RNA-guided DNA insertion with CRISPR-associated transposases

Abstract

CRISPR-Cas nucleases are powerful tools for manipulating nucleic acids; however, targeted insertion of DNA remains a challenge, as it requires host cell repair machinery. Here we characterize a CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST) that consists of Tn7-like transposase subunits and the type V-K CRISPR effector (Cas12k). ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 to 66 base pairs downstream of the protospacer. ShCAST integrates DNA into targeted sites in the Escherichia coli genome with frequencies of up to 80% without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion.

Figure 1.. Targeting requirements for CRISPR-associated transposase (CAST) systems

A. Schematic of the Scytonema hofmanni CAST locus containing Tn7-like proteins, the CRISPR-Cas effector Cas12k, and a CRISPR array. B. Fluorescent micrograph of the cyanobacteria S. hofmanni. Scale bar, 40 uM. C. Alignment of small RNA-Seq reads from S. hofmanni. The location of the putative tracrRNA is marked. D. Schematic of experiment to test CAST system activity in E. coli. E. PAM motifs for insertions mediated by ShCAST and AcCAST. F. ShCAST and AcCAST insertion positions identified by deep sequencing. G. Insertion frequency of ShCAST system in E. coli with pTarget substrates as determined by ddPCR. Error bars represent s.d. from n=3 replicates.

Figure 2.. Genetic requirements for RNA-guided insertions

A. Genetic requirement of tnsB, tnsC, tniQ, Cas12k, and tracrRNA on insertion activity. Deleted components are indicated by a dashed outline. B. Insertion activity of 6 tracrRNA variants expressed with the pJ23119 promoter. C. Schematic of tracrRNA and crRNA base pairing and two sgRNA designs highlighting the linker sequence (blue). D. Insertion activity into pTarget containing ShCAST transposon ends relative to activity into pTarget without previous insertion.

Figure 3.. In vitro reconstitution of an RNA-guided transposase.

A. Schematic of in vitro transposition reactions with purified ShCAST proteins and plasmid donor and targets. B. RNA requirements for in vitro transposition. pInsert was detected by PCR for LE and RE junctions. All reactions contained pDonor and pTarget. Schematics indicate the location of primers and the expected product sizes for all reactions. C. Targeting specificity of ShCAST in vitro. All reactions contained ShCAST proteins and sgRNA. D. Protein requirements for in vitro transposition. All reactions contained pDonor, pTarget, and sgRNA. E. CRISPR-Cas effector requirements for in vitro transposition. All reactions contained ShCAST proteins, pDonor, and pTarget. F. Chromatograms of pInsert reaction products following transformation and extraction from E. coli. LE and RE elements are highlight and the duplicated insertion sites denoted. G. For all panels, ShCAST proteins were used at a final concentration of 50 nM, and n=3 replicates for all reactions were performed with a representative image shown.

Figure 4.. ShCAST mediates genome insertions in E. coli

A. Schematic of experiment to test for genome insertions in E. coli. B. Insertion frequency at 10 tested protospacers following ShCAST transformation. Insertion frequency was determined by ddPCR on extracted genomic DNA. Error bars represent s.d. from n=3 replicates. C. Flanking PCR of 3 tested protospacers in a population of E. coli following ShCAST transformation. Schematics indicate the location of primers and the expected product sizes. D. Insertion site position as determined by deep sequencing following ShCAST transformation. E. Insertion positions determined by unbiased donor detection. The location of each protospacer is annotated along with the percent of total donor reads that map to the target.

Figure 5.. Model for RNA-guided DNA transposition

The ShCAST complex that consists of Cas12k, TnsB, TnsC, and TniQ mediates insertion of DNA 60–66 bp downstream of the PAM. Transposon LE and RE sequences along with any additional cargo genes are inserted into DNA resulting in the duplication of 5 bp insertion sites.