Superior Fidelity and Distinct Editing Outcomes of SaCas9 Compared with SpCas9 in Genome Editing

Biochemistry Genetics Bioengineering Hematology Life Sciences Medicine Cell Biology
genome editing sacas9 spcas9 PAM sequence insertion efficiency off-target effects

Comparison of SaCas9 and SpCas9 in High-Fidelity and Different Gene Editing Outcomes

Research Background

CRISPR systems based on Cas9 protein have become powerful tools for genome editing, widely used in basic research and clinical gene therapy. Currently, the most commonly used Cas9 variants are SpCas9 from Streptococcus pyogenes and SaCas9 from Staphylococcus aureus. Although these two Cas9 proteins have been extensively validated in many studies, a systematic comparison of their specific gene editing effects is still lacking. Therefore, this paper aims to systematically compare the efficiency and accuracy of SpCas9 and SaCas9 in gene editing, and explore their editing performance under different cleavage lengths.

Research Source

This paper was written by researchers from several research institutions, including the Institute of Hematology, Chinese Academy of Medical Sciences, including Zhi-Xue Yang, Ya-Wen Fu, Jian-Ping Zhang, etc. The research results were published in the journal “Genomics, Proteomics & Bioinformatics” in 2023.

Research Process

The research process can be divided into the following steps:

  1. Research Design and Genomic Target Sequence Selection:

    • Using ChopChop tool to design sgRNA sequences targeting human genes such as AAVS1, ALB, B2M1
    • Identifying PAM sequences (NGGRRT) recognized by both Staphylococcus aureus Cas9 (SaCas9) and Streptococcus pyogenes Cas9 (SpCas9)

  2. Cell Culture and Electroporation:

    • The study included human induced pluripotent stem cells (iPSCs) and K562 leukemia cells, both of which underwent standardized culture and electroporation
    • Using the Lonza 2b system for electroporation to introduce Cas9-sgRNA plasmids into cells

  3. Gene Editing Efficiency Evaluation:

    • Amplifying target sequences through primary and secondary PCR, followed by Illumina deep sequencing
    • Using CRISPResso2 tool to analyze editing frequency, HDR efficiency, and dsODN insertion rate

  4. High-throughput Sequencing and Data Analysis:

    • Utilizing Guide-seq analysis to assess genome-wide specificity detection
    • Conducting precise analysis of Cas9-induced double-strand break repair results, focusing on NHEJ, MMEJ, and HDR repair mechanisms

Research Results

Editing Efficiency of SpCas9 and SaCas9 at Different Spacer Lengths

The study shows:

  1. Optimal Spacer Length for SpCas9 is 20 nt, with 18, 19, and 21 nt spacer lengths showing higher activity in a few cases (such as AAVS1c and B2M1).

  2. SaCas9 is More Sensitive to Spacer Length changes, with the most effective spacer length being 21-22 nt. Editing efficiency significantly decreases when spacer length reaches 23 nt.

Differences in Gene Editing Results and Repair Types

  1. Insertion-Deletion Frequency (Indel):

    • In iPSCs and K562 cells, SaCas9 showed higher Indel frequencies at most target sites.

  2. NHEJ Repair Characteristics:

    • SaCas9 produced about 10 times fewer +1 insertion mutations (especially +T) under NHEJ repair compared to SpCas9, reducing mutations caused by Staggered Cut. This provides better conditions for DSODN insertion and HDR editing.

  3. HDR Efficiency and Insertion Efficiency:

    • On average, SaCas9 showed significantly higher editing efficiency in gene insertion (e.g., DSODN) and HDR (e.g., AAV6-donor) compared to SpCas9.

Off-target Effects

  • Through Guide-seq analysis, SaCas9 showed significantly fewer total off-target sites in iPSCs and K562 cells compared to SpCas9, indicating higher specificity of SaCas9 in in vitro gene editing. SaCas9 editing using 22-nt spacers showed lower off-target activity.

Conclusion

The research results of this paper demonstrate the superiority of SaCas9 over SpCas9 in gene editing in multiple aspects. Particularly in terms of Staggered Cut preference, NHEJ repair outcomes, and off-target effects, SaCas9 demonstrated higher editing efficiency and accuracy. This study provides important theoretical support and application guidance for tool selection and use in gene editing, especially in clinical gene therapy.

Research Highlights

  • Significantly Lower Off-target Effects: High-throughput sequencing analysis through Guide-seq showed that the SaCas9 system has significantly reduced off-target risks in gene editing compared to SpCas9.
  • Methods to Improve Editing Efficiency: This paper designed and validated an optimized Cas9 fusion protein, significantly enhancing gene editing efficiency by using NLS and HMGA2 elements.
  • Detailed Experimental Design and Analysis: The paper conducted a detailed assessment of the knockout efficiency and repair mechanisms of Cas9-sgRNA at different spacer lengths, providing important references for future gene editing experimental designs.

Research Significance

This study systematically compared the performance of SpCas9 and SaCas9 in gene editing, which is of great significance for the development of precise gene editing and gene therapy. The SaCas9 protein is highly specific and prefers to generate blunt-end termini, making it more advantageous in clinical gene therapy applications, especially in some treatment areas sensitive to off-target effects.