Poly(A) RNA Selection Kit

CRISPR-Cas9 Editing in Maize: Systematic Evaluation of Off-target Activity and Its Relevance in Crop Improvement

Joshua Young, Gina Zastrow-Hayes, Stéphane Deschamps, Sergei Svitashev, Mindaugas Zaremba, Ananta Acharya, Sushmitha Paulraj, Brooke Peterson-Burch, Chris Schwartz, Vesna Djukanovic, Brian Lenderts, Lanie Feigenbutz, Lijuan Wang, Clara Alarcon, Virginijus Siksnys, Gregory May, N. Doane Chilcoat & Sandeep Kumar

Scientific Reports, doi:10.1038/s41598-019-43141-6

CRISPR-Cas9 enabled genome engineering has great potential for improving agriculture productivity, but the possibility of unintended off-target edits has evoked some concerns. Here we employ a three-step strategy to investigate Cas9 nuclease specificity in a complex plant genome. Our approach pairs computational prediction with genome-wide biochemical off-target detection followed by validation in maize plants. Our results reveal high frequency (up to 90%) on-target editing with no evidence of off-target cleavage activity when guide RNAs were bioinformatically predicted to be specific. Predictable off-target edits were observed but only with a promiscuous guide RNA intentionally designed to validate our approach. Off-target editing can be minimized by designing guide RNAs that are different from other genomic locations by at least three mismatches in combination with at least one mismatch occurring in the PAM proximal region. With well-designed guides, genetic variation from Cas9 off-target cleavage in plants is negligible, and much less than inherent variation.

Features Poly(A) RNA Selection Kit

While in eukaryotes three RNA polymerases are involved in ribosome production [1] under usual growth conditions, the 18S and 25S ribosomal RNA (rRNA) components are thought to be exclusively the products of transcription by RNA polymerase I (Pol I) followed by processing [2]. We have observed recently, in Candida albicans during nutritional depletion and with TOR inhibition, the appearance of 18S and 25S rRNA molecules, resisting digestion by a 5′-phosphate-dependent exonuclease, indicating that they were different from the usual processed rRNA transcripts [3]. Candida albicans, a eukaryotic yeast, is a major cause of invasive fungal disease especially in immune compromised patients [4]. Ribosomes of eukaryotic cells are assembled from four individual rRNAs and 79 proteins [5]. As in Saccharomyces cerevisiae, genes coding for rRNA (rDNA) in C. albicans are repeated multiple times in tandem [6], allowing for efficient transcription by Pol I. Like other eukaryotes, the current accepted mechanism of the production of the 18S and 25S components of the ribosome in this yeast, is transcription of a 35S copy of the rDNA, followed by post and co-transcriptional processing of the nascent RNA [7]. Typically, processed RNA molecules will have a single phosphate on their 5’-end making them vulnerable to processive 5′→3′ exonucleases (P53E) that digests only RNA that has a 5′-monophosphate end [8]. Therefore, after digestion by such an exonuclease, it was unexpected to find 18S and 25S rRNA molecules in total RNA isolated from C. albicans entering its stationary phase [3]. Similar molecules were appearing also in yeast, whose TOR was inhibited by rapamycin [3]. This background information is illustrated in Fig 1A. Pyrophosphatase digestion which separates linked phosphates, made these resistant 18S and 25S molecules vulnerable again to 5’-exonulease digestion [3]. This indicated that these molecules contained more than a single phosphate at their 5’-end. This in turn raised the possibility that they were newly transcribed rather than processed, as polymerases use triphosphate nucleotides when they initiate transcription. Another serial enzyme digestion included alkaline phosphatase (AP) followed by P53E digestion, in this case the rRNAs remained protected [3]. This could have been due to either more than one phosphate being digested by AP resulting in 5′-OH, or further modification in rRNA, such as a 5’-cap protecting against both AP and P53E, again preventing exonuclease digestion. Additionally, we have previously found that C. albicans grown overnight, polyadenylates some of its 18S [9] and 25S [10] rRNA molecules, a feature associated with Pol II transcription [11]. These features prompted us to see whether Pol II is involved with ribosomal rRNA transcription.

Features Poly(A) RNA Selection Kit