PgmNr M282: Easy-(Isi)-CRISPR; a method to efficiently knock-in long DNA inserts.

Authors:
CB Gurumurthy 1 ; Rolen Quadros 1 ; Don Harms 1 ; Guy Richardson 2 ; Suzanne Mansour 3 ; Masato Ohtsuka 4


Institutes
1) University of Nebraska Medical Center, Omaha, NE; 2) University of Sussex, UK; 3) University of Utah; 4) Tokai University, Japan.


Abstract:

CRISPR/Cas9 technology efficiently produces indels (insertions or deletions) and inserts short sequences when single stranded oligonucleotides are used as repair templates. However, insertion of longer stretches using double stranded DNA (dsDNA) templates is proven to be less efficient and challenging. There are a few reports that employed strategies to enhance homology directed repair mechanism of longer dsDNA templates. Here we describe a novel strategy called Isi-CRISPR (IvTRT-ssDNA-insertion-CRISPR; pronounced Easy-CRISPR) that uses in vitro synthesized ssDNAs up to 1.6 kb and show that they get inserted at the Cas9 cut sites very efficiently.