Genome engineering has revolutionized our ability to understand and treat human diseases. This guide explores the landscape of programmable nucleases, focusing on the widely used CRISPR/Cas9 system and emerging alternatives like NgAgo. This technology holds immense promise for both basic research and therapeutic applications.
With rapid progress in genome sciences, effective genome engineering offers exciting possibilities for understanding the molecular basis of human diseases and developing treatments for disorders involving identifiable genomic alterations. The development of zinc-finger nuclease-based TALEN (transcription activator-like effector nucleases) technology marked an early step, but the search for simpler and more effective genome manipulation approaches continued. The CRISPR/Cas9 system quickly rose to prominence. CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are prokaryotic DNA segments with short sequence repetitions. The bacterial endonuclease Cas9 is programmed by a small guide RNA (gRNA) to induce a double-strand break at a specific genomic location followed by a protospacer-adjacent motif (PAM).
Alt: Illustration of the CRISPR-Cas9 system cleaving DNA at a targeted site, highlighting the gRNA and PAM sequence.
The resulting double-strand break can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ), which often leads to small insertions or deletions (indels). This technology has been extensively used for rapid gene knockouts in various organisms. Ongoing efforts focus on optimizing the CRISPR/Cas9 system and discovering Cas9-like nucleases with improved efficiency and specificity.
NgAgo: A Novel Alternative for Genome Editing
A more recent discovery may be poised to change the genome editing field. Gao F., et al. identified a member of the Argonaute endonuclease family, Natronobacterium gregoryi Argonaute (NgAgo), which effectively performs DNA-guided genome editing in mammalian cells. Argonautes are a family of endonucleases that use 5′-phosphorylated short single-stranded nucleic acids to guide the cleavage of target DNA. Argonaute proteins are critical in RNA interference and microRNA processing. Several Argonautes from thermophiles use single-stranded (ss) DNA guides to cleave DNA targets at high temperatures.
Gao F., et al., showed that the NgAgo endonuclease binds 5′-phosphorylated single-stranded guide DNA (gDNA) of approximately 24 nucleotides and efficiently generates gDNA sequence-specific DNA double-stranded breaks at 37 °C. Importantly, this editing process does not require a PAM sequence. NgAgo removes several nucleotides in the target region, although it lacks exonuclease activity. Testing 47 different ssDNA guides targeting eight human genes, the authors reported targeting efficiencies ranging from 21% to 41%. The NgAgo–gDNA system displayed low tolerance for guide–target mismatches; mismatches between the ssDNA guide and target reduced cleavage efficiency. A comprehensive analysis of single mismatches at all 24 positions revealed that all positions were crucial, particularly positions 8–11, and that three consecutive mismatches abolished activity. Unlike CRISPR/Cas9, NgAgo efficiently edits almost any sequence, including G/C-rich genomic sequences.
Alt: Diagram illustrating NgAgo using a single-stranded DNA guide to target and cleave DNA without the need for a PAM sequence.
CRISPR/Cas9 vs. NgAgo: Key Distinctions
While still in early development, NgAgo presents several potential advantages over CRISPR/Cas9:
- NgAgo utilizes 5′-phosphorylated ssDNA guides (∼24 bp) to cleave supercoiled DNA.
- Mammalian cells have very low levels of 5′P-ssDNA, reducing the potential for off-target guides.
- NgAgo exhibits higher specificity than Cas9, losing activity with only three mismatches.
- NgAgo’s DNA cleavage effectiveness in mammalian cells is comparable to or even surpasses that of Cas9.
- NgAgo is a smaller protein, facilitating easier expression.
- NgAgo does not require a PAM sequence, enabling targeting of GC-rich genomic loci with high efficiency.
Future Directions and Applications
Further research is needed to fully understand NgAgo’s potential applications. The use of 5′-phosphorylated ssDNA as a guide simplifies in vitro cell-based genome editing, but may limit in vivo utility unless effective and targeted ssDNA guide delivery methods are developed. Whether NgAgo can effectively mediate homology-directed recombination with similar or better efficiency than CRISPR/Cas9 requires further investigation. Structure-function studies of NgAgo may also enhance its genome editing activity. The discovery of additional NgAgo-like proteins is also conceivable.
Conclusion
Regardless of whether NgAgo surpasses CRISPR/Cas9, the scientific community welcomes another valuable tool in the genome engineering field. The continued development of programmable nucleases like CRISPR/Cas9 and NgAgo will further advance our understanding of gene function, disease mechanisms, and therapeutic interventions.
