Scientists Reveal Mechanisms of CRISPR/Cas System Induced Mutations in Rice

Aug 19, 2014     Email"> PrintText Size

CRISPR (clustered regularly interspersed short palindromic repeats)/Cas (CRISPR-associated) systems are parts of adaptive immune system found in bacterial and archaeal. Engineered CRISPR/Cas system has been employed as a tool to induce sequence-specific double strand breaks (DSBs) and targeted genome modifications in a number of organisms, including rice. However, it still is unclear about the mutation patterns, efficiency, heritability and specificity of targeted gene editing by CRISPR/Cas9. 

A team of researchers, led by Professor ZHU Jiankang of the Shanghai Center for Plant Stress Biology (PSC), Chinese Academy of Sciences, tested in 2 rice subspecies, Nipponbare (O. sativa L. ssp. japonica) and Kasalath (O. sativa L. ssp. indica), 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 all 11 target genes mutated and the mutation efficiency as high as 66.7%. Homozygous mutations were found in the T0 plants with over half of the targets sites (6/11). The CRISPR/Cas9 generated mutations were passed to the next generation (T1) following classic Mendelian law.  

In a detailed study in rice, over 70% mutations resulting from CRISPR/Cas9 were only changed 1 bp and most of them (53.9%) were 1 bp insertion. All but one 1-bp-deletions occurred right upstream of this DSB position, at the 4^th base from the PAM (protospacer adjacent motif) site, and 100% of the 1 bp insertions were also at this position. By in-depth whole genome sequencing and 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 one bp difference from the intended target. So, CRISPR/Cas9 system was highly specific in plants. It’s shown that the CRISPR/Cas9 system can induce highly efficient, inheritable and specify target gene mutation in rice.  

This work advances the application of CRISPR/Cas gene editing technology in rice and thus facilitates this technology to fundamentally change rice productivity, stress-tolerant and quality by modifying the gene functions.  

The finding entitled "The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation " was selected as Cover Article for Plant Biotechnology Journal and published on the recent issue (Plant Biotechnology Journal, 2014, 12, 798-807). 

 

This article describes the application of the CRISPR-Cas9 genome editing technology in rice. The Cas9 nuclease is directed by a guide RNA to target DNA through base-pairing. The targeting also requires the presence of a PAM sequence. Cas9 generates a double stranded break (indicated by the yellow x) three base pairs upstream of the NGG PAM sequence. Cellular repair of the double stranded break then leads to a wide variety of gene modifications at the break site (Image by ZHANG Hui and Rebecca Ann Stevenson).