GENOME HOMING and GENOME ENGINEERING

Many naturally occurring TRANSCRIPTION FACTORS contain a "ZINC-FINGER" domain. "Zinc" refers to the ion that stabilizes the protein motif, but beyond that Zinc Finger (ZF) proteins can be quite diverse.  Researchers have identified a number of common ZFs. For example, the C2H2 ZF group is very common among mammalian TRANSCRIPTION FACTORS. 

After extensive study researchers have identified tandem repeats of multiple fingers that corresponded to actual DNA sequences these proteins were binding. These " modules" can be arranged together to bind typically 3 bases at a time, where multiple modules are required to denote unique sequence binding within the GENOME (shown right).  Researchers introduced the nuclease (N) component as DNA "scissors." 

For years, knowledge of ZF binding was expanded and developed, and ZFs became the best GENOME targeting tool available. Although ZFs remain in use today and even in new clinical trials, see below for the the simpler and more specific modular design of TALES. These have accelerated the pace of gene editing discovery. Even simpler in denoting target sequence binding, the CRISPR/CAS is redefining the capacity of gene and GENOME EDITING.

Viewed by many as the original gene editing revolution, Transcription Activator Like Effector Nucleases (TALENS) offer several advantages over ZFs. These include a much smaller "intellectual property wall," increased production speed, and simpler modular design approach. As with ZFNs, researchers introduced the nuclease (N) domain as DNA "scissors."

Originally identified as naturally occurring TRANSCRIPTION FACTORS in Xanathomonas bacteria, these sequence specific HOMING PROTEINS aid in host plant colonization by homing to, and changing gene-specific transcription. Through extensive effort, researchers deciphered the repetitive DNA sequence code which governs TALE DNA binding. 

Briefly, the central region of a TALE protein is highly conserved and consists of 33-34 amino acid repeats (termed "modules.” Potentially divergent at position 12 and 13, these special amino acids, the so called "repeat variable diresidue," are responsible for binding a particular DNA letter. That is, change amino acids 12 and/or 13 and you change which DNA base the protein will bind to. By stringing together modules in a user-defined manner, one can assemble a TALE to bind virtually any DNA sequence. This simple code is displayed in the image to the right.

Clustered Regularly Spaced Interspaced Short Palindromic Repeats (CRISPR)/Crispr Associated Protein (CAS) is currently by the most effective and efficient gene-editing system available. It represents a transition to facile GENOME EDITING and is revolutionizing gene research and medicine. Originally identified as an adaptive immune system in many types of bacteria, the key protein and RNA "guiding" components have been adapted to function in higher cell types. 

In short, a family of DNA cutting proteins associated with crispr RNAs (crRNAs) and called CRISPR Associated Protein (CAS), can be "guided" to unique DNA locations for cutting. In contrast to the repetitive modular designs of ZFs and TALEs, a newly assembled CAS protein does not need to be designed for each DNA binding event. Rather, In the CRISPR/CAS system it is the gRNA that determines DNA binding specificity. A new set of delivery instructions, therefore, means simply changing the crRNA or "guide" RNA sequence. 

As related to EPIGENETIC ENGINEERING, researchers have recently mutated Cas9 cutting capacity (by D10A and H841A mutations) to create a "dead" Cas9. Lacking DNA cutting capacity, DNA HOMING is still in tact...

Simple in general concept, there are several important details for properly using CRISPR/CAS. For more information on natural CRISPR/CAS functions and its use in GENOME ENGINEERING click here.