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, 112 (11), 3570-5

Boosting CRISPR/Cas9 Multiplex Editing Capability With the Endogenous tRNA-processing System

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Boosting CRISPR/Cas9 Multiplex Editing Capability With the Endogenous tRNA-processing System

Kabin Xie et al. Proc Natl Acad Sci U S A.

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA-gRNA architecture were efficiently and precisely processed into gRNAs with desired 5' targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits.

Keywords: CRISPR/Cas9; genome editing; multiplex; tRNA processing.

Conflict of interest statement

Conflict of interest statement: The Pennsylvania State University filed a provisional patent application related to this study.

Figures

Fig. 1.
Fig. 1.
Engineering the endogenous tRNA system for multiplex genome editing with CRISPR/Cas9. (A) The eukaryotic pre-tRNA with 5′ leader and 3′ trailer is cleaved by RNase P and RNase Z at specific sites. (B) Transcription of tRNA gene with RNA polymerase III (Pol III). The box A and box B elements in the tRNA gene function as internal transcriptional elements and are bound by transcription factor IIIC (TFIII C), which recruits TFIIIB and Pol III to start transcription. (C) Schematic depiction of the PTG/Cas9 method for simultaneously targeting multiple sites. The synthetic PTG consists of tandemly arrayed tRNA-gRNA units, with each gRNA containing a target-specific spacer (labeled as a diamond with different color) and conserved gRNA scaffold (rectangle). The tRNA containing box A and B elements is shown as round rectangles. The primary transcript of PTG is cleaved by endogenous RNase P and RNase Z (labeled as scissors) to release mature gRNAs and tRNA (red lines of cloverleaf structure). The excised mature gRNAs direct Cas9 to multiple targets.
Fig. 2.
Fig. 2.
Precise excision of functional gRNAs in vivo from synthetic PTG genes. (A) The architecture of two sgRNA genes and four PTGs to produce one gRNA. (B) Sequence and predicted secondary structure of tRNA–gRNA–tRNA fusion of PTG gene. The bases of the tRNA region are indicated with red color and the tRNA 5′ leader is shown in lowercase. The gRNA is indicated in black, and the gRNA spacer sequence is shown as N. (CF) Examination of mature gRNAs produced from sgRNA or PTGs with cRT-PCR. Total RNAs from the protoplasts expressing empty vector were used as control (CK). Arrows indicate mature gRNAs amplified by cRT-PCR, and asterisks indicate the nonspecifically amplified rRNA. (G) Summary of excision sites in PTG according to mapped gRNA ends from cRT-PCR (SI Appendix, Figs. S3–S5). Arrows indicate the cleavage sites in PTG to release gRNA. The mature gRNA 5′ ends were excised from PTG exactly at the tRNA–gRNA fusion site in all cRT-PCR results whereas its 3′ ends shifted 1–4 nt within the tRNA 5′ leader (lowercase). (H) gRNA produced from U3p:sgRNA. All detected U3p:sgRNA-produced gRNA started with ribonucleotide A and terminated with multiple Us. (I) Introduction of indels at the desired sites by PTG1:Cas9 or PTG2:Cas9 in rice protoplasts as shown by PCR/RE. Arrows indicate mutated fragments resistant to RE digestion. The indel frequency is indicated at the bottom. (J) Relative expression of sgRNA1/2 and PTG1/2 in rice protoplasts. Data represent mean ± SD. ND, not detected. CK, empty vector control.
Fig. 3.
Fig. 3.
Simultaneous editing of multiple genomic sites in rice protoplasts expressing PTG:Cas9. (A) Architecture, gRNA components, and targets of PTGs for multiplex genome editing. (B) PCR detection of chromosomal fragment deletion at targeted loci in rice protoplasts expressing respective PTGs with Cas9. Successful deletion is shown as truncated PCR product (indicated with arrows). The chromosomal fragment deletion frequency (del %) is indicated at the bottom of each lane. The protoplast samples expressing an empty vector were used as control (CK). (C) Representative sequences of chromosomal fragment deletion aligned with that of WT. The gRNA paired region is labeled with green color, and the PAM region is shown in red color letters. The number at the end indicates deleted (−) or inserted (+) bases between two Cas9 cuts. The total length between two Cas9 cut sites (labeled with scissor) is indicated on the top. Short lines in the aligned sequences indicate deletions.
Fig. 4.
Fig. 4.
Highly efficient targeted mutagenesis in transgenic rice expressing PTG:Cas9. (A and B) Chromosomal fragment deletion in PTG7:Cas9 plant at T0 generation. Of note, only mpk1 with 358-bp deletion (Δ358) was detected in genomic DNA. Sequence analysis of the PCR products (the number in parentheses) reveals an identical deletion pattern in the transgenic plant. (C) Albino seedlings were regenerated from calli transformed with PTG10:Cas9. Most T0 seedlings (87%, n = 15) exhibited a similar photo-bleach phenotype, suggesting a very high efficiency of knocking out PDS with PTG10:Cas9. Vec, control plants transformed with empty vector. (Scale bar: 5 cm.)

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