Current Technology
There are currently three ways to edit the sequence of the genome, the TALEN method, the zinc-finger method, and the CRISPR method. Scientists are able to program the nucleases to cut and edit the DNA at a precise location in the genome.
TALENs- Also known as transcription activator like effector proteins, the TALEN method of genome editing is comprised of proteins produced by bacteria. These proteins have two specific domains, an effector domain and a DNA binding domain. The formation of the DNA binding domain can be altered to produce a protein domain that binds to a specified sequence of the genome. These select proteins can then be linked to a chosen effector domain, such as a nuclease, in order to create a protein able to edit DNA. (1) When the TALEN technology enters the body it must first locate the altered gene in the DNA using the plant bacterial gene, TAL. Once the gene is recognized, the endonuclease genes cut the DNA creating a double strand break in the DNA. Scientists can then add the right DNA sequence and the cell will repair itself. (2)
Zinc-Finger Nucleases- ZFNs are able to edit the genome using a single- step procedure. The alterations to the cell line are permanent and inheritable. The ZFNs are also made up of two working domains. The first domain is a chain of proprietary zinc finger protein modules that recognize and bind to a specific 6 base pair DNA sequence. Linking multiple modules together creates a protein that recognizes target DNA sequences, (what is going to be altered.) The second domain, an endonuclease isolated from the FOC-1 restriction enzyme, delivers DNA splitting functionality. When delivered as a pair, ZFNs dimerize surround a target DNA strand to create an extremely specific pair of molecular scissors. The ZFN then causes a precise double strand break in the DNA, which causes the DNA to start a repair process. Since this is an unnatural process, random insertions or deletions of DNA may occur. In that case, the DNA will be mis repaired and a localized mutation has a high possibility of forming. (3)
CRISPR- This is the most recently developed form of genetic engineering. The CRISPR method is a RNA guided nuclease system used by bacteria and archaea. It uses the nuclease CAS9 to make a double strand break at a specific location of DNA, determined by the guide RNA, (that scientists can alter). After the DNA is broken, scientists can add a correct DNA sequence in place of the irregular one. CRISPR is different than the TALEN and ZFN methods because it can be used on many cells at once using multiple guide RNAs, a huge breakthrough in genome editing field. The CRISPR method is also simple, inexpensive, very efficient, and the guide RNAs can be easily programed. (4)
(1) "TALEN-Based Genome Editing." TALEN-Based Genome Editing. N.p., n.d. Web. 25 Jan. 2015.
(2) Wirt, Stacey. "Editing Our DNA with Molecular Scissors." Stanford at The Tech. N.p., n.d. Web. .
(3) "What Is Zinc Finger Nuclease (ZFN) Technology?" Sigma-Aldrich. N.p., n.d. Web. 25 Jan. 2015.
(4) Perkel, Jeffrey M. "Genome Editing with CRISPRs, TALENs, and ZFNs."Biocompare. N.p., 27 Aug. 2013. Web. 25 Jan. 2015.
TALENs- Also known as transcription activator like effector proteins, the TALEN method of genome editing is comprised of proteins produced by bacteria. These proteins have two specific domains, an effector domain and a DNA binding domain. The formation of the DNA binding domain can be altered to produce a protein domain that binds to a specified sequence of the genome. These select proteins can then be linked to a chosen effector domain, such as a nuclease, in order to create a protein able to edit DNA. (1) When the TALEN technology enters the body it must first locate the altered gene in the DNA using the plant bacterial gene, TAL. Once the gene is recognized, the endonuclease genes cut the DNA creating a double strand break in the DNA. Scientists can then add the right DNA sequence and the cell will repair itself. (2)
Zinc-Finger Nucleases- ZFNs are able to edit the genome using a single- step procedure. The alterations to the cell line are permanent and inheritable. The ZFNs are also made up of two working domains. The first domain is a chain of proprietary zinc finger protein modules that recognize and bind to a specific 6 base pair DNA sequence. Linking multiple modules together creates a protein that recognizes target DNA sequences, (what is going to be altered.) The second domain, an endonuclease isolated from the FOC-1 restriction enzyme, delivers DNA splitting functionality. When delivered as a pair, ZFNs dimerize surround a target DNA strand to create an extremely specific pair of molecular scissors. The ZFN then causes a precise double strand break in the DNA, which causes the DNA to start a repair process. Since this is an unnatural process, random insertions or deletions of DNA may occur. In that case, the DNA will be mis repaired and a localized mutation has a high possibility of forming. (3)
CRISPR- This is the most recently developed form of genetic engineering. The CRISPR method is a RNA guided nuclease system used by bacteria and archaea. It uses the nuclease CAS9 to make a double strand break at a specific location of DNA, determined by the guide RNA, (that scientists can alter). After the DNA is broken, scientists can add a correct DNA sequence in place of the irregular one. CRISPR is different than the TALEN and ZFN methods because it can be used on many cells at once using multiple guide RNAs, a huge breakthrough in genome editing field. The CRISPR method is also simple, inexpensive, very efficient, and the guide RNAs can be easily programed. (4)
(1) "TALEN-Based Genome Editing." TALEN-Based Genome Editing. N.p., n.d. Web. 25 Jan. 2015.
(2) Wirt, Stacey. "Editing Our DNA with Molecular Scissors." Stanford at The Tech. N.p., n.d. Web. .
(3) "What Is Zinc Finger Nuclease (ZFN) Technology?" Sigma-Aldrich. N.p., n.d. Web. 25 Jan. 2015.
(4) Perkel, Jeffrey M. "Genome Editing with CRISPRs, TALENs, and ZFNs."Biocompare. N.p., 27 Aug. 2013. Web. 25 Jan. 2015.