Anna Azvolinsky, The Scientist, 4 August 2014, http://www.the-scientist.com/?articles.view/articleNo/46724/title/CRISPR–No-Cutting-Required/
Researches out of Japan have modified the CRISPR/Cas9 system to contain a deaminase enzyme, allowing Cas9 to modify single base pairs without introducing a double stranded break. Double stranded breaks result in deleterious NHEJ mutations and are toxic to cells, allowing this new method to potentially introduce mutations more efficiently. The deaminase was attached to either dead Cas9 or to a “nickase” version. The deaminase substitutes a uracil for cytosine which is replaced with a thymine in subsequent rounds of DNA replication. This new tool can modify a cytosine within a 3-5 base pair window of the target site and works in mammalian cell lines. The team is now working on developing Cas9 systems capable of making any of the four nucleotide substitution combinations.