You can manage bookmarks using lists, please log in to your user account for this.
Media type:
E-Article
Title:
Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair
Contributor:
Schubert, Mollie S.;
Thommandru, Bernice;
Woodley, Jessica;
Turk, Rolf;
Yan, Shuqi;
Kurgan, Gavin;
McNeill, Matthew S.;
Rettig, Garrett R.
imprint:
Springer Science and Business Media LLC, 2021
Published in:Scientific Reports
Language:
English
DOI:
10.1038/s41598-021-98965-y
ISSN:
2045-2322
Origination:
Footnote:
Description:
<jats:title>Abstract</jats:title><jats:p>CRISPR–Cas proteins are RNA-guided nucleases used to introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing an exogenous DNA template during repair allows for the intentional, precise incorporation of a desired mutation via the HDR pathway. However, rates of repair by HDR are often slow compared to the more rapid but less accurate NHEJ-mediated repair. Here, we describe comprehensive design considerations and optimized methods for highly efficient HDR using single-stranded oligodeoxynucleotide (ssODN) donor templates for several CRISPR–Cas systems including <jats:italic>S.p.</jats:italic> Cas9, <jats:italic>S.p.</jats:italic> Cas9 D10A nickase, and <jats:italic>A.s.</jats:italic> Cas12a delivered as ribonucleoprotein (RNP) complexes. Features relating to guide RNA selection, donor strand preference, and incorporation of blocking mutations in the donor template to prevent re-cleavage were investigated and were implemented in a novel online tool for HDR donor template design. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines. Tool availability: <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://www.idtdna.com/HDR">https://www.idtdna.com/HDR</jats:ext-link></jats:p>