Advancements in CRISPR/Cas9 Technology

Introduction to CRISPR/Cas9

Hu and colleagues have reported significant enhancements to CRISPR/Cas9, a groundbreaking technology that facilitates direct genome editing in living cells. Historically, researchers had to conduct extensive cross-breeding and mutation experiments to study specific genes, which was a time-consuming process. However, advancements over the past decade have led to the development of the CRISPR system, providing a more efficient approach to genetic research.

Mechanism of CRISPR/Cas9

CRISPR/Cas9 operates by cutting the DNA strand at designated locations identified by a protospacer adjacent motif (PAM) sequence—a short 2-6 base-pair motif that aligns with the Cas9 sequence. Despite its effectiveness, the occurrence of PAM sequences in the genome is relatively rare, which restricts the application of CRISPR/Cas9 for editing regions lacking these sequences.

New Methodology Introduced

In a recent publication in *Nature*, Hu and colleagues introduced a novel method that expands the range of PAM sequences recognized by CRISPR/Cas9, thereby increasing the proportion of the genome that can be edited. This method, known as phage-assisted continuous evolution (PACE), enables the generation of new Cas9 variants capable of recognizing a broader array of PAM sequences.

Understanding PACE

PACE involves the use of specialized viruses, or phages, hosted within E. coli cells. These phages harbor various Cas9 sequences, and those that exhibit a wider range of PAM matches are more likely to survive within the E. coli environment. This process allows evolution to occur autonomously, without direct intervention from researchers.

Development of xCas9 Variants

Through a modified PACE approach, Hu and colleagues successfully produced several Cas9 variants, referred to as ‘xCas9’. These new variants demonstrate an effectiveness of binding to PAM sequences in human cells that is 1.5 to 8 times greater than that of the original Cas9 versions.

Implications for Research and Medicine

The newly developed xCas9 variants hold the potential to broaden the applications of CRISPR technology across various fields, particularly in medical treatments. These variants enable researchers and healthcare professionals to more precisely identify and modify genes in gene therapies for human applications. Additionally, xCas9 could facilitate the exploration of diverse research questions in model organisms like mice and allow for targeted mutations in significant agricultural crops such as corn and soybeans.

Conclusion

The work of Hu and colleagues marks a notable advancement in genome editing technology, paving the way for enhanced precision and versatility in genetic research and applications.

Reference

JH Hu, SM Miller, MH Geurts, et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. *Nature*, 2018.