The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases

Abstract

IscB proteins are putative nucleases encoded in a distinct family of IS200/IS605 transposons and are likely ancestors of the RNA-guided endonuclease Cas9, but the functions of IscB and its interactions with any RNA remain uncharacterized. Using evolutionary analysis, RNA sequencing, and biochemical experiments, we reconstructed the evolution of CRISPR-Cas9 systems from IS200/IS605 transposons. We found that IscB uses a single noncoding RNA for RNA-guided cleavage of double-stranded DNA and can be harnessed for genome editing in human cells. We also demonstrate the RNA-guided nuclease activity of TnpB, another IS200/IS605 transposon-encoded protein and the likely ancestor of Cas12 endonucleases. This work reveals a widespread class of transposon-encoded RNA-guided nucleases, which we name OMEGA (obligate mobile element–guided activity), with strong potential for developing as biotechnologies.

Fig. 1.. IscBs are associated with ncRNAs of unknown function.

(A) Comparison of IscB and Cas9 domains and previously described ncRNAs. (B) Phylogenetic analysis of the RuvC, BH, and HNH domains of Cas9 and IscB clusters using IQ-Tree 2. Genomic association shows 16/603 IscB clusters have strong association to CRISPR, occurring independently in multiple clades. (C) Small RNA-seq of a heterologously expressed CRISPR-associated IscB locus (top) and RNP pulldown (bottom). (D) Sequence logo for the PAM as determined by a plasmid depletion assay. (E) In vitro cleavage by IscB-single guide RNA RNP complex. (F) (Top) Conservation analysis of regions upstream of N=563 non-redundant IscB loci. (Bottom) Small RNA-seq of an IscB locus in K. racemifer strain SOSP1-21. (G) Secondary structure predictions of CRISPR-associated IscB ncRNA and IscB ωRNA. Guiding function of ωRNAs was inferred by comparison of the two structures. TE: transposon end.

Fig. 2.. IscB is an RNA-guided DNA endonuclease.

(A) Design of an IVTT-based TAM screen. (B) KraIscB-1 endogenous target and reprogrammed target sequences used in IVTT TAM screens. (C) dsDNA cleavage by KraIscB-1 and ωRNA targeting sequence flanked by ATAAA 3’ TAM. (D) dsDNA clevage by AwaIscB and ωRNA targeting sequence flanked by ATGA 3’ TAM. (E) In vitro-reconstituted AwaIscB-ωRNA RNP cleavage of dsDNA substrates in the presence or absence of a target and/or TAM. TS: target strand; NTS: non-target strand; nt: nucleotides. (F) In vitro dsDNA cleavage of AwaIscB with selectively inactivated nuclease domains. (G) Sequencing of cleavage products generated by AwaIscB.

Fig. 3.. Guide-encoding mechanisms of IscB

(A) Example loci for each major mechanism of encoding multiple guides: entire ωRNAs arrays associate with IscB, ωRNAs duplicate or insert into CRISPRs, transposition expansion results in multiple nearly identical loci that each express different guides, and standalone trans-acting ωRNAs form independently of adjacent IscBs. (B) K. racemifer encodes 48 IscB loci with cis ωRNAs and 10 standalone trans-acting ωRNAs. (C) Small RNA-seq of a standalone ωRNA locus in K. racemifer. (D) KraIscB-1, in complex with cis or trans ωRNAs with the same guide sequence, mediate cleavage of dsDNA in a TAM- and target-dependent manner. Reactions were performed in IVTT using 5’ strand-specific labeled linear targets. TS: Target strand; NTS: non-target strand. Contig accession and position information for all displayed loci are listed in Table S6.

Fig. 4.. Diversity and evolution of IscB

(A) Phylogenetic tree of IsrB, IscB, and Cas9. Associations with IS200/605 TnpA, ωRNA, CRISPR arrays, anti-repeats (where applicable), and Cas acquisition genes. ORF size of cluster representative is shown on the second outermost ring. Notable groups are shown as colored arcs on the outermost ring. First occurrences of evolutionary events in each clade are marked by colored circles/squares, as described in (B). CR: CRISPR array. (B) Parsimonious evolutionary timeline linking IsrB to Cas9 with exemplifying loci. Colors of protein of interest indicate distinct stages in the evolution of IsrB to Cas9. (C) Structural diversity and evolution of ωRNAs in IsrB and IscB systems.

Fig. 5.. Exploration of the diversity of IS200/605 superfamily nucleases

(A) Evolution between IS200/605 transposon superfamily-encoded nucleases and associated RNAs. Dashed lines reflect tentative/unknown relationships. (B) Locations of IscB loci and fragments in the I. tetrasporus genome. Intact locus is labeled as “ChlorIscB.” (C) Small RNA-seq of I. tetrasporus. (D) Weblogo of ChlorIscB cleavage TAM using a reprogrammed guide in an IVTT TAM screen. (E) Weblogo of OgeuIscB TAM using a reprogrammed guide in an IVTT TAM screen. (F) Targeted OgeuIscB-mediated indel formation at the VEGFA locus in HEK293FT cells ordered by abundance, with indel size on the left. (G) OgeuIscB-mediated indel formation at multiple sites in HEK293T cells, *P < 0.05). (H) Small RNA-seq of ωRNA from IsrB locus in K. racemifer strain SOSP1-21. (I) Weblogo of Desulfovigula thermocuniculi (DthIsrB) TAM using a reprogrammed guide in an IVTT TAM screen. (J) DthIsrB mediates ωRNA-guided non-target strand nicking in a TAM- and target-dependent manner in an IVTT cleavage assay using 5’ strand-specific labeled targets. (K) Small RNA-seq of ωRNA from TnpB locus in K. racemifer strain SOSP1-21. (L) Comparison of ωRNAs from K. racemifer IscB and TnpB loci. (M) Secondary structure prediction of KraTnpB-associated ωRNA. (N) Weblogo of A. macrosporangiidus TnpB (AmaTnpB) TAM using a reprogrammed guide in an IVTT TAM screen. (O) In vitro-reconstituted AmaTnpB cleavage of dsDNA substrates in the presence or absence of ωRNA, target, and/or TAM. (P) AmaTnpB performs ωRNA-guided TAM-independent target-dependent cleavage of 3’ Cy5.5-labeled ssDNA substrates. (Q) AmaTnpB cleaves a 3’ Cy5.5-labeled collateral ssDNA substrate in the presence of TAM- and target-containing dsDNA or target-containing ssDNA substrates. Contig accession and position information for all displayed loci are listed in Table S6.

Fig. 6.. Naturally occurring RNA-guided DNA-targeting systems

Comparison of Ω (OMEGA) systems with other known RNA-guided systems. In contrast to CRISPR systems, which capture spacer sequences and store them in the locus within the CRISPR array, Ω systems may transpose their loci (or trans-acting loci) into target sequences, converting targets into ωRNA guides.