Supplementary material for the paper Plant cell reports 38(4):487-501 (2019)
CRISPR-Cas9 has become the method of choice for targeted gene disruption, but its implementation in maize (Zea mays) requires adapting to a large, repetitive genome, a lengthy transformation protocol, and a strong interest in multiplexed editing given the widespread genetic redundancy that results from the ancient polyploid history of this species.
In this study, we report the systematic application of CRISPR-Cas9 mutagenesis to 20 maize genes selected for their putative roles in kernel development. Two types of multi-guide RNA vectors were used: RDP vectors developed in-house (plasmids L1609 and L1611), and Iowa vectors derived from a Gateway-compatible construct. Both support the simultaneous expression of multiple sgRNAs, enabling the co-targeting of independent genes, paralogous gene families, or multiple sites within a single gene in a single transformation event.
Analysis of 93 mutant alleles across 18 of the 20 targeted genes confirmed that CRISPR-Cas9 is robust in maize. Small insertions or deletions at or near the predicted cleavage site were the most frequent outcome (82%), with biallelic mutations observed in 19% of edited plants. Importantly, the multiplex approach enabled the generation of double and triple mutants in a single step, including tightly linked paralogous genes (ZmEsr1, ZmEsr2, ZmEsr3) that would otherwise have required thousands of crosses. Off-target effects were minimized through stringent sgRNA design, and Sanger sequencing chromatograms from primary transformants proved reliable for distinguishing fully edited plants, which transmit mutations to offspring following Mendelian segregation, from chimeric events that do not.
The downloadable files below correspond to Online Resource 1 of the paper: a genome-wide catalog of all CRISPR-Cas9 target sites identified in the Zea mays B73 reference genome, listed as 23-mers ending with NGG. The original file was generated against genome version 3.26; an updated version covering the more recent assembly (v4) was subsequently produced. Together these files contain over 15 million target sequences covering nearly 19 million genomic loci, and can be queried to design sgRNAs for any gene of interest in the maize B73 genome. The two CRISPR-Cas9 vectors described in the paper (L1609 and L1611) are deposited on GenBank under accession numbers MH662439 and MH662440.
