Effects of sgRNA length and number on gene editing efficiency and predicted mutations generated in rice

CRISPR-Cas9 is a common tool for gene editing, and appropriate sg RNAs are the key factor for successful editing. In this study, the effect of sg RNA length and number on editing efficiency was analyzed in rice using CYP81 A6 as the target gene. A series of CRISPR-Cas9 plant expression vectors containing single sg RNAs with different lengths(17, 18, 19, 20, 21, 22, 23 nt) or two sg RNAs were constructed and introduced into rice cultivar Zhonghua11 by Agrobacterium-mediated transformation. Analysis of the editing status of 1283 transgenic rice plants showed that 371 were successfully edited with base preference.Single A or T insertions were the most frequent among the six edited types. The editing efficiency of transgenic rice with two sg RNAs was higher than that with a single sg RNA. Editing efficiency and sg RNA length showed a normal distribution with 20 nt sg RNA(25%) being the most efficient. The editing efficiency decreased slightly with decreases of 1–2 bases(19 nt 20%, 18 nt 21%), but decreased significantly with a decrease of 3 bases(17 nt 4.5%). Editing efficiency was significantly reduced by adding 1 to 3 bases(21 nt 16.8%, 22 nt 13%, 23 nt 13%) to the sg RNA. These results provide data for successful gene editing or rice by CRISPR-Cas9.

Improving the efficiency of the CRISPR-Cas12a system with tRNA-crRNA arrays

CRISPR-Cas12a offers a convenient tool for multiplex genome editing in rice. However, the CRISPR-Cas12a system displays variable editing efficiency across genomic loci, with marked influence by CRISPR RNAs(crRNAs). To improve the efficiency of the CRISPR-Cas12a system for multiplex genome editing, we identified various architectures and expression strategies for crRNAs. Transformation of binary vectors loaded with engineered CRISPR-Cas12a systems into rice calli and subsequent sequencing revealed that a modified tRNA-crRNA array not only efficiently achieved rice multiplex genome editing, but also successfully edited target sites that were not edited by the crRNA array. This improvement contributes to the application of the CRISPR-Cas12a system in plant genome editing, especially for genomic loci that have hitherto been difficult to edit.

Highly efficient CRISPR-SaKKH tools for plant multiplex cytosine base editing

Base editing, as an expanded clustered regularly interspaced short palindromic repeats(CRISPR)-Cas genome editing strategy, permits precise and irreversible nucleotide conversion. SaKKH, an efficient variant of a Cas9 ortholog from Staphylococcus aureus(SaCas9), is important in genome editing because it can edit sites with HHHAAT protospacer adjacent motif(PAM) that the canonical Streptococcus pyogenes Cas9(SpCas9) or its variants(e.g. xCas9, Cas9-NG) cannot. However, several technical parameters of SaKKH involved base editors have not been well defined and this uncertainty limits their application. We developed an effective multiplex cytosine base editor(SaKKHn-pBE) and showed that it recognized NNARRT, NNCRRT, NNGRGT, and NNTRGT PAMs. Based on 27 targets tested, we defined technical parameters of SaKKHn-pBE including the editing window, the preferred sequence context, and the mutation type. The editing efficiency was further improved by modification of the SaKKH sgRNA. These advances can be applied in future research and molecular breeding in rice and other plants.

An optimized prime editing system for efficient modification of the pig genome

Prime editing(PE)is a recent gene editing technology that can mediate insertions or deletions and all twelve types of base-tobase conversions.However,its low efficiency hampers the application in creating novel breeds and biomedical models,especially in pigs and other important farm animals.Here,we demonstrate that the pig genome is editable using the PE system,but the editing efficiency was quite low as expected.Therefore,we aimed to enhance PE efficiency by modulating both exogenous PE tools and endogenous pathways in porcine embryonic fibroblasts(PEFs).First,we modified the peg RNA by extending the duplex length and mutating the fourth thymine in a continuous sequence of thymine bases to cytosine,which significantly enhanced PE efficiency by improving the expression of peg RNA and targeted cleavage.Then,we targeted SAMHD1,a deoxynucleoside triphosphate triphosphohydrolase(d NTPase)that impedes the reverse transcription process in retroviruses,and found that treatment with its inhibitor,cephalosporin C zinc salt(CPC),increased PE efficiency up to 29-fold(4-fold on average),presumably by improving the reverse transcription process of Moloney murine leukemia virus reverse transcriptase(M-MLV RT)in the PE system.Moreover,PE efficiency was obviously improved by treatment with a panel of histone deacetylase inhibitors(HDACis).Among the four HDACis tested,panobinostat was the most efficient,with an efficiency up to 122-fold(7-fold on average),partly due to the considerable HDACi-mediated increase in transgene expression.In addition,the synergistic use of the three strategies further enhanced PE efficiency in PEFs.Our study provides novel approaches for optimization of the PE system and broadens the application scope of PE in agriculture and biomedicine.

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

Since the discovery that nucleases of the bacterial CRISPR(clustered regularly interspaced palindromic repeat)-associated(Cas) system can be used as easily programmable tools for genome engineering,their application massively transformed different areas of plant biology. In this review, we assess the current state of their use for crop breeding to incorporate attractive new agronomical traits into specific cultivars of various crop plants. This can be achieved by the use of Cas9/12 nucleases for double-strand break induction,resulting in mutations by non-homologous recombinatr e-tion. Strategies for performing such experiments à from Rthe design of guide RNA to the use of different transformation technologies à are evaluated. Furtherweive-more, we sum up recent developments regarding the use of nuclease-deficient Cas9/12 proteins, as DNAbinding moieties for targeting different kinds of enzyme activities to specific sites within the genome. Progress in base deamination, transcriptional induction and transcriptional repression, as well as in imaging in plants, is also discussed. As different Cas9/12 enzymes are at hand, the simultaneous application of various enzyme activities, to multiple genomic sites, is now in reach to redirect plant metabolism in a multifunctional manner and pave the way for a new level of plant synthetic biology.

Efficient allelic replacement in rice by gene editing： A case study of the NRT1.1B gene

Summary Precise replacement of an existing allele in commercial cultivars with an elite allele is a major goal in crop breeding. A single nucleotide polymorphism in the NRT1.1B gene between japonica and indica rice is responsible for the improved nitrogen use efficiency in indica rice. Herein, we precisely replaced the japonica NRT1.1B allele with the indica allele, in just one generation, using CRISPR/Cas9 gene-editing technology. No additional selective pressure was needed to enrich the precise replacement events.

Shortened snRNA promoters for efficient CRISPR/Cas-based multiplex genome editing in monocot plants

Dear Editor,CRISPR(clustered regularly interspaced short palindromic repeats)/Cas genome editing is a powerful tool for introducing specific mutations in organisms including plants.The system is composed of a nuclease such as Cas9 or Cas12a and an engineered single-guide RNA(sgRNA)incorporating a target sequence(Li et al.,2019).A Cas9/sgRNA complex recognizes its target site in the genome,resulting in a mutation at that site.

Highly efficient genome editing using oocyte-specific zcas9 transgenic zebrafish

Since its first application to induce mutations in mammalian cells （Cong et al., 2013： Mall et al., 2013）, CRISPR/Cas9 has rapidly become a routine technique to perform genome editing in a variety of biological systems due to its facile, robust, and multiplexable fea- tures （Hwang et al, 2013; Wang et al., 2013; Guo et al., 2014; Liu, 2017）.

Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice

Most of the important agronomic traits in crop plants, such as yield, quality and stress response, are quantitative and jointly controlled by many genomic loci or major genes. Improving these complex traits depends on the combination of beneficial alleles at the quantitative trait loci （QTLs）. However, the conventional cross breeding method is extremely time-consuming and laborious for pyramiding multiple QTLs. In certain cases, this approach might be technically difficult because of close linkage between genes separately responsible for desirable and undesirable traits.