An alternative TALEN/CRISPR-mediated gene insertion technique described in detail

December 17, 2015

A streamlined protocol for an alternative gene insertion method using genome editing technologies, the PITCh (Precise Integration into Target Chromosome) system, has been reported in Nature Protocols by Specially Appointed Lecturer Tetsushi Sakuma, Professor Takashi Yamamoto, Specially Appointed Associate Professor Ken-Ichi T Suzuki, and their colleagues at Hiroshima University, Japan.

The PITCh system is more convenient and effective than existing methods for inserting foreign DNA into targeted genomic loci by using genome-editing tools. This new versatile technique can aid the rapid progression of research in fields such as screening of new drug candidates and creating cell or animal models of human diseases.

Genome editing is an innovative technique used in genetic engineering that enables researchers to modify the genome not at random but at a particular target. In this technique, researchers employ engineered nucleases as "molecular scissors," which create DNA breaks at desired locations in the genome. When DNA breaks are repaired by repair pathways, genetic modifications including insertion of foreign DNA into the genome (knock-in) and replacement or removal of a targeted genomic locus are induced.

"The PITCh system is an alternative knock-in method that is independent of homologous recombination (HR), one of DNA-break repair pathways, unlike existing knock-in techniques that use genome editing tools like TALEN or CRISPR-Cas9, which mainly utilize HR," said Dr. Sakuma. "The existing knock-in techniques cannot be applied to every cell type and organism owing to variable HR frequencies. Therefore, we aimed at another repair pathway, microhomology-mediated end-joining (MMEJ), and developed the PITCh system."

In this article, we describe detailed procedures for constructing a desired vector, transfecting it into cells, selecting knocked-in cells, and checking after insertion together with an actual successful example. Furthermore, a simplified method of gene insertion in frog embryos is also described. This article will allow researchers to use this powerful tool easily, and will contribute to the progress of not only basic but also applied research in the life science.
-end-
Details about the article:

[Title]

MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems

[Journal]

Nature Protocols

[Authors]

Tetsushi Sakuma, Shota Nakade, Yuto Sakane, Ken-Ichi T Suzuki & Takashi Yamamoto

Hiroshima University

Related DNA Articles from Brightsurf:

A new twist on DNA origami
A team* of scientists from ASU and Shanghai Jiao Tong University (SJTU) led by Hao Yan, ASU's Milton Glick Professor in the School of Molecular Sciences, and director of the ASU Biodesign Institute's Center for Molecular Design and Biomimetics, has just announced the creation of a new type of meta-DNA structures that will open up the fields of optoelectronics (including information storage and encryption) as well as synthetic biology.

Solving a DNA mystery
''A watched pot never boils,'' as the saying goes, but that was not the case for UC Santa Barbara researchers watching a ''pot'' of liquids formed from DNA.

Junk DNA might be really, really useful for biocomputing
When you don't understand how things work, it's not unusual to think of them as just plain old junk.

Designing DNA from scratch: Engineering the functions of micrometer-sized DNA droplets
Scientists at Tokyo Institute of Technology (Tokyo Tech) have constructed ''DNA droplets'' comprising designed DNA nanostructures.

Does DNA in the water tell us how many fish are there?
Researchers have developed a new non-invasive method to count individual fish by measuring the concentration of environmental DNA in the water, which could be applied for quantitative monitoring of aquatic ecosystems.

Zigzag DNA
How the cell organizes DNA into tightly packed chromosomes. Nature publication by Delft University of Technology and EMBL Heidelberg.

Scientists now know what DNA's chaperone looks like
Researchers have discovered the structure of the FACT protein -- a mysterious protein central to the functioning of DNA.

DNA is like everything else: it's not what you have, but how you use it
A new paradigm for reading out genetic information in DNA is described by Dr.

A new spin on DNA
For decades, researchers have chased ways to study biological machines.

From face to DNA: New method aims to improve match between DNA sample and face database
Predicting what someone's face looks like based on a DNA sample remains a hard nut to crack for science.

Read More: DNA News and DNA Current Events
Brightsurf.com is a participant in the Amazon Services LLC Associates Program, an affiliate advertising program designed to provide a means for sites to earn advertising fees by advertising and linking to Amazon.com.