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Efficient Human Genome Editing Using SaCas9 Ribonucleoprotein Complexes.

Yufei Wang1,2, Bang Wang1,2, Haihua Xie1,2

  • 1School of Ophthalmology and Optometry, Eye Hospital, State Key Laboratory of Ophthalmology, Optometry and Vision Science, Wenzhou Medical University, 270 Xueyuan West Road, Wenzhou, Zhejiang, 325027, China.

Biotechnology Journal
|March 31, 2019
PubMed
Summary
This summary is machine-generated.

This study demonstrates Staphylococcus aureus Cas9 nuclease (SaCas9) ribonucleoprotein (RNP) genome editing in human cells. SaCas9-RNP efficiently edits genes and corrects mutations, showing potential for gene therapy with no detectable off-target effects.

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Biotechnology

Background:

  • Ribonucleoprotein (RNP) form of RNA-guided nucleases offers advantages for gene editing, including reduced toxicity and no exogenous DNA integration.
  • Staphylococcus aureus Cas9 (SaCas9) nuclease has a distinct protospacer adjacent motif (PAM) requirement (5'-NNGRRT-3') compared to Streptococcus pyogenes Cas9 (SpCas9) (5'-NGG-3').
  • SaCas9 in RNP form has not been previously reported for genome editing in human cells.

Purpose of the Study:

  • To investigate and report SaCas9 nuclease genome editing using the ribonucleoprotein (RNP) complex in human cells.
  • To evaluate the efficiency of SaCas9-RNP in editing both reporter genes and endogenous human genes.
  • To assess the potential of SaCas9-RNP for therapeutic applications, including the correction of genetic mutations.

Main Methods:

  • Formation of SaCas9-RNP complexes for delivery into human cells.
  • Genome editing assays targeting the enhanced green fluorescent protein (EGFP) gene.
  • Targeted genome editing of endogenous human genes: OPA1, RS1, and VEGFA.
  • Assessment of SaCas9-RNP for correcting a specific pathogenic mutation in the RS1 gene.
  • Analysis of off-target effects using targeted deep sequencing.

Main Results:

  • SaCas9-RNP demonstrated efficient genome editing activity in human cells.
  • Editing was observed in the EGFP coding gene and the endogenous OPA1, RS1, and VEGFA genes.
  • SaCas9-RNP successfully corrected a pathogenic mutation associated with X-linked juvenile retinoschisis (RS1).
  • No detectable off-target mutations were identified by targeted deep sequencing.

Conclusions:

  • SaCas9-RNP is an effective tool for genome editing in human cells.
  • This approach holds promise for therapeutic applications, including the correction of genetic disorders like X-linked juvenile retinoschisis.
  • The lack of detectable off-target effects suggests a favorable safety profile for SaCas9-RNP-mediated gene editing.