Advancements in CRISPR/Cas9 Technology

Introduction to CRISPR/Cas9

Hu and colleagues have reported significant enhancements to the CRISPR/Cas9 technology, which facilitates direct genome editing in living cells. Traditionally, researchers seeking to investigate the functions of specific genes faced lengthy processes involving cross-breeding and mutation experiments to isolate various gene variants. Fortunately, technological advancements in the past decade have led to the emergence of the CRISPR system, a powerful tool enabling researchers to insert or remove DNA sequences directly within a genome.

Mechanism of CRISPR/Cas9

CRISPR/Cas9 operates by cleaving the DNA strand at predetermined locations marked by a PAM (Protospacer Adjacent Motif) sequence, a short motif consisting of 2-6 base pairs that corresponds with the Cas9 sequence. However, the infrequency of PAM sequences within the genome poses a limitation on the effectiveness of CRISPR/Cas9 for editing regions lacking the necessary PAM sequences.

New Methodology for PAM Recognition

In a recent publication in *Nature*, Hu and colleagues introduce a novel methodology that enhances CRISPR/Cas9’s ability to recognize a broader spectrum of PAM sequences, thereby expanding the range of the genome available for editing. They employed a technique known as phage-assisted continuous evolution (PACE) to create new variants of Cas9 capable of identifying these additional PAM sequences.

Understanding Phage-Assisted Continuous Evolution (PACE)

PACE involves the use of specialized viruses, called phages, which are housed within E. coli cells. These phages carry a diverse array of Cas9 sequences. Variants of Cas9 that demonstrate a broader recognition of PAM sequences are more likely to survive within the E. coli environment, allowing evolution to occur without direct intervention from researchers.

Development of xCas9 Variants

Utilizing a modified PACE approach, Hu and colleagues successfully generated several Cas9 variants known as ‘xCas9’. These new variants exhibit enhanced binding effectiveness to PAM sequences in human cells, showing improvements ranging from 1.5 to 8 times greater than those of the original Cas9.

Implications for Research and Medicine

The introduction of the xCas9 variants by Hu and colleagues presents new opportunities for researchers to apply CRISPR technology across a wider array of applications, including medical treatments. These variants can enhance gene therapy precision, enabling researchers and clinicians to more accurately target and modify genes in human patients. Furthermore, xCas9 will broaden the scope of CRISPR applications in model organisms such as mice, and facilitate the induction of specific mutations in crucial agricultural species like corn and soybeans.

Conclusion

The advancements in CRISPR/Cas9 technology, particularly through the development of xCas9 variants, mark a significant step forward in genetic research and therapeutic applications.

Reference

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