The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation

Abstract

Summary The CRISPR/Cas9 system has been demonstrated to efficiently induce targeted gene editing in a variety of organisms including plants. Recent work showed that CRISPR/Cas9‐induced gene mutations in Arabidopsis were mostly somatic mutations in the early generation, although some mutations could be stably inherited in later generations. However, it remains unclear whether this system will work similarly in crops such as rice. In this study, we tested in two rice subspecies 11 target genes for their amenability to CRISPR/Cas9‐induced editing and determined the patterns, specificity and heritability of the gene modifications. Analysis of the genotypes and frequency of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in rice, with target genes edited in nearly half of the transformed embryogenic cells before their first cell division. Homozygotes of edited target genes were readily found in T0 plants. The gene mutations were passed to the next generation (T1) following classic Mendelian law, without any detectable new mutation or reversion. Even with extensive searches including whole genome resequencing, we could not find any evidence of large‐scale off‐targeting in rice for any of the many targets tested in this study. By specifically sequencing the putative off‐target sites of a large number of T0 plants, low‐frequency mutations were found in only one off‐target site where the sequence had 1‐bp difference from the intended target. Overall, the data in this study point to the CRISPR/Cas9 system being a powerful tool in crop genome engineering.

Generation of targeted mutant rice using a CRISPR‐Cpf1 system

“…Fragment‐specific PCR showed that the T‐DNA insertion could be segregated out in certain T 1 mutants (Fig. S5), suggesting that similar to the CRISPR‐Cas9 system, CRISPR‐Cpf1 could rapidly generate transgene‐free, genome‐modified crops (Zhang et al ., 2014). …”

Consumer acceptance of food crops developed by genome editing

“…The unprecedented introduction of artificial nucleases could induce off-target DSBs and result in unwanted, off-target mutations in resultant plants. Although the off-target effect would be reduced if the nucleases are introduced in the form of a ribonucleoprotein, rather than plasmid DNA (Woo, et al 2015), the risk of off-target mutations can be substantial, depending on the selection of the target site and the design of the targeting molecules (Shan, et al 2013;Zhang, et al 2014). Higher-fidelity CRISPR/Cas9 variants have been developed and some of them displayed no detectable genome-wide off-target effects (Kleinstiver, et al 2016;Ran, et al 2013;Zetsche, et al 2015).…”

“…However, the nucleases could simultaneously create off-target DSBs at non-target sites. Despite an extremely low frequency, off-target DSBs could lead to indels of various lengths, including point mutations, in crop genomes (Shan, et al 2013;Zhang, et al 2014). There is a possibility that multiplex genome editing would increase off-target effects if improperly designed nucleases or guide RNAs (gRNAs, in the case of CRISPR/Cas9) are simultaneously introduced into plant cells.…”

Selective gene dosage by CRISPR‐Cas9 genome editing in hexaploid Camelina sativa

“…Highly efficient, stable mutagenesis was subsequently demonstrated in Arabidopsis, as well as in several crops: rice, wheat, barley, Brassica oleracea , potato, maize and soybean (Brooks et al ., 2014; Butler et al ., 2015; Čermák et al ., 2015; Fauser et al ., 2014; Feng et al ., 2014; Ito et al ., 2015; Jiang et al ., 2014; Lawrenson et al ., 2015; Li et al ., 2015; Svitashev et al ., 2015; Wang et al ., 2014; Zhang et al ., 2014; Zhou et al ., 2014). The CRISPR‐Cas9 system can be used to achieve multiple mutagenesis by the expression of several sgRNAs targetting different genes (Lowder et al ., 2015; Ma et al ., 2015).…”