Improving Plant Genome Editing with High-Fidelity xCas9 and Non-canonical PAM-Targeting Cas9-NG

Two recently engineered SpCas9 variants, namely xCas9 and Cas9-NG, show promising potential in improving targeting specificity and broadening the targeting range. In this study, we evaluated these Cas9 variants in the model and crop plant, rice. We first tested xCas9-3.7, the most effective xCas9 variant in mammalian cells, for targeted mutagenesis at 16 possible NGN PAM (protospacer adjacent motif) combinations in duplicates. xCas9 exhibited nearly equivalent editing efficiency to wild-type Cas9 (Cas9-WT) at most canonical NGG PAM sites tested, whereas it showed limited activity at non-canonical NGH (H = A, C, T) PAM sites. High editing efficiency of xCas9 at NGG PAMs was further demonstrated with C to T base editing by both rAPOBECI and PmCDAI cytidine deaminases. With mismatched sgRNAs, we found that xCas9 had improved targeting specificity over the Cas9-WT. Furthermore, we tested two Cas9-NG variants, Cas9-NGv1 and Cas9-NG, for targeting NGN PAMs. Both Cas9-NG variants showed higher editing efficiency at most non-canonical NG PAM sites tested, and enabled much more efficient editing than xCas9 at AT-rich PAM sites such as GAT, GAA, and CAA. Nevertheless, we found that Cas9-NG variants showed significant reduced activity at the canonical NGG PAM sites. In stable transgenic rice lines, we demonstrated that Cas9-NG had much higher editing efficiency than Cas9-NGv1 and xCas9 at NG PAM sites. To expand the base-editing scope, we developed an efficient C to T base-editing system by making fusion of Cas9-NG nickase (D10A version), PmCDAI, and UGI. Taken together, our work benchmarked xCas9 as a high-fidelity nuclease for targeting canonical NGG PAMs and Cas9-NG as a preferred variant for targeting relaxed PAMs for plant genome editing.

The effects of person-organization fit on lending behaviors:Empirical evidence from Kiva

Donation-based crowdfunding,as part of impact investment,plays a vital role in promoting economic development and alleviating poverty.In order to enhance the lender's enthusiasm for lending behavior,some platforms,for example Kiva,have introduced groups to facilitate lending.This study examines how the group environment can affect the lenders’behaviors in crowdfunding.It has been found that lenders who join groups contribute 1.2 more loans(about$30-$42)per month than those who do not,but the theoretical mechanism of these differences is unclear.To understand in depth how the group environment affects lending behaviors,we introduce and develop the PersonOrganization fit theory and Free-rider theory in this study.Combining machine-learning techniques with empirical analysis,the results show that the matching degree of motivation between group and lender has a positive effect on the lender behavior,i.e.,lending to loans,and this relationship is weakened by free-riding in large groups.In addition,the group openness can have different effects on lenders of different group sizes.Our research enriches the existing crowdfunding literature and fills the gap in the research on new lending models in crowdfunding,and it will also be useful for crowdfunding platforms in setting the rules for building groups.

Plant Genome Editing Using FnCpfl and LbCpf Nucleases at Redefined and Altered PAM-Sites

Dear Editor CRISPR from Prevotella and Francisella 1 （Cpfl） is an emerging RNA-guided endonuclease system that relies on thymidine-rich protospacer adjacent motif （PAM） for DNA targeting （Zetsche et al., 2015）. CRISPR-Cpfl has unique features that could be advantageous over the CRISPR-Cas9 system. For example, Cpfl requires only a 42 nt crRNA, while Cas9 uses 100 nt gRNA. While Cas9 generates blunt ends of DNA breaks, the Cpfl cleavage results in 5＇ overhangs distal from the protospacer, which may improve efficiency for NHEJ-based gene insertion. Interestingly, Cpfl proteins also have RNase activity （Fonfara et al., 2016）, which was utilized to process crRNA arrays for multiplexed genome editing in both mammalian systems and plants （Wang et al., 2017; Zetsche et al., 2017）.