A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice

Abstract

We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing.

Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida

“…The mutation patterns of FnCpf1 were similar to those of TALENs, ZFNs and paired nickases (Cas9)33363738. FnCpf1 generates DNA ends with 5′ overhangs; TALENs, ZFNs and paired nickases also generate sticky DNA ends after cleavage of target sequences.…”

“…This difference may due to the difference in the transformation process as described above. Rice plants could be regenerated mostly from genetically homogeneous callus, and SSN-induced mutation rarely occurs in regenerated rice plants33, whereas tobacco might accumulate mutation events in going from regeneration to the reproductive stage. As a result, constitutively occurring mutations could create genetically chimeric plants.…”

Biallelic Gene Targeting in Rice

“…Notably, HDR activity in Lig4 knockdown rice calli was two-to three-fold higher than that in control calli, and suppression of Lig4 leads to decreased stable transformation in rice (NishizawaYokoi et al, 2012). Furthermore, Lig4 deficiency increases mutation frequency and produce longer indels at the target site of TALENs (Nishizawa-Yokoi et al, 2015a). Another study reported that HDR-based GT was 5-to 16-fold and 3-to 4-fold enhanced in Arabidopsis ku70 and lig4 mutants, respectively .…”

In planta gene targeting can be enhanced by the use of CRISPR/Cas12a

“…Using biolistic delivery of Cas9 as well as a single‐stranded repair template and subsequent regeneration of immature embryos, precise gene modifications and insertions were achieved in maize (Svitashev et al ., ), rice (Sun et al ., ), and soybean (Li et al ., ). Other attempts to shift the balance between NHEJ and HR include repression or knockout of NHEJ factors (Jia et al ., ; Qi et al ., ; Endo et al ., ; Nishizawa‐Yokoi et al ., ), or employment of NHEJ inhibiting or HR promoting small molecules (van Chu et al ., ; Maruyama et al ., ; Yu et al ., ; Song et al ., ; Shao et al ., ). However, no study has been published to date on performing GT in plant cells and showing enhanced HR efficiency due to mutations in factors suppressing genomic instability.…”