A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 (Cas12a) nucleases in rice

Abstract

BackgroundTargeting specificity has been a barrier to applying genome editing systems in functional genomics, precise medicine and plant breeding. In plants, only limited studies have used whole-genome sequencing (WGS) to test off-target effects of Cas9. The cause of numerous discovered mutations is still controversial. Furthermore, WGS-based off-target analysis of Cpf1 (Cas12a) has not been reported in any higher organism to date.ResultsWe conduct a WGS analysis of 34 plants edited by Cas9 and 15 plants edited by Cpf1 in T0 and T1 generations along with 20 diverse control plants in rice. The sequencing depths range from 45× to 105× with read mapping rates above 96%. Our results clearly show that most mutations in edited plants are created by the tissue culture process, which causes approximately 102 to 148 single nucleotide variations (SNVs) and approximately 32 to 83 insertions/deletions (indels) per plant. Among 12 Cas9 single guide RNAs (sgRNAs) and three Cpf1 CRISPR RNAs (crRNAs) assessed by WGS, only one Cas9 sgRNA resulted in off-target mutations in T0 lines at sites predicted by computer programs. Moreover, we cannot find evidence for bona fide off-target mutations due to continued expression of Cas9 or Cpf1 with guide RNAs in T1 generation.ConclusionsOur comprehensive and rigorous analysis of WGS data across multiple sample types suggests both Cas9 and Cpf1 nucleases are very specific in generating targeted DNA modifications and off-targeting can be avoided by designing guide RNAs with high specificity.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1458-5) contains supplementary material, which is available to authorized users.

Whole genome sequencing reveals rare off‐target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9‐edited cotton plants

“…To investigate pre‐existing mutations in the three WT cotton plants, we identified 28 054 SNPs and four indels with high confidence. The data suggested that the pre‐existing mutation ratio is ~2.8 × 10 −6 , which is much higher than in rice (Tang et al ., ). However, there is no significant difference of variation number when comparing to Cas9‐edited lines and negative lines, whereas there is considerable genetic variation among different individual WT plants (Table ).…”

“…After filtering the pre‐existing and Cas9‐induced mutations, 466 SNPs and 77 indels were identified as spontaneous mutations from T0 (s1) to T1 (s20, s23) in this experiment. The inherited mutation rate was ~5.43 × 10 −8 per site per tetraploid genome from T0 to T1, which is consistent with previous report in rice with the spontaneous mutation rates (~5.4 × 10 −8 ; Tang et al ., ) and maize (2.17~3.87 × 10 −8 ; Yang et al ., ).…”

“…Since it shows higher on‐target editing efficiency and undetectable off‐target effects in cotton genome editing. We demonstrated that the most variations in Cas9‐edited cotton plants were due either to somaclonal variation or/and pre‐existing/natural variation from maternal plants, but not off‐target effects, which is consistent with previous report in rice (Tang et al ., ). To investigate pre‐existing mutations in the three WT plants, we get high confidence 28 054 SNPs and four indels, the pre‐existing mutations is ~2.8 × 10 −6 , but much higher that the pre‐existing mutation rate in rice (Tang et al ., ).…”

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

“…Interestingly, hardly any off‐target events were identified so far in plants by sequencing respective sites (Feng et al ). Likewise, large‐scale whole‐genome sequencing analysis showed barely any off‐target events for SpyCas9 and LbCas12a (Tang et al ). Nevertheless, off‐target activity remains a concern to be considered, especially for targeted nuclease applications in crops with large and partly polyploid genomes, such as maize, rice and soybean (Scheben et al ), where highly repetitive sequences represent special challenges for site‐specific mutagenesis or GT.…”

Application and future perspective of CRISPR/Cas9 genome editing in fruit crops

“…Targeting specificity was recently investigated comprehensively using whole genome sequencing (WGS) of Cas9 and Cpf1 edited rice plants as well as a large number of control plants (Tang et al ). Significantly, the work showed that most mutations in edited plants are generated by the tissue‐culture process not by CRISPR.…”