An Effective Strategy for Reliably Isolating Heritable and Cas9-Free Arabidopsis Mutants Generated by CRISPR/Cas9-Mediated Genome Editing

Abstract

ORCID ID: 0000-0002-7224-8449 (Y.Z.).Mutations generated by CRISPR/Cas9 in Arabidopsis (Arabidopsis thaliana) are often somatic and are rarely heritable. Isolation of mutations in Cas9-free Arabidopsis plants can ensure the stable transmission of the identified mutations to next generations, but the process is laborious and inefficient. Here, we present a simple visual screen for Cas9-free T2 seeds, allowing us to quickly obtain Cas9-free Arabidopsis mutants in the T2 generation. To demonstrate this in principle, we targeted two sites in the AUXIN-BINDING PROTEIN1 (ABP1) gene, whose function as a membrane-associated auxin receptor has been challenged recently. We obtained many T1 plants with detectable mutations near the target sites, but only a small fraction of T1 plants yielded Cas9-free abp1 mutations in the T2 generation. Moreover, the mutations did not segregate in Mendelian fashion in the T2 generation. However, mutations identified in the Cas9-free T2 plants were stably transmitted to the T3 generation following Mendelian genetics. To further simplify the screening procedure, we simultaneously targeted two sites in ABP1 to generate large deletions, which can be easily identified by PCR. We successfully generated two abp1 alleles that contained 1,141-and 711-bp deletions in the ABP1 gene. All of the Cas9-free abp1 alleles we generated were stable and heritable. The method described here allows for effectively isolating Cas9-free heritable CRISPR mutants in Arabidopsis.

Production of Guide RNAs in vitro and in vivo for CRISPR Using Ribozymes and RNA Polymerase II Promoters

“…We use pHDE-35S-Cas9-mCherry-UBQ plasmid as our CRISPR vector (Gao et al , 2016), which has been used for editing genes in Arabidopsis (the plasmid can be obtained through Addgene ).…”

“…The RGR unit has led to successful isolation of several null alleles of abp1 in Arabidopsis (Gao et al , 2015 and 2016). …”

“…The transgenic T1 plants were identified either as hygromycin-resistant or producing mCherry fluorescence (Gao et al , 2015 and 2016). T1 plants were screened for editing events using PCR and restriction digestion as described in Gao et al , 2015 and 2016.…”

Precise Genome Modification via Sequence-Specific Nucleases-Mediated Gene Targeting for Crop Improvement

“…At last, regeneration and transformation efficiency, and issues of regulation must also be taken into consideration when selecting a transformation strategy for a given crop species (Altpeter et al, 2016). Both the CRISPR/Cas9 array and the donor templates could be tagged by fluorescence proteins and tracked and eliminated following segregation in the progeny to avoid the random integration of donor fragments and obtain Cas9-free lines (Gao et al, 2016). However, this tagging strategy will only feasible for the integrations of intact constructs.…”

Revolutionize Genetic Studies and Crop Improvement with High-Throughput and Genome-Scale CRISPR/Cas9 Gene Editing Technology

“…Moreover, the technology can be used to generate multiple mutants with a single plasmid that produces several gRNAs as shown in the tomato study (Jacobs et al, 2017). Undoubtedly, more features such as fluorescence markers for transgene identification (Gao et al, 2016), different types of nucleases including Cpf1 (Tang et al, 2017), and diversified gRNA production methods (Gao and Zhao, 2014) will further improve CRISPR-mediated mutagenesis and broaden the applications of CRISPR gene editing technologies in genetic studies and crop improvement.…”