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 rAPOBEC1 and PmCDA1 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), PmCDA1, 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.
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.
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