Recent advancements in genome editing have pushed the boundaries of possibilities in therapeutics and diagnostics. At the forefront of this innovation is developing novel CRISPR-Cas9 variants engineered to achieve enhanced precision and range of targets within the genome. While Streptococcus pyogenes Cas9 (SpCas9) has been instrumental in genome editing, its superior cleavage activity comes at the cost of limited cellular targeting efficiency due to strict PAM requirements and many off-targets.
A recent article published by Dr Debojyoti Chakraborty’s lab at CSIR-IGIB in Nature Communications, titled “PAM-flexible Engineered FnCas9 Variants for Robust and Ultra-Precise Genome Editing and Diagnostics,” showcases pioneering research focused on enhancing the capabilities of the Francisella novicida Cas9 (FnCas9) protein. This study has produced variants of FnCas9 that offer superior performance, addressing key limitations of existing genome editing tools while maintaining the high specificity necessary for clinical applications.
Rational engineering of FnCas9 by modifying its WED-PI domain and phosphate-lock loop (PLL), led to the creation of 49 different variants with single amino acid substitutions, all of which were tested for DNA cleavage activities. Among these, three kinetically enhanced, PAM-flexible enFnCas9 variants — en1, en15, and en31 — were identified and characterised. These variants demonstrated significantly higher cleavage rates than wild-type FnCas9 and maintained the intrinsic DNA interrogation specificity of the original protein.
The engineered FnCas9 variants (enFnCas9) proved PAM-flexible and highly specific, expanding their utility in genome editing and diagnostics. These variants exhibited increased flexibility in recognising non-canonical PAMs while preserving high specificity, effectively expanding the PAM recognition landscape from 5’-NGG-3’ to 5’-NRG/NGR-3’. This expansion increased the accessibility across the human genome by approximately 3.5 times, marking a significant advancement in genome editing technology.
In the context of CRISPR diagnostics (CRISPRDx), the enFnCas9 variants were tested on platforms like FELUDA and RAY, demonstrating a two-fold increase in the coverage of Mendelian SNVs across the human genome. Furthermore, these variants exhibited improved specificity and signal discrimination in diagnostic assays for diseases such as Sickle Cell Anemia and the SARS-CoV-2 Alpha variant.
The en1 variant, in particular, showed higher editing rates than SpCas9-HF1 and eSpCas9, with no detectable off-target effects. This variant demonstrated robust editing at non-canonical PAM sites in therapeutically relevant loci, including those associated with Sickle Cell Anemia. Additionally, the engineered variants outperformed SpCas9 in Homology-directed Repair (HDR)-mediated knock-in assays, highlighting their potential for therapeutic genome editing.
The en31 variant showcased its ability to perform precise base editing at specific loci in the human genome. This was further validated in a proof-of-concept experiment for therapeutic base editing using en31-ABEmax8.17d in patient-specific induced Pluripotent Stem Cells (iPSCs) derived from a Leber Congenital Amaurosis (LCA2) patient. The study concluded by demonstrating the restoration of full-length protein expression in patient-specific iPSC-derived retinal pigmented epithelium using the en31-ABEmax8.17d variant, successfully correcting a point mutation in the RPE65 gene.
Overall, the engineered FnCas9 variants represent a significant leap forward in genome editing, offering enhanced precision, flexibility, and potential for therapeutic applications. With a growing need to address genetic disorders affecting millions across India, these enhanced gene editors could revolutionise treatment approaches, offering highly precise and efficient methods for editing disease-causing genes.
