognition is intervened by an arginine-rich motif present in Cas9. Genome altering biological tools likewise bring healing chances. For instance, ZFN-interceded gene interruption has been taken to the clinic, particularly for the treatment of glioblastoma and HIV by Sangamo biosciences. ZFNs focused to the HIV co-receptor CCR5 for the medication of HIV/AIDS are in stage I clinical trials have been finished currently and are in advancement). In these clinical investigations, the security and possibility of autologous infusion of ex vivo extended CD4+ T cells treated with CCR5- specific ZFNs are assessed in patients with HIV/AIDS. Genome altering itself likewise holds huge potential for treating the fundamental hereditary causes for specific infections. Thusly, the point of this survey is to sum up the vital standards of genome altering, focusing a considerable lot of the designing advances that have laid the foundation for the creation, refinement, and usage of the current set-up of genome-changing biological tools.

CRISPRbuilder-TB: “CRISPR-builder for tuberculosis”. Exhaustive reconstruction of the CRISPR locus in mycobacterium tuberculosis complex using SRA

Mycobacterium tuberculosis complex (MTC) CRISPR locus diversity has long been studied solely investigating the presence/absence of a known set of spacers. Unveiling the genetic mechanisms of its evolution requires a more exhaustive reconstruction in a large amount of representative strains. In this article, we point out and resolve, with a new pipeline, the problem of CRISPR reconstruction based directly on short read sequences in M. tuberculosis. We first show that the process we set up, that we coin as “CRISPRbuilder-TB” (https://github.com/cguyeux/CRISPRbuilder-TB), allows an efficient reconstruction of simulated or real CRISPRs, even when including complex evolutionary steps like the insertions of mobile elements. Compared to more generalist tools, the whole process is much more precise and robust, and requires only minimal manual investigation. Second, we show that more than 1/3 of the currently complete genomes available for this complex in the public databases contain largely erroneous CRISPR loci. Third, we highlight how both the classical experimental in vitro approach and the basic in silico spoligotyping provided by existing analytic tools miss a whole diversity of this locus in MTC, by not capturing duplications, spacer and direct repeats variants, and IS6110 insertion locations. This description is extended in a second article that describes MTC-CRISPR diversity and suggests general rules for its evolution. This work opens perspectives for an in-depth exploration of M. tuberculosis CRISPR loci diversity and of mechanisms involved in its evolution and its functionality, as well as its adaptation to other CRISPR locus-harboring bacterial species.

Using CRISPR-Cas9 for Therapeutic Protein Production (Review Article)

Existence of CRISPR/Cas9 systems in bacteria and archaea has been noted to be the reason for these organisms’ ability to disarm invading nucleic acids. Such immunity is noted to arise from the targeting of the invading nucleic acids by guiding RNAs (sgRNAs), their cleavage by Cas9 (an endonuclease), and their subsequent integration into CRISPR locus. Recent studies have shown that the CRISPR/Cas9 tool can be adopted for gene editing in eukaryotic cells and thus offering potential for its use to treat genetic conditions. In this review, CRISPR/Cas9 has been shown to be an effective genome-editing tool with studies showing efficacy in zygote editing, in-vivo editing of somatic cells and ex-vivo editing of somatic cells. Occurrence of off-target effects however make zygote editing in human cells ethically questionable due to possibility of introducing unwanted mutations that may be passed on to the progeny. Nevertheless, observations that such off-target effects arise mainly from the promiscuity of sgRNAs rather that errors in CRISPR/Cas9 system show promise for increased specificity by developing better sgRNAs. Such increased specificity will facilitate the adoption of CRISPR/Cas9 for clinical use in treatment of conditions such as β-thalassemia, cystic fibrosis, Duchenne muscular dystrophy and HIV.

Mechanisms of spacer acquisition by sequential assembly of the adaptation module in Synechocystis

CRISPR–Cas immune systems process and integrate short fragments of DNA from new invaders as spacers into the host CRISPR locus to establish molecular memory of prior infection, which is also known as adaptation in the field. Some CRISPR–Cas systems rely on Cas1 and Cas2 to complete the adaptation process, which has been characterized in a few systems. In contrast, many other CRISPR–Cas systems require an additional factor of Cas4 for efficient adaptation, the mechanism of which remains less understood. Here we present biochemical reconstitution of the Synechocystis sp. PCC6803 type I-D adaptation system, X-ray crystal structures of Cas1–Cas2–prespacer complexes, and negative stained electron microscopy structure of the Cas4–Cas1 complex. Cas4 and Cas2 compete with each other to interact with Cas1. In the absence of prespacer, Cas4 but not Cas2 assembles with Cas1 into a very stable complex for processing the prespacer. Strikingly, the Cas1-prespacer complex develops a higher binding affinity toward Cas2 to form the Cas1–Cas2–prespacer ternary complex for integration. Together, we show a two-step sequential assembly mechanism for the type I-D adaptation module of Synechocystis, in which Cas4–Cas1 and Cas1–Cas2 function as two exclusive complexes for prespacer processing, capture, and integration.

Unexpected diversity of CRISPR unveils some evolutionary patterns of repeated sequences in Mycobacterium tuberculosis

Background Diversity of the CRISPR locus of Mycobacterium tuberculosis complex has been studied since 1997 for molecular epidemiology purposes. By targeting solely the 43 spacers present in the two first sequenced genomes (H37Rv and BCG), it gave a biased idea of CRISPR diversity and ignored diversity in the neighbouring cas-genes. Results We set up tailored pipelines to explore the diversity of CRISPR-cas locus in Short Reads. We analyzed data from a representative set of 198 clinical isolates as evidenced by well-characterized SNPs. We found a relatively low diversity in terms of spacers: we recovered only the 68 spacers that had been described in 2000. We found no partial or global inversions in the sequences, letting always the Direct Variant Repeats (DVR) in the same order. In contrast, we found an unexpected diversity in the form of: SNPs in spacers and in Direct Repeats, duplications of various length, and insertions at various locations of the IS6110 insertion sequence, as well as blocks of DVR deletions. The diversity was in part specific to lineages. When reconstructing evolutionary steps of the locus, we found no evidence for SNP reversal. DVR deletions were linked to recombination between IS6110 insertions or between Direct Repeats. Conclusion This work definitively shows that CRISPR locus of M. tuberculosis did not evolve by classical CRISPR adaptation (incorporation of new spacers) since the last most recent common ancestor of virulent lineages. The evolutionary mechanisms that we discovered could be involved in bacterial adaptation but in a way that remains to be identified.

Molecular Mechanisms of CRISPR-Cas Immunity in Bacteria

Prokaryotes have developed numerous defense strategies to combat the constant threat posed by the diverse genetic parasites that endanger them. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas loci guard their hosts with an adaptive immune system against foreign nucleic acids. Protection starts with an immunization phase, in which short pieces of the invader's genome, known as spacers, are captured and integrated into the CRISPR locus after infection. Next, during the targeting phase, spacers are transcribed into CRISPR RNAs (crRNAs) that guide CRISPR-associated (Cas) nucleases to destroy the invader's DNA or RNA. Here we describe the many different molecular mechanisms of CRISPR targeting and how they are interconnected with the immunization phase through a third phase of the CRISPR-Cas immune response: primed spacer acquisition. In this phase, Cas proteins direct the crRNA-guided acquisition of additional spacers to achieve a more rapid and robust immunization of the population.

Integration of prophages into CRISPR loci remodels viral immunity in Streptococcus pyogenes

SummaryCRISPR loci are composed of short DNA repeats separated by sequences that match the genomes of phages and plasmids, known as spacers. Spacers are transcribed and processed to generate RNA guides used by CRISPR-associated nucleases to recognize and destroy the complementary nucleic acids of invaders. To counteract this defense, phages can produce small proteins that inhibit these nucleases. Here we demonstrate that the ΦAP1.1 temperate phage utilizes an alternate approach to antagonize the type II-A CRISPR response in Streptococcus pyogenes. Immediately after infection this phage expresses a canonical anti-CRISPR, AcrIIA23 that prevents Cas9 function, allowing ΦAP1.1 to integrate into the direct repeats of the CRISPR locus and neutralizing immunity. However, acrIIA23 is not transcribed during lysogeny and phage integration/excision cycles can result in the deletion and/or transfer of spacers, enabling a complex modulation of the type II-A CRISPR immune response.