Hoping to increase the gene-insertion efficiency of already-powerful genome-engineering techniques, researchers based at Hiroshima University have worked out a new approach, the Precise Integration into Targeted Chromosome (PITCh) system. It relies on microhomology-mediated end joining (MMEJ) instead of homologous recombination (HR). PITCh, the researchers report, has been used with two different kinds of programmable nucleases, transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases, the latter of which features in the much-celebrated clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system.

According to the researchers, HR-mediated knock-in technology presents a couple of difficulties. First, labor is required to construct targeting vectors containing homology arms. Second, HR in some cell types and organisms is often low and can be difficult to induce.

Casting about for an alternative to HR, the researchers realized that unlike HR, which is active during late S/G2 phases of the cell cycle, the MMEJ repair mechanism is active during the G1/early S phases. Also, the researchers noticed that other investigators had found that MMEJ-dependent mutations have been found in programmable nuclease-mediated gene disruption without exogenous donors.

These observations prompted the researchers to devise PITCh, an MMEJ-mediated gene knock-in strategy that they say enables efficient targeted integration of large DNA fragments in a wide range of cells and organisms, even those with low HR activity. The researchers presented PITCh November 20 in Nature Communications, in an article entitled “Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9.”

“TALEN-mediated PITCh, termed TAL-PITCh, enables efficient integration of exogenous donor DNA in human cells and animals, including silkworms and frogs,” wrote the authors. “We further demonstrate that CRISPR/Cas9-mediated PITCh, termed CRIS-PITCh, can be applied in human cells without carrying the plasmid backbone sequence.”

According to a press release issued by Hiroshima University, the PITCh system may prove useful in a variety of applications, including creation of disease-modeling cells and animals for drug screening and therapy development. PITCh, the release continued, could also be used to increase the production efficiency of recombinant proteins such as pharmaceutical materials in cultured animal cells. Finally, it was suggested that in silkworms, PITCh strategies could be applied to production of functional recombinant silk proteins.

Details in the Nature Communications article included ways to adapt the PITCh system so that it could be used with CRISPR/Cas9 instead of TALENs: “The principles of inducing [double-strand breaks] with TALENs and CRISPR/Cas9 are totally different; therefore, we modified the targeting strategy. In CRIS-PITCh, three guide RNAs (gRNAs) and Cas9 nuclease should be coexpressed, and two different gRNA target sites should be added to the CRIS-PITCh vector.”

“To the best of our knowledge, this is the first report to show that targeted insertions can occur via very short microhomologies, both in cultured cells and in animals,” the authors concluded. “In addition, we demonstrated successful CRIS-PITCh-mediated gene knock-in in human cells without carrying over a vector backbone sequence. We anticipate that our PITCh systems will enhance the usefulness of genome engineering techniques in a variety of cells and organisms, especially in those in which gene knock-in is difficult because of low HR efficiency.”








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