Finding a needle in the Haystack

Some special proteins and RNAs have the natural ability to HOME to, and bind a unique DNA position among millions. In the human genome, this typically entails locating ~20 unique DNA bases among 6,000,000,000.

Delivering Molecular "Scissors"

Cas9 nuclease in complex with gRNA

Such HOMING PROTEINS innately possess, or can be be modified to deliver, nuclease activity. Efficient homing, therefore, provides a pair of "molecular scissors" to cut DNA any GENOME COORDINATE.

Repairing the DNA DAMAGE

Even a single unrepaired DNA double-strand break (DSB) is lethal to cells. Therefore, cells possess DNA repair proteins and use 2 primary pathways to cope with the damage. NHEJ results in small deletions, but homologous recombination (HR) allows accurate replacement of the broken DNA.

Restriction Enzyme Homing and Cutting

RESTRICTION ENZYMES are the original genome homing enzyme. Their natural function is to scan, locate, and cut a unique sequence among millions.

Key video events:

1) free floating plasmid DNA

2) T=14S, blue restriction enzyme binds  and scans the helix

3) T=25S, homing is complete and the enzyme cuts the DNA

4) T=29S, As related to DNA repair, a new piece of DNA can be delivered that perfectly fits into the break point

5) T=42S, inserted DNA is enzymatically "glued" into place by a ligase (green)

Imagining the Epigenomic Context

In above video a simple prokaryotic chromosome is displayed. For DNA homing in eukaryotic cells, one should imagine linear DNA packaged into higher order chromatin, such as modified histones, nucleosome positioning, and looped DNA.


The first promising user-defined custom homing enzymes

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.

Zinc Finger Nuclease. Copyright J Tompkins, PhD

Zinc Finger Nuclease. Copyright J Tompkins, PhD


Nature Method of the Year, 2011. TALENS

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.

TALENS. Copyright J Tompkins, PhD

TALENS. Copyright J Tompkins, PhD


The ongoing gene editing revolution.

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

Cas9 with gRNA. Click for related video and information.

Cas9 with gRNA. Click for related video and information.


...unlike the TALE and ZF modular designs which require a new protein to be generated for each DNA binding attempt, CAS just needs a new gRNA for each location. Multple gRNAs=Cas9 gene editing in multiple locations simultanesouly!

IF THERE'S ONE COMMON FEATURE AMONG SYSTEMS TO REMEMBER... that all result in a DNA break that must be repaired, or targeted cells will die. DNA DOUBLE-STRAND BREAK (DSB) repair occurs via pathways that either introduce DNA deletions or can be used to introduce replacement DNA.