Application of CRISPR/Cas9 Genome Editing in Genetic Disorders: A Systematic Review Up to Date

Pandey, Vivek; Tripathi, Anima; Bhushan, Ravi; Ali, Akhtar; Dubey, Pawan K.

doi:10.4172/2157-7412.1000321

Cited by 15 publications

(15 citation statements)

References 68 publications

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“…Now it is reported from a large number of eubacteria and almost in all the archaebacteria as well [2][3][4][5]. CRISPR-Cas9 system also holds great promise in stem cell and tissue engineering, translational medicine, next-generation gene therapy and engineering trait enhancement and diseaseresistant plants [6,7].…”

Section: Introductionmentioning

confidence: 99%

Analyses of Homing Endonucleases and Mechanism of Action of CRISPR-Cas9 HNH Endonucleases

Palanivelu

2020

IJBcRR

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Aim: To analyze different HNH endonucleases from various sources including the HNH endonuclease regions of CRISPR-Cas9 proteins for their conserved motifs, metal-binding sites and catalytic amino acids and propose a plausible mechanism of action for HNH endonucleases, using CRISPR-Cas9 as the model enzyme. Study Design: Multiple sequence analysis (MSA) of homing endonucleases including the CRISPR-Cas9 using Clustal Omega was studied. Other biochemical, Site-directed mutagenesis (SDM) and X-ray crystallographic data were also analyzed. Place and Duration of Study: School of Biotechnology, Madurai Kamaraj University, Madurai, India, between 2007 and 2013. Methodology: Bioinformatics, Biochemical, SDM and X-ray crystallographic data of the HNH endonucleases from different organisms including CRISPR-Cas9 enzymes were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of different HNH endonucleases from various sources. The conserved motifs identified by the bioinformatics analysis were analyzed further with the data already available from biochemical and SDM and X-ray crystallographic analyses of this group of enzymes and to confirm the possible amino acids involved in the active sites and catalysis. Results: Different types of homing endonucleases from various sources including the HNH endonuclease regions of CRISPR-Cas9 enzymes exhibit different catalytic regions and metal-binding sites. However, the catalytic amino acid, i.e., the proton acceptor histidine (His), is completely conserved in all homing endonucleases analyzed. From these data, a plausible mechanism of action for HNH endonucleases, using CRISPR-Cas9 from Streptococcus pyogenes, as the model enzyme is proposed. Furthermore, multiple sequence alignment (MSA) of various homing endonucleases from different organisms showed many highly conserved motifs also among them. However, some of the HNH endonucleases showed consensus only around the active site regions. Possible catalytic amino acids identified among them belong to either -DH---N or -HH--N types. There are at least two types of metal-binding sites and bind Mg2+ or Zn2+ or both. The CRISPR-Cas9 enzyme from S. pyogenes belongs to the -DH- based HNH endonucleases and possesses –DxD- type metal-binding site where it possibly binds to a Mg2+ ion. The other HNH enzymes possess one or two invariant Zn binding CxxC/ CxxxC motifs. Conclusions: The CRISPR-Cas9 enzymes are found to be -DH- type where the first D is likely to involve in metal-binding and the second invariant H acts as the proton acceptor and the N in –HNH- Cas9 confers specificity by interacting with the nucleotide near the catalytic region. In this communication, a metal-bound water molecule is shown as the nucleophile initiating catalysis. Homing endonucleases may be used as novel DNA binding and cleaving reagents for a variety of genome editing applications and Zinc finger nucleases have already found applications in genome editing.

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Section: Introductionmentioning

confidence: 99%

Analyses of Homing Endonucleases and Mechanism of Action of CRISPR-Cas9 HNH Endonucleases

Palanivelu

2020

IJBcRR

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“…Several molecular tools that introduce DNA double-strand breaks (DSBs) at specific sites have been developed and utilized in biomedical research [4]. Of these, a clustered regularly interspaced short palindromic repeat-associated 9 (CRISPR/Cas9) system, which is able to achieve highly flexible and specific targeting, has superior advantages and represents a powerful tool for potential clinical application.…”

Section: Introductionmentioning

confidence: 99%

“…The DSB subsequently is repaired by either nonhomologous end joining (NHEJ) or homology-directed repair (HDR). During this DSB repair process, pathogenic mutations can be replaced with a donor DNA homologous template containing a corrected gene sequence [4].…”

Section: Introductionmentioning

confidence: 99%

Induced Pluripotent Stem Cell Derivation and Ex Vivo Gene Correction Using a Mucopolysaccharidosis Type 1 Disease Mouse Model

Miki

Vazquez

Yanuaria

et al. 2019

Stem Cells International

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Mucopolysaccharidosis type 1 (MPS-1), also known as Hurler’s disease, is a congenital metabolic disorder caused by a mutation in the alpha-L-iduronidase (IDUA) gene, which results in the loss of lysosomal enzyme function for the degradation of glycosaminoglycans. Here, we demonstrate the proof of concept of ex vivo gene editing therapy using induced pluripotent stem cell (iPSC) and CRISPR/Cas9 technologies with MPS-1 model mouse cell. Disease-affected iPSCs were generated from Idua knockout mouse embryonic fibroblasts, which carry a disrupting neomycin-resistant gene cassette (Neor) in exon VI of the Idua gene. Double guide RNAs were used to remove the Neor sequence, and various lengths of donor templates were used to reconstruct the exon VI sequence. A quantitative PCR-based screening method was used to identify Neor removal. The sequence restoration without any indel mutation was further confirmed by Sanger sequencing. After induced fibroblast differentiation, the gene-corrected iPSC-derived fibroblasts demonstrated Idua function equivalent to the wild-type iPSC-derived fibroblasts. The Idua-deficient cells were competent to be reprogrammed to iPSCs, and pluripotency was maintained through CRISPR/CAS9-mediated gene correction. These results support the concept of ex vivo gene editing therapy using iPSC and CRISPR/Cas9 technologies for MPS-1 patients.

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“…In addition to these infectious diseases, hereditary diseases are congenital and show peculiar phenotypic characters 4 . These diseases are consequent of genome alteration by inducing mutations by chemical, physical or biological agents such as viruses, bacteria or fungi 5 .…”

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confidence: 99%

CRISPR: A new paradigm of theranostics

Yadav

Narang

Chhillar

et al. 2021

Nanomedicine: Nanotechnology, Biology and Medicine

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Application of CRISPR/Cas9 Genome Editing in Genetic Disorders: A Systematic Review Up to Date

Cited by 15 publications

References 68 publications

Analyses of Homing Endonucleases and Mechanism of Action of CRISPR-Cas9 HNH Endonucleases

Analyses of Homing Endonucleases and Mechanism of Action of CRISPR-Cas9 HNH Endonucleases

Induced Pluripotent Stem Cell Derivation and Ex Vivo Gene Correction Using a Mucopolysaccharidosis Type 1 Disease Mouse Model

CRISPR: A new paradigm of theranostics

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