The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied plant research.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 has emerged as an effective genome editing tool in animals. Here we compare the activity of Cpf1 from Acidaminococcus sp. BV3L6 (As) and Lachnospiraceae bacterium ND2006 (Lb) in plants, using a dual RNA polymerase II promoter expression system. LbCpf1 generated biallelic mutations at nearly 100% efficiency at four independent sites in rice T0 transgenic plants. Moreover, we repurposed AsCpf1 and LbCpf1 for efficient transcriptional repression in Arabidopsis, and demonstrated a more than tenfold reduction in miR159b transcription. Our data suggest promising applications of CRISPR-Cpf1 for editing plant genomes and modulating the plant transcriptome.

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.

BackgroundCRISPR-Cas12a (formerly Cpf1) is an RNA-guided endonuclease with distinct features that have expanded genome editing capabilities. Cas12a-mediated genome editing is temperature sensitive in plants, but a lack of a comprehensive understanding on Cas12a temperature sensitivity in plant cells has hampered effective application of Cas12a nucleases in plant genome editing.ResultsWe compared AsCas12a, FnCas12a, and LbCas12a for their editing efficiencies and non-homologous end joining (NHEJ) repair profiles at four different temperatures in rice. We found that AsCas12a is more sensitive to temperature and that it requires a temperature of over 28 °C for high activity. Each Cas12a nuclease exhibited distinct indel mutation profiles which were not affected by temperatures. For the first time, we successfully applied AsCas12a for generating rice mutants with high frequencies up to 93% among T0 lines. We next pursued editing in the dicot model plant Arabidopsis, for which Cas12a-based genome editing has not been previously demonstrated. While LbCas12a barely showed any editing activity at 22 °C, its editing activity was rescued by growing the transgenic plants at 29 °C. With an early high-temperature treatment regime, we successfully achieved germline editing at the two target genes, GL2 and TT4, in Arabidopsis transgenic lines. We then used high-temperature treatment to improve Cas12a-mediated genome editing in maize. By growing LbCas12a T0 maize lines at 28 °C, we obtained Cas12a-edited mutants at frequencies up to 100% in the T1 generation. Finally, we demonstrated DNA binding of Cas12a was not abolished at lower temperatures by using a dCas12a-SRDX-based transcriptional repression system in Arabidopsis.ConclusionOur study demonstrates the use of high-temperature regimes to achieve high editing efficiencies with Cas12a systems in rice, Arabidopsis, and maize and sheds light on the mechanism of temperature sensitivity for Cas12a in plants.Electronic supplementary materialThe online version of this article (10.1186/s12915-019-0629-5) contains supplementary material, which is available to authorized users.

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Prime editing events revealed by next-generation sequencing (NGS). (D) Quantification of prime editing frequencies by PPE3b-V01 at five target sites. (E) Editing events revealed by NGS reads. (F) Schematics of the expression vectors of Plant Prime Editor 3 or 2-Version 2 (PPE3/2-V02). (G-I) Comparison of multiple PBS-RT pairs of different lengths for directing TCA insertion at the OsPDS target site, directing C to A base change at the OsDEP1 target site and directing TGA insertion at the OsDEP1 target site, respectively, by PPE3-V02. (J and K) Validation of prime editing outcomes by NGS at the OsPDS-pegR15, OsDEP1-pegR03, and OsDEP1-pegR10 target sites. (M) Comparison of PPE3-V02 and PPE2-V02 for precise editing at five target sites. (N) PPE2-V02 based prime editing events revealed by NGS for the OsPDS-sgRNA01 3T ins construct (for insertion of a T 3 nt downstream of the PBS). (O) PPE2-V02 based prime editing at another site with multiple PBS-RT pairs of different lengths. The experiments were done in rice protoplasts.Three biological replicates were used to assess the PPE3-V01 system (B-E), and two biological replicates were used to assess PPE3-V02 and PPE2-V02 systems (G-O). Error bars represent standard deviations of the biological replicates. For NGS-based genotyping data presentations (C, E, J, K, L, and N), the sequences (from top to bottom) include the wild-type (WT) sequence (protospacer underlined and PAM in bold), the expected prime editing outcome (Reference), confirmed precise prime editing events matching the expected prime editing outcome (PE_Ref), precise prime editing plus additional single nucleotide polymorphisms (e.g., PE_h01; h stands for haplotype) and deletions resulted from the NHEJ repair. The prime edited DNA nucleotides are highlighted in red.

Summary CRISPR ‐Cas9 and Cas12a are two powerful genome editing systems. Expression of CRISPR in plants is typically achieved with a mixed dual promoter system, in which Cas protein is expressed by a Pol II promoter and a guide RNA is expressed by a species‐specific Pol III promoter such as U6 or U3. To achieve coordinated expression and compact vector packaging, it is desirable to express both CRISPR components under a single Pol II promoter. Previously, we demonstrated a first‐generation single transcript unit ( STU )‐Cas9 system, STU ‐Cas9‐ RZ , which is based on hammerhead ribozyme for processing single guide RNA s (sg RNA s). In this study, we developed two new STU ‐Cas9 systems and one STU ‐Cas12a system for applications in plants, collectively called the STU CRISPR 2.0 systems. We demonstrated these systems for genome editing in rice with both transient expression and stable transgenesis. The two STU ‐Cas9 2.0 systems process the sg RNA s with Csy4 ribonuclease and endogenous tRNA processing system respectively. Both STU ‐Cas9‐Csy4 and STU ‐Cas9‐ tRNA systems showed more robust genome editing efficiencies than our first‐generation STU ‐Cas9‐ RZ system and the conventional mixed dual promoter system. We further applied the STU ‐Cas9‐ tRNA system to compare two C to T base editing systems based on rAPOBEC 1 and Pm CDA 1 cytidine deaminases. The results suggest STU ‐based Pm CDA 1 base editor system is highly efficient in rice. The STU ‐Cas12a system, based on Cas12a’ self‐processing of a CRISPR RNA (cr RNA ) array, was also developed and demonstrated for expression of a single cr RNA and four cr RNA s. Altogether, our STU CRISPR 2.0 systems further expanded the CRISPR toolbox for plant genome editing and other applications.