CRISPR-Cereal: a guide RNA design tool integrating regulome and genomic variation for wheat, maize and rice

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas) genome editing system (CRISPR-Cas) is revolutionizing agriculture. In this system, a guide sequence that matches to a particular genomic DNA is placed in front of a synthetic RNA that consists of a scaffold sequence necessary for Cas-binding to form a guide RNA (gRNA). gRNA/Cas complex binds to the target DNA that contains a protospacer adjacent motif (PAM) via base-paring and generates a double strand break (DSB) by Cas protein. Mutations will be created when the DSB cannot be perfectly repaired. Among kinds of Cas variants, Cas9 and Cas12a (also termed Cpf1) are the two major nucleases with highest edit efficiency. NGG (N=A, T, G or C) for SpCas9 from Streptococcus pyogenes, TTTN for Cpf1 from Acidaminococcus or Lachnospiraceae, is necessary for recruiting the nuclease to produce DSBs.