Systematic high-throughput evaluation reveals FrCas9's superior specificity and efficiency for therapeutic genome...

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Biotechnology

Background:

• CRISPR-Cas9 technology has transformed genome editing but faces limitations due to off-target effects.
• Clinical applications of CRISPR are hindered by the need for highly specific and safe editing tools.

Purpose of the Study:

• To systematically evaluate the performance of FrCas9, a CRISPR variant from *Faecalibaculum rodentium*, for cell and gene therapy (CGT).
• To compare FrCas9's on-target activity and off-target effects against SpCas9 and OpenCRISPR-1, a novel LLM-synthesized CRISPR system.
• To assess the impact of TREX2 fusion with FrCas9 on genomic stability.

Main Methods:

• Utilized Assays for *in vivo* DNA Editing (AID-seq), Amplicon sequencing, and Genome-wide Unbiased Identification of DSBs Enabled by Sequencing (GUIDE-seq) to evaluate editing outcomes.
• Assessed on-target efficiency and off-target profiles across multiple genomic loci.
• Screened 1903 single-guide RNAs (sgRNAs) targeting 21 CGT-relevant genes using a sequential high-throughput approach.

Main Results:

• FrCas9 exhibited significantly higher on-target efficiency and markedly reduced off-target effects compared to SpCas9 and OpenCRISPR-1.
• TREX2 fusion with FrCas9 effectively minimized large deletions and translocations, thereby improving genomic stability.
• Identified optimal sgRNAs for 21 CGT-relevant genes through comprehensive screening.

Conclusions:

• FrCas9, especially when fused with TREX2, represents a highly specific and efficient genome editing tool for precise therapeutic applications.
• This study provides a robust high-throughput screening platform for identifying superior CRISPR systems and sgRNAs for CGT.
• FrCas9 demonstrates significant potential to advance the safety and efficacy of genome editing in clinical settings.