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Zhang, Liyang; Li, Gen; Zhang, Yingxiao; Cheng, Yanhao; Roberts, Nathaniel; Glenn, Steve E.; DeZwaan-McCabe, Diane; Rube, H. Tomas; Manthey, Jeff; Coleman, Gary; et al(
, Genome Biology)
AbstractBackground
Cas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants.
Results
To improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis inE. coliand identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency inT0plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites.
Conclusions
Our results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential ofmore »these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.
Atmospheric conditions affect the release of anemophilous pollen, and the timing and magnitude will be altered by climate change. As simulated with a pollen emission model and future climate data, warmer end-of-century temperatures (4–6 K) shift the start of spring emissions 10–40 days earlier and summer/fall weeds and grasses 5–15 days later and lengthen the season duration. Phenological shifts depend on the temperature response of individual taxa, with convergence in some regions and divergence in others. Temperature and precipitation alter daily pollen emission maxima by −35 to 40% and increase the annual total pollen emission by 16–40% due to changes in phenology and temperature-driven pollen production. Increasing atmospheric CO2may increase pollen production, and doubling production in conjunction with climate increases end-of-century emissions up to 200%. Land cover change modifies the distribution of pollen emitters, yet the effects are relatively small (<10%) compared to climate or CO2. These simulations indicate that increasing pollen and longer seasons will increase the likelihood of seasonal allergies.
Abstract CRISPR/Cas systems have been widely used for genome engineering in many plant species, while their potentials have remained largely untapped in fruit crops, particularly in pear, due to the high levels of genomic heterozygosity and difficulties in tissue culture and stable transformation. To date, only few reports on application of CRISPR/Cas9 system in pear have been documented with a very low editing efficiency. Here, we report a highly efficient CRISPR toolbox for loss-of-function and gain-of-function research in pear. We compared four different CRISPR/Cas9 expression systems for loss-of-function analysis and identified a potent system that showed nearly 100% editing efficiency for multi-site mutagenesis. To expand targeting scope, we further tested different CRISPR/Cas12a and Cas12b systems in pear for the first time, albeit with low editing efficiency. In addition, we established a CRISPR activation (CRISPRa) system for multiplexed gene activation in pear calli for gain-of-function analysis. Furthermore, we successfully engineered the anthocyanin and lignin biosynthesis pathways using both CRISPR/Cas9 and CRISPRa systems in pear calli. Taken together, we build a highly efficient CRISPR toolbox for genome editing and gene regulation, paving the way for functional genomics studies as well as molecular breeding in pear.
CRISPR-mediated genome editing has been widely applied in plants to make uncomplicated genomic modifications including gene knockout and base changes. However, the introduction of many genetic variants related to valuable agronomic traits requires complex and precise DNA changes. Different CRISPR systems have been developed to achieve efficient sequence insertion and replacement but with limited success. A recent study has significantly improved NHEJ- and HDR-mediated sequence insertion and replacement using chemically modified donor templates. Together with other newly developed precise editing systems, such as prime editing and CRISPR-associated transposases, these technologies will provide new avenues to further the plant genome editing field.
Abstract CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis . This study greatly expands the targeting scope of Cas12a for crop genome engineering.
