Delivered as Promised: A non-expert review of genetic editing

                                         Genetically Editing the New You with CRISPR and TALEN

                                                                           ByThomas Irvine

Your Choices

            If you could choose between 1)changing your eye color to any color of the rainbow and possibly losing all ofyour hair or 2) changing your eye color to onlya select variety of colors, but with no side effects. Which would you choose? Butdo not choose too quickly, because your choice is slightly more complicatedthan you might realize. In regard to genetic editing tools,accuracy is the ability to find and alter the desired section of DNA, andefficiency relates to the speed of change to all the DNA in all your cells. Genetic editing with Transcription Activator-Like Effector Nucleases, or TALENs, allowspeople to either add or remove information from virtually any part of any genein their DNA, but there’s a catch. With TALEN’s versatility, or ability totarget any section of DNA comes the price of reduced cutting accuracy andefficiency. Clustered Regularly Inter-spaced ShortPalindromic Repeats, or CRISPRs, on the other hand, require a very specificsection of DNA, like a specialized landing pad, called the Protospacer-Adjacent Motif, or PAM site, in order to attach to theDNA. The requirement of a PAM site causes CRISPR to behave extremely accurately.

Backdrop

            In order for you tounderstand the differences between the two systems, you’ll need to know thebasic way the two systems operate. Because both CRISPR and TALEN work verysimilarly, details of operation are only required for one type.

           Originally, scientistsassumed CRISPR elements in the bacteria E. coliwere the remnants of viruses that the bacteria had previously encountered.Researchers thought the viruses were implanting a part of themselves into thebacteria in order to rise again in the future (similar to the chicken pox viruscausing shingles later in life in humans). Further research revealed that theremnant elements were located in specific sections of DNA, and researchersfound this to be true in many other types of bacteria as well.Consequently, these findings indicated that the bacteria created the remnantelements with its own cellular machinery. The scientific community’s currentunderstanding is that bacteria use these remnant elements as part of aninternal anti-viral defense. As shown in Figure 1, bacteriause these CRISPR elements, as they are now known, to create a seek-and-destroytargeting sequence. With the help of a Casprotein, which we’ll talk about in asecond, thetargeting sequence attaches to the virus and literally cuts the virus in half.

 

Scientistshave now adopted CRISPR’s seek-and-destroy ability to perform DNA editing withthe same precision as targeting a specific virus in a cell. I see how this canbe confusing because DNA and viruses don’t seem the same, but in fact DNA makesup a lot of the structure of a virus. Therefore, scientists have adopted thesame strategy to perform genetic editing in plants, fish, mice, and human cellsthat were grown in test tubes.

            So what kinds of things can genetic editing tools, suchas CRISPR or TALEN, do for humans? Well, I’m glad you asked reader.

Scenario:

            -Let’s say your significant other tells you he or she has HumanPapillomavirus (HPV). Unfortunately, you never received the vaccine. Now youhave the possibility of developing tumors in your groin and neck (for which nocure exists) from the insertion of tumor causing genes into your DNA from HPV. How can this condition befixed? If the inserted genes could somehow be disabled or destroyed you wouldno longer have to fear developing tumors in the future. Therefore, you decideto undergo an experimental test to treat HPV, in which the scientists useCRISPR with a seek-and-destroy sequence targeted to the tumor causing genes.

           If you would kindly refer to Figure2 as we go through this section, I will explain how the genetic editing tool knownas CRISPR works.

 

The process of curing your condition starts at Figure 2a. Acircular piece of DNA, known as a Tiplasmid, is inserted into the cells that contain tumor-causing genes. Allthe necessary information to create CRISPR elements is housed in the Tiplasmid, similar to blueprints at a construction site. Once inside the cell,the Ti plasmid will produce copies of the CRISPR complex.

            Figure2b shows an artist’s representation of the final, active form of a CRISPRelement. All CRISPR elements contain a 20 nucleotide (A’s, G’s, T’s, and C’s)seek-and-destroy targeting sequence known as single-guide RNA (sgRNA) and a Cas protein that holds the sgRNA andcuts the target DNA. The sgRNA works like a key that will only open a singlelock. The lock in this metaphor represents the specific DNA segment thatperfectly matches the sgRNA, and the 20 nucleotides are the teeth of the key.

            Next,figure 2c shows how the sgRNA attaches to the section of DNA that it matches. SgRNA/Cascomplex is produced near the genomic DNA. This relative proximity allows theCas protein to quickly find the matching DNA segment to the sgRNA and attach toit. Each individual nucleotide in the sgRNA, as shown in Figure 2c, will attachto its matching nucleotide in the DNA. Once attached, the Cas protein will cutstraight through both strands of your double-helixed DNA causing a double-strand break (DSB).  DNA repair proteins will attempt to repairthe broken segment, but even if the segment is repaired another sgRNA/Cas willattach and cut the DNA again. The DNA repair mechanism soon becomes overwhelmeddue to repeated cutting, and an error is made. An example of an error could bea deletion or insertion of a nucleotide in the middle of the original sequenceof A’s, T’s, G’s, and C’s targeted by the sgRNA.

           Figure2d shows the aftermath of CRISPR gene destruction. The alteration will be onlyto the tumor causing genes that the scientists wanted to target. The product ofthe gene will no longer be made. This alteration is permanent, and therepercussion, in this case, is the loss of tumor inducing genes. So now you arecured, how do you feel?

           Nowthat you are more informed about how genetic editing works, you can understandthe differences between the CRISPR and TALEN systems. While many criterion canbe considered, versatility, efficiency, and accuracy show the greatestdifferences when comparing TALEN and CRISPR. Although TALEN possesses moreversatile genetic editing power, CRISPR edits with exceptional efficiency whilemaintaining superior accuracy.

The Skinny about Versatility

           Dueto the way TALEN finds and attaches to DNA targets, the TALEN system is muchmore versatile than the CRISPR system. Let’s again define versatility to be theability to select any DNA target with a genetic editing tool. The TALEN systemdoes not require a PAM site like CRISPR does. This means any region of a genomeis fair game to be targeted by TALEN, while only certainregions of genes containing PAM sites can be targeted by CRISPR. Increasedversatility means that genes can be fine-tuned to give you exactly the effectyou want. For example, TALEN could be targeted to the genes that control eyecolor in order to produce the perfect shade of emerald green. In Figure 3a and3b you can see that more than one gene is required to produce the color of youriris, therefore, manipulation of each of these genes can produce differentcolors.

 

In contrast, CRISPR’s requirement of a PAM site means that changes togenes could only occur at a select number of locations. As seen in Figure 3c,some genes may not have a PAM site in a location suitable for editing. WithCRISPR you may only have the option of dramatically changing your eye color,like from green to yellow. Therefore with the greater versatility of TALEN, aperson has more options of what they can change in their DNA and to what degreethey change it.

The 411 on Efficiency

            However, TALEN fails tobe as effective a genetic editing tool as CRISPR. The amount of informationneeded to create TALEN complexes inside of a cell requires the production ofvery long seek-and-destroy guide segments. Unlike the 20 nucleotidematching method, used by CRISPR, TALEN requires at least 99 nucleotides in itstarget sequence. The larger target sequence required by TALEN means the processof seeking a target sequence requires more time. Cells have ways of recognizingand destroying proteins that are hanging out for too long. Increased seekingtime causes some of TALEN complexes to be destroyed, therefore reducing theefficiency of the system. Reduced efficiency means that if you choose toutilize TALEN to seek-and-destroy a deadly virus you have contracted, like theEbola virus, you might die from the effects of the virus before TALEN couldseek-and-destroy all the virus particles in your body. Conversely, the sgRNAutilized by the CRISPR system can quickly find its target DNA.CRISPR’s exceptional efficiency give it the power to edit genes moreeffectively than TALEN.

The Info for Accuracy

            Consequently, CRISPR’s PAMsite requirement means that can count on CRISPR to provide accurate geneticediting every time it’s used. Even if another site in your DNA matched thesgRNA, chances are slim that the matching segment would be adjacent to a PAMsite. Whereas, TALEN does not require a special landing pad, and therefore, canattach to any matching DNA segment. Also, the length of thetargeting segments of TALEN allow the complex to accidentally attach to DNA andcause a DSB in unintended target areas. This is known as an off-target effect, or more simply as anoff-target. An example of an off-target effect would be accidentally turningoff the gene that controls hair growth when you were originally trying to onlytarget a gene that controls eye color. Researchers have essentially lowered thechance for off-targets by CRISPR to zero with the advent of paired nickases. Pairednickases are almost the same as a normal CRISPR complex, but instead of requiringa single PAM site and 1 sgRNA nickases require 2 PAM sites and 2 sgRNA. Thiscondition requires both sgRNAs to locate the target segment in order to cause aDSB. You can think of this like a vault door that requires 2 keys to be usedsimultaneously in order to open the door. Similarly, the 2 sgRNA must attach andcut at the same time in order to cause a DSB in your gene of interest. Pairednickases have the ability to provide humanity the necessary genetic editingaccuracy to treat and cure many genetically based diseases such as sickle cellanemia, certain cancers, and cystic fibrosis with very little fear ofoff-target effects.

Bibliography

1: Barrangou, R. et al. “CRISPR provides acquiredresistance against viruses in prokaryotes”. 2007. Science.

2: Duan, J. et al. “Genome-wide identification of CRISPR/Cas9 off-targets in human genome”.2014. Cell Research.

3: Kennedy, E. et al.“Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinomacells using a bacterial CRISPR/Cas RNA-guided endonuclease”. 6Aug2014. JVI Accepts.

4: Wang, X. et al. “Precise Gene ModificationMediated by TALEN and Single-Stranded Oligodeoxynucleotides in Human Cells”.1Apr2014. PLOS one.

5: Wright, D. et al.“TALEN-mediated genomic editing: prospects and perspectives”. 2014. Biochem. J.

6:  Zhen, S. et al. “Invitro and in vivo growth suppression of human papillomavirus 16-positivecervical cancer cells by CRISPR/Cas9”. 17Jul2014. BBRC.