CRISPR-Cas in <i>Streptococcus pyogenes</i>

Rhun, Anaïs Le; Escalera-Maurer, Andrés; Bratovič, Majda; Charpentier, Emmanuelle

doi:10.1080/15476286.2019.1582974

Cited by 101 publications

(71 citation statements)

References 127 publications

(168 reference statements)

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“…The CRISPR/Cas system is composed of functionally modular sgRNAs and Cas proteins, which can recognize target nucleotide sequences and degrade target DNAs or RNAs. The CRISPR/Cas9 system derived from Streptococcus pyogenes, selected from among diverse CRISPR systems, is commonly used as a genome editing tool, because it contains a simple complex composed of a single Cas9 polypeptide and sgRNA, which causes the double strand breakage (DSB) of target DNAs (Makarova et al 2015;Makarova et al 2018;Le Rhun et al 2019).…”

Section: Introductionmentioning

confidence: 99%

CRISPR-Cas9-mediated pinpoint microbial genome editing aided by target-mismatched sgRNAs

2020

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Genome editing has been revolutionized by the CRISPR/Cas9 system. CRISPR/Cas9 is composed of single molecular guide RNA (sgRNA) and a proteinaceous Cas9 nuclease, which recognizes a specific target sequence and a PAM (protospacer adjacent motif) sequence and, subsequently, cleaves the targeted DNA sequence. This CRISPR/Cas9 system has been used as an efficient negative-selection tool to cleave unedited or unchanged target DNAs during site-specific mutagenesis and, consequently, obtain microbial cells with desired mutations. This study aimed to investigate the genome editing efficiency of the CRISPR/Cas9 system for in vivo oligonucleotide-directed mutagenesis in bacteria. This system successfully introduced 2-to 4-base mutations in galK in E. coli with high editing efficiencies (8186%). However, single point mutations (T504A or C578A) were rarely introduced with very low editing efficiencies (<3%), probably owing to mismatch tolerance. To resolve this issue, we designed 1-or 2-base mismatches in the sgRNA sequence to recognize target sequences in galK in E. coli. A single point nucleotide mutation (T504A or C578A in the galK gene) was successfully introduced in 3695% of negatively selected E. coli cells on using single base-mismatched sgRNAs. Sixteen targets were randomly selected through genome-wide single-base editing experiments using mismatched sgRNAs. Consequently, out of 48 desired single base mutations, 25 single bases were successfully edited, using mismatched sgRNAs. Finally, applicable design rules for target-mismatched sgRNAs were provided for single-nucleotide editing in microbial genomes.

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Section: Introductionmentioning

confidence: 99%

CRISPR-Cas9-mediated pinpoint microbial genome editing aided by target-mismatched sgRNAs

2020

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“…150 As a result, when the two conditions of sgRNA-targeting PAM and correct base pair complementary are satisfied, the sgRNA:Cas9 complex will be able to discontinue the scanning process in order to induce a DNA-DSB at the proper position. 137,143 Thereupon, similarly to ZFNs and TALENs approaches, Cas9-generated sitespecific DNA-DSBs are later repaired by homologous directed repair (HDR) if the homologous sequences are available or otherwise by non-homologous endjoining (NHEJ), depending on the therapeutical purpose. 92,97,102 Particularly in HIV treatment, the rationale for gRNA selection must consider having a strict homology to the CCR5-gene ORF in order to successfully lead to predictable Cas9-DSB and subsequent cellular repair mechanisms on the target site, which can therefore promote a frameshift mutation in the CCR5 sequence and thereby result in complete gene disruption.…”

Section: Efficient Nuclease Delivery and Expressionmentioning

confidence: 99%

“…For example, new types of Cas9 from differing species have been studied and revealed increased specificity without compromising the on-target effectivity, when compared to SpCas9. 143,161 As example, scientists emphasized the Cas9 protein from Staphylococcus aureus (SaCas9), Neisseria meningitidis (NmCas9) and Streptococcus thermophilus (St1Cas9), which all interact with variable PAM sequences and then lead to distinct gene-editing activity. 128 Specific Cas9 ortholog selection may guarantee value-added gene editing efficiency and so should be considered as part of a gene editing system design.…”

Section: Development Of Cas9 Variantsmentioning

confidence: 99%

“…On the other hand, type II is the simplest among the CRISPR/Cas systems and only requires Cas9 gene to obtain a single endonuclease for the interference with foreign and target DNA 141,142. Therefore, Cas9 gene, the CRISPR/Cas type II signature gene, is originated from Streptococcus pyogenes and codifies for the multidomain protein Cas9,143 which encompasses both the HNH and RuvC nuclease domains: the HNH domain is a single domain that cleaves the DNA-complementary strand, while the RuvC domain involves three subdomains that, oppositely, are responsible for the noncomplementary DNA-strand cleavage in the target site.…”

mentioning

confidence: 99%

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Designer Nucleases: Gene-Editing Therapies using CCR5 as an Emerging Target in HIV

Almeida

Matos

2019

CHR

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Acquired Immunodeficiency Syndrome (AIDS), caused by the Human Immunodeficiency Virus (HIV), is a life-threatening disorder that persists worldwide as a severe health problem. Since it was linked with the HIV attachment process, the Chemokine receptor, CCR5, has been at the development leading edge of several gene-based therapies. Given the shortcomings of the current antiretroviral treatment procedure and the non-availability of a licensed vaccine, the aptitude to modify complex genomes with Designer Nucleases has had a noteworthy impact on biotechnology. Over the last years, ZFN, TALEN and CRISPR/Cas9 gene-editing technology have appeared as a promising solution that mimics the naturally occurring CCR5/Δ32 mutation and permanently guarantees the absence of CCR5-expression on the surface of HIV target-cells, leading to a continuous resistance to the virus entry and, ultimately, proving that cellular immunization from infection could be, in fact, a conceivable therapeutic approach to finally achieve the long-awaited functional cure of HIV.

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“…Class 2 systems: connected to pathogenicity and a treasure trove for genome-editing tools A comprehensive overview of the type II-A and I-C systems from the pathogenic Streptococcus pyogenes is given in the manuscript of Le Rhun et al [11]. This paper summarises the current knowledge about the biochemical and functional features of these systems and highlights the biotechnological applications developed for the S. pyogenes type II-A system, which encodes the tracrRNA (trans-activating crRNA):crRNA-Cas9 enzymatic complex that is commonly used for genome editing tools in a large variety of applications.…”

Section: Guest Editorialmentioning

confidence: 99%