A CRISPR/Cas9 Tutorial

This post comes from Fred Nelson, MD, an orthopaedic surgeon in the Department of Orthopedics at Henry Ford Hospital and a clinical associate professor at Wayne State Medical School. Some of Dr. Nelson’s tips go out weekly to more than 3,000 members of the Orthopaedic Research Society (ORS), and all are distributed to more than 30 orthopaedic residency programs. Those not sent to the ORS are periodically reposted in OrthoBuzz with the permission of Dr. Nelson.

Understanding recent gene technology can be very daunting. The CRISPR/Cas9 method for gene editing is a prominent example. CRISPR stands for Clustered Regularly Interspersed Short Palindromic Repeats, and Cas9 is an acronym for the CRISPER-Associated Protein 9. Scientists became aware of CRISPR in E. coli in 1987, but they only recently realized that CRISPR constituted an adaptive immune system for bacteria and archae, which are primitive bacteria-like cells.

When infected by a virus (phage), a bacteria’s Cas genes are activated. Cas gene products cut viral DNA sequence sites called protospacers and then insert those sequences into the bacterial DNA. The host bacterium identifies the viral sequences by a protospacer adjacent motif (PAM), which is rarely seen in the host genome. Hence, replication of this sequence will not adversely affect the host. In the event of a second phage attack, Cas genes are activated and they generate CRISPR RNA (crRNA), which recognizes the phage sequence.  crRNA associates with Cas nucleases to cleave both DNA strands of the invader.

There are numerous CRISPR modules. Type II CRISPR is one of an expanding number of naturally existing CRISPR families that has have been used for gene editing in eukaryotes. The type II CRISPR family uses crRNA and an additional tracrRNA to target specific DNA sequences. These have been combined to create a single guide RNA (gRNA) to direct sequence-specific Cas9 double-stranded DNA cleavage. The result is a simple, programmable RNA method that has been used for genome targeting and genome editing in eukaryotes.

The accuracy of this system has been markedly enhanced to avoid unwanted mutations. The system is being fashioned to block existing gene expression, modify gene expression by inserting DNA sequences, and activate expression of single or multiple genes. CRISPR technology enables researchers to develop mouse models of disease much more quickly and less expensively than traditional approaches. Larger animal models of disease can also now be produced.

Successful treatment of mouse models of human diseases with CRISPR suggests that the technology can be applied to directly treat human diseases in the future. Preclinical research is underway using CRISPR-ed stem cells or mouse models to study human diseases such as retinitis pigmentosa, Fanconi anemia, Duchenne muscular dystrophy, sickle-cell anemia, and cystic fibrosis.

Thanks to Dr. Gary Gibson for his help with this tip.

Reference

Gibson GJ, Yang M. What rheumatologists need to know about CRISPR/Cas9. Nat Rev Rheumatol. 2017 Apr;13(4):205-216. doi: 10.1038/nrrheum.2017.6. Epub 2017 Feb 9.

 

 

Tags: , ,

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out /  Change )

Google photo

You are commenting using your Google account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s

%d bloggers like this: