Genome-wide analyses of PAM-relaxed Cas9 genome editors reveal substantial off-target effects by ABE8e in rice

Abstract

PAM-relaxed Cas9 nucleases, cytosine base editors and adenine base editors are promising tools for precise genome editing in plants. However, their genome-wide off-target effects are largely unexplored. Here, we conduct whole-genome sequencing (WGS) analyses of transgenic plants edited by xCas9, Cas9-NGv1, Cas9-NG, SpRY, nCas9-NG-PmCDA1, nSpRY-PmCDA1 and nSpRY-ABE8e in rice. Our results reveal that Cas9 nuclease and base editors, when coupled with the same guide RNA (gRNA), prefer distinct gRNA-dependent off-target sites. De novo generated gRNAs by SpRY editors lead to additional, but insubstantial, off-target mutations. Strikingly, ABE8e results in ~500 genome-wide A-to-G off-target mutations at TA motif sites per transgenic plant. ABE8e's preference for the TA motif is also observed at the target sites. Finally, we investigate the timeline and mechanism of somaclonal variation due to tissue culture, which chiefly contributes to the background mutations. This study provides a comprehensive understanding on the scale and mechanisms of off-target and background mutations occurring during PAM-relaxed genome editing in plants.

Keywords: PAM-relaxed Cas9 nucleases; adenine base editor; cytosine base editor; genome editing; off-target effect; rice; whole-genome sequencing.

Figure 1

Assessment of PAM‐less genome editing in rice by whole‐genome sequencing. (a‐c) Genome editing frequencies in T₀ lines by PAM‐relaxed Cas9‐NGv1, Cas9‐NG and SpRY (a) by PAM‐relaxed cytosine base editors based on nCas9‐NG and nSpRY (b), and by PAM‐less nSpRY‐ABE8e adenine base editor (c). (d) Summary of plants used for whole‐genome sequencing. (e) The bioinformatic pipeline for analysis of whole‐genome sequencing (WGS) data. NA, editing frequency in T₀ lines was not scored for the constructs xCas9‐OsDEP1‐gR02‐GGG and nSpRY‐PmCDA1‐OsALS‐gR21‐GCA. Different editing systems targeting the same target site are indicated by using the same sgRNA name (e.g., OsDEP1‐gR02‐CGC for SpCas9‐NG, SpCas9‐NGv1 and nSpCas9‐NG‐PmCDA1).

Figure 2

Different sequence preference of gRNA‐dependent potential off‐target editing by Cas9‐NG nucleases and cytosine base editors. (a‐c) gRNA‐dependent off‐target mutations in edited T₀ lines at the OsDEP1‐gR02‐CGC site by SpCas9‐NGv1 (a), SpCas9‐NG (b) and nSpCas9‐PmCDA1 (c). Off‐target sites that were shared between SpCas9‐NGv1 and SpCas9‐NG are marked in red. Top panel, sequence comparison of target gRNA and potential off‐target sites. Middle panel, the genotype of the off‐target sites. Bottom panel, the number of potential off‐target sites in two T₀ plants. (d) Venn diagram depicting many shared off‐target sites induced by the OsDEP1‐gR02‐CGC gRNA in SpCas9‐NGv1 and SpCas9‐NG, while not in nCas9‐NG‐PmCDA1.

Figure 3

Genome‐wide landscape of gRNA‐dependent off‐target mutations by de novo generated new sgRNAs by SpRY editors. (a, d) Off‐target analysis for de novo generated new gRNAs due to on‐target editing by SpRY nuclease, nSpRY‐PmCDA1 and nSpRY‐ABE8e. The number of off‐target sites overlapping identified mutation (SNVs+INDELs) versus the number of all potential off‐target sites that predicted by Cas‐OFFinder. (b‐c), gRNA‐dependent off‐target mutations in T₀ lines by de novo generated new gRNAs by SpRY at the OsDEP1‐gR01‐CGC site (b) and the OsDEP1‐gR04‐CGC‐1 site (c). Top panel, sequence comparison of new gRNA and potential off‐target sites. Middle panel, sequence comparison of target gRNA and potential off‐target sites. Bottom panel, the genotype of the off‐target sites. (e‐f) gRNA‐dependent off‐target mutations by de novo generated new gRNAs by nSpRY‐ABE8e at the OsPDS‐gR01‐TTG‐2 site (e) and OsPDS‐gR04‐TAA‐4 site (f).

Figure 4

Genome‐wide sgRNA‐independent off‐target effects by PAM‐relaxed nucleases, cytosine base editors and adenine base editors. (a) Number of single nucleotide variation (SNV) mutations in all sequenced samples. (b) Average number of SNV mutations in per 1 Mbp genomic region. (c) Fractions of different nucleotide substitutions in different samples. (d) Genome‐wide distribution of A‐to‐G SNVs in all sequenced samples. (a‐c) Error bars represent s.e.m. and dots represent individual plants.

Figure 5

ABE8e favours A‐to‐G conversion at TA motifs at both off‐target and on‐target sites. (a) Preference of a TA motif by ABE8e at gRNA‐independent off‐target A‐to‐G base editing in Watson strand, 0 indicates the A‐to‐G SNV position. (b) Base editing frequencies at different protospacer positions by ABE8e at a target site in rice protoplasts, n represents biological replicates. Data reanalysed from ref (Ren et al., 2021c). Error bars represent s.e.m. P‐value was calculated by the one‐sided paired Student’s t‐Test, *P < 0.05, **P < 0.01. (c) The genotype of mutation alleles in T₀ stable transformation plants. (d) Base editing frequencies at different protospacer positions by ABE8e at a target site in rice T₀ lines. (e) Presence of TA motifs at the target site appears to increase gRNA‐dependent off‐target A‐to‐G editing. (f) The frequency of A‐to‐G SNV with different di‐nucleic acids in T₀ stable transformation plants.

Figure 6

Investigation of somaclonal variation production in rice tissue culture. (a) A model that divides the generation of somaclonal variation into two phases, which points to potential of minimizing Phase II somaclonal variation with the use of morganic factors to accelerate plant regeneration. (b) Genome‐wide mapping of T‐DNA integration sites for all T₀ lines. Constructs that contain more than one T‐DNA integration site are highlighted in red. The two T₀ lines that carry the same T‐DNA integration site were grouped by a solid line on the right, indicating they are from the same transgenic event. (c) Four examples for the analysis of T₀ lines for shared mutations revealed by WGS. The T₀ lines resulting from the same transgenic event (highlighted in red) share a significant portion of mutations (termed Phase I somaclonal variation). (d) T₀ lines with the same T‐DNA integration sites share an average of 98 mutations, while T₀ lines with different T‐DNA integration sites barely share any mutations. (e) The frequency of A‐to‐G SNVs in shared SNVs and whole‐genome SNVs from the nSpRY‐ABE8e T₀ lines with the same transgenic events, the number above of each bar represents A‐to‐G SNVs versus all SNVs in a pair of T₀ lines. P‐value was calculated by the Wilcoxon rank sum test, *P < 0.05, **P < 0.01, NS represents not significant.