STAR Methods

Rapid cell-free characterization of multi-subunit CRISPR effectors and transposons

Plasmid construction

Standard cloning methods Gibson Assembly, Site Directed Mutagenesis (SDM) and Golden Gate were used to clone plasmids used in TXTL experiments. pPAMlibrary containing a PAM library with five randomized nucleotides was generated by SDM on p70a-deGFP_PacI with primers FW531 and FW532 (Table S5). Single-spacer CRISPR arrays were generated either with Golden Gate adding spacer sequences in a plasmid containing two repeat sequences interspaced by two BaeI or BbsI restriction sites or by SDM on pEc_gRNA1, pEc_gRNA2 or pEc_gRNAnt to change the repeat sequences to match the tested CRISPR systems. Plasmids harboring different PAM sequences for PAM validation assays were generated by SDM on p70a-deGFP_PacI. To generate plasmids encoding _X. albilineans type I-C and type I-F1 Cas proteins, genomic DNA isolated from Xanthomonas albilineans CFBP7063 was PCR amplified using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and cloned into pET28a using Gibson Assembly. All other plasmids were generated with Gibson Assembly or SDM (Table S5). All constructed plasmids were verified with Sanger sequencing.

For the VcCAST in vivo transposition experiments we cloned into the previously described pSL0284 vector (Klompe et al., 2019) two spacers targeting the lacZ gene of the E. coli BL21 (DE3) genome, yielding the pQCas_CAA and pQCas_AAA vectors. The protospacer targeted by the former vector has a 5’CAA PAM, whereas the protospacer targeted by the latter vector has a 5’AAA PAM.

For the RoCAST in vivo transposition experiments, genes encoding the Rippkaea orientalis tnsAB, tnsC, tnsD and tniQ were synthesized (Twist Bioscience) and cloned in the pET24a vector in various combinations, resulting in the construction of the pRoTnsABC, pRoTnsABCD, pRoTnsABCQ, pRoTnsABCDQ vectors (Table S5). The Rippkaea orientalis Cascade operon (cas6, cas8, cas7, cas5) was synthesized (Twist Bioscience) and cloned into the pCDFDuet-1 vector together with a gfp gene flanked by two BsaI restriction sites and the corresponding CRISPR direct repeats. Into the resulting pRoCascadegfp vector we cloned a spacer targeting the _lacZ gene of the E. coli BL21 (DE3) genome and a non-targeting control spacer, constructing the pRoCascadeT (targeting) and pRoCascade_NT (non-targeting) vectors, respectively (Table S5). DNA fragments encoding the right and left RoCAST ends were synthesized (IDT) and cloned into the pUC19 vector flanking a _gfp gene, yielding pRoDonor (Table S5). A 105-bp long DNA fragment from the Rippkaea orientalis genome, encoding the region which is located right upstream of the left end of RoCAST and includes the last 74 bp of the tRNA-Leu gene, was synthesized (IDT) and cloned into the pCDFDuet-1 vector, resulting in the construction of the pRoTarget vector (Table S5).

PAM-DETECT

A plasmid with five randomized nucleotides flanking a target site covering a PacI restriction enzyme recognition site was constructed as described before. If Cas proteins required for Cascade formation were encoded on separate plasmids, a MasterMix with the required Cas protein encoding plasmids in their stoichiometric amount was prepared beforehand. Thereby, a stoichiometry of Cas8e1-Cse22-Cas76-Cas51-Cas61 was used for all Type I-E systems. A 6 µL TXTL reaction was assembled consisting of 3 nM (high Cascade) or 0.25 nM (low Cascade) of the Cascade-encoding plasmid or the Cascade MasterMix, 4.5 µL myTXTL Sigma 70 Master Mix, 0.2 nM pET28aT7RNAP, 0.5 mM IPTG, 1 nM gRNA-encoding plasmid and 1 nM pPAM_library. A negative control containing all components from the reaction besides the Cascade plasmids and the gRNA-expressing plasmid was included. PAM-DETECT assays assessing either the type I-C or the type I-F1 system in _X. albilineans were lacking IPTG in their reactions. TXTL reactions were incubated at 29°C for 6 h or 16 h. The samples were diluted 1:400 in nuclease-free H2O. 500 µL were digested at 37°C with PacI (NEB) at 0.09 units/µL in 1x CutSmart Buffer (NEB) for 1 h and 500 µL were used as a “non-digested” control by adding nuclease-free H2O instead of PacI. After inactivation of PacI at 65°C for 20 min, 0.05 mg/mL Proteinase K (GE Healthcare) was added and incubated at 45°C for 1 h. After inactivation of Proteinase K at 95 °C for 5 min, remaining plasmids were extracted via standard EtOH precipitation. Illumina adapters with unique dual indices were added by two amplification steps with KAPA HiFi HotStart Library Amplification Kit (KAPA Biosystems) and purified by Agencourt AMPure XP (Beckman Coulter) after every PCR reaction. The first PCR reaction adds the Illumina sequencing primers with primers that can be found in Table S5 using 15 µL of the EtOH-purified samples in a 50 µL reaction and 19 cycles. The second PCR adds the unique dual indices and the flow cell binding sequence using 1 ng purified amplicons generated with the first PCR using 18 cycles. The samples were submitted for next-generation sequencing with 50 bp paired-end reads with 1.25 or 2.0 million reads per sample on an Illumina NovaSeq 6000 sequencer. PAM wheels were generated according to Leenay et al. (Leenay et al., 2016). Nucleotide enrichment plot generation was adapted to the script from Marshall et al. (Marshall et al., 2018) by changing the script to visualize the probability of a given nucleotide at a given position by depicting the percentage of the nucleotide in that position. All PAM-DETECT assays were done in duplicates and PAM wheel and nucleotide enrichment plots show averages. The generated NGS data have been deposited in NCBI’s Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE179614 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE179614 ). The following token can be used to access the data prior to publication: exexiqgyhrcblgj.

qPCR Reactions

To assess the remaining amount of PAM-library containing plasmid after conducting PAM-DETECT, quantitative PCR (qPCR) was performed using SsoAdvanced Universal SYBR Green Supermix (Biorad) in 10 µL reactions. The reactions were quantified using a QuantStudio Real-Time PCR System (Thermo Fisher) with an annealing temperature of 68 °C according to manufacturers’ instructions. All samples were prepared by using the liquid handling machine Echo525 (Beckman Coulter).

deGFP repression assays in TXTL

To assess activity of CRISPR-Cas systems, deGFP-repression assays in 3 µL or 5 µL TXTL reactions were conducted, measuring deGFP-expression over time in a 96-well V-bottom plate with BioTek Synergy H1 plate reader (BioTek) at 485/528 nm excitation/emission (Shin and Noireaux, 2012). All TXTL samples were either prepared by hand or by using the liquid handling machine Echo525 (Beckman Coulter).

3 µL TXTL reactions for PAM validation assays were prepared containing Cascade plasmid concentrations according to Table S2. If Cas proteins required for Cascade formation were encoded on separate plasmids, a MasterMix with the required Cas protein encoding plasmids in their stoichiometric amount was prepared beforehand. Thereby, a stoichiometry of Cas51-Cas81-Cas77 was used for X. albilineans Type I-C, Cas8f11-Cas5f11-Cas7f16-Cas6f1 was used for X. albilineans Type I-F1 and Cas8e1-Cse22-Cas76-Cas51-Cas61 was used for all Type I-E systems. Other components included in the TXTL reactions were 2.25 µL myTXTL Sigma 70 Master Mix, 0.2 nM p70aT7RNAP, 0.5 mM IPTG and 1 nM gRNA-encoding plasmid. After a 4 h pre-incubation at 29 °C or 37 °C that allowed the ribonucleoprotein complex of Cascade and crRNA to form, 1 nM reporter plasmid (pGFP_XXXXX) with various PAM sequences in close proximity to the promoter driving deGFP expression was added to the reaction to ensure Cascade-binding would lead to deGFP inhibition. The reactions were incubated for additional 16 h at 29 °C or 37 °C while measuring deGFP expression. The gRNAs were constructed to target a protospacer within the _degfp promoter located adjacent to the various PAM sequences.

To test the cleavage and/or binding ability of the type I-C and the type I-F1 systems in X. albilineans, 3 µL TXTL assays were conducted containing Cascade-encoding plasmids in the stoichiometry as mentioned before. To test binding ability, 2.25 µL myTXTL Sigma 70 Master Mix, 0.2 nM p70a_T7RNAP, 0.5 mM IPTG, 1 nM gRNA1-, gRNA2, or gRNAnt-encoding plasmid and 1 nM or 0.25 nM Cascade MasterMix was added to a TXTL reaction for the type I-C and type I-F1 system, respectively. To test cleavage ability, 2.25 µL myTXTL Sigma 70 Master Mix, 0.2 nM p70a_T7RNAP, 0.5 mM IPTG, 1 nM gRNA1-, gRNA2, or gRNAnt-encoding plasmid, 1 nM Cascade MasterMix and 0.5 nM or 0.25 nM pXalb_IC_Cas3 or pXalb_IF_Cas2-3 was added to a TXTL reaction for the type I-C and type I-F1 system, respectively. After 4 h pre-expression at 29°C, 1 nM p70a_deGFP reporter plasmid was added to the reactions and incubated for additional 16 h at 29°C while measuring deGFP-fluorescence. gRNA1 is designed to target a protospacer within the promoter driving deGFP expression adjacent to a type I-C TTC or a type I-F1 CC PAM to ensure Cascade-binding would lead to deGFP-inhibition. gRNA2 is designed to target a protospacer adjacent to a type I-C TTC or a type I-F1 CC PAM upstream of the promoter to ensure cleavage of the targeted plasmid would result in deGFP-inhibition whereas binding-only would result in deGFP-production. gRNAnt represents a non-targeting control.

5 µL TXTL reactions assessing dispensability of TniQ for V. cholerae I-F CAST Cascade-binding were performed with reactions containing 3.75 µL myTXTL Sigma 70 Master Mix, 0.2 nM p70a_T7RNAP, 0.5 mM IPTG and 0.5 nM pVch_IF_CasQ_gRNA3/nt or 0.5 nM pVch_IF_Cas_gRNA3/nt. After a 4 h pre-incubation step at 29 °C, the reporter plasmid p70a_deGFP was added and the reactions were incubated for additional 16 h at 29 °C while measuring deGFP-fluorescence. gRNA3 is designed to target a protospacer within the promoter driving deGFP expression adjacent to a CC PAM. gRNAnt represents a non-targeting control.

Transposition in TXTL

To assess crRNA-dependent transposition of the Vibrio cholerae Tn6677 I-F CAST in TXTL, 5 µL TXTL reactions containing 3.75 µL myTXTL Sigma 70 Master Mix, 0.2 nM p70a_T7RNAP, 0.5 mM IPTG, 1 nM of the previously described donor plasmid (pSL0527), 2 nM of the previously described TnsABC-plasmid (pSL0283) (Klompe et al., 2019), 1 nM p70a_deGFP and 1 nM pVch_IF_CasQ_gRNA3 or pVch_IF_CasQ_gRNAnt were prepared. The reactions were incubated at 29 °C for 16 h. Transposition events were detected in a 1:400 dilution of the TXTL reaction by PCR amplification using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and combinations of donor DNA and genome specific primers. Transposition was verified by Sanger sequencing (Table S5).

crRNA-dependent transposition of RoCAST in TXTL was performed in 3 µL TXTL reactions consisting of 2.25 µL myTXTL Sigma 70 Master Mix, 0.2 nM p70a_T7RNAP, 0.5 mM IPTG, 1 nM pRoCascade, 1 nM pRo_gRNA2/nt, 1 nM pGFP_CAATG, 1 nM pRoDonor or pRoDonor_extended and 1 nM pRoTnsABC, pRoTnsABCD, pRoTnsABCQ or pRoTnsABCDQ. The reactions were incubated at 29 °C for 16 h. Transposition events were detected in a 1:100 dilution of the TXTL reaction by PCR amplification using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and combinations of donor DNA and genome specific primers (Table S5). Transposition was verified by Sanger sequencing.

crRNA-independent transposition of RoCAST in TXTL was performed in 3 µL TXTL reactions consisting of 2.25 µL myTXTL Sigma 70 Master Mix, 0.2 nM p70a_T7RNAP, 0.5 mM IPTG, 1 nM pRoTarget, 1 nM pRoDonor and 1 nM pRoTnsABC, pRoTnsABCD, pRoTnsABCQ or pRoTnsABCDQ. The reactions were incubated at 29 °C for 16 h. Transposition events were detected in a 1:100 dilution of the TXTL reaction by PCR amplification using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and combinations of donor DNA and genome specific primers (Table S5). Transposition was verified by Sanger sequencing.

Transposition in vivo

For the crRNA-dependent transposition in vivo using the I-F CAST from Vibrio cholerae Tn6677, we employed the previously described transposition system (Klompe et al., 2019). We electroporated 30 ng of the pSL0283 vector with 30 ng of the pSL0527 vector and 30 ng of either the pQCasCAA or pQCas_AAA vector into _E. coli BL21(DE3) electrocompetent cells. We plated a fraction of each electroporation mixture on 100 mg/ml ampicillin, 50 mg/ml spectinomycin, 50 mg/ml kanamycin, 0.1 mM IPTG and 100 µg/ml X-gal containing LB-agar plates. The plates were incubated for 24 h at 30°C and the formed colonies were subjected to blue/white screening. Transposition events were identified by colony PCR using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and genome specific primers (Table S5).

For the crRNA-dependent transposition in vivo using RoCAST, we electroporated 30 ng of either pRoCascadeT or pRoCascade_NT vector with 30 ng of pRoDonor and 30 ng of either pRoTnsABC, pRoTnsABCD, pRoTnsABCQ or pRoTnsABCDQ vector into _E. coli BL21(DE3) electrocompetent cells. We plated a fraction of each electroporation mixture on 100 mg/ml ampicillin, 50 mg/ml spectinomycin, and 50 mg/ml kanamycin containing LB-agar plates. The plates were incubated for 20 h at 37°C and the formed colonies were scraped and resuspended in LB liquid medium. A fraction of each cell suspension was re-plated on LB-agar plates supplemented with 100 mg/ml ampicillin, 50 mg/ml spectinomycin, 50 mg/ml kanamycin and 0.01 mM IPTG for induction of the expression of the Cascade and transposase proteins. The plates were incubated 20 h at 37°C and all the formed colonies were scraped and resuspended in LB liquid medium. A fraction of each cell suspension was subjected to gDNA isolation using the illustra Bacteria genomicPrep Mini Spin Kit (GE Healthcare). Transposition events were identified by PCR using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and combinations of donor DNA and genome specific primers (Table S5).

For the crRNA-independent in vivo transposition using RoCAST, we electroporated 30 ng of the pRoTarget with 30 ng of pRoDonor and 30 ng of either the pRoTnsABC, pRoTnsABCD, pRoTnsABCQ or pRoTnsABCDQ vector into E. coli BL21(DE3) electrocompetent cells. We plated a fraction of each electroporation mixture on 100 mg/ml ampicillin, 50 mg/ml spectinomycin, and 50 mg/ml kanamycin containing LB-agar plates. The plates were incubated for 20 h at 37°C and the formed colonies were scraped and resuspended in LB liquid medium. A fraction of each cell suspension was re-plated on LB-agar plates supplemented with 100 mg/ml ampicillin, 50 mg/ml spectinomycin, 50 mg/ml kanamycin and 0.01 mM IPTG for induction of the expression of the transposase proteins . The plates were incubated 20 h at 37°C and all the formed colonies were scraped and resuspended in LB liquid medium. A fraction of each cell suspension was subjected to gDNA isolation using the illustra Bacteria genomicPrep Mini Spin Kit (GE Healthcare). Transposition events were identified by PCR using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and combinations of donor DNA and pRoTarget specific primers (Table S5).

Assessing transposition insertion point

To assess the exact insertion point of Rippkaea orientalis I-B2.2 CAST, in vivo and in vitro, transposition assays were conducted as previously described and the transposition products were PCR amplified and sent for next-generation sequencing. Illumina adapters with unique dual indices were added by two amplification steps with KAPA HiFi HotStart Library Amplification Kit (KAPA Biosystems) and each amplicon was purified by Agencourt AMPure XP (Beckman Coulter). The first PCR reaction adds the Illumina sequencing primer sites with primers that can be found in Table S5, the second PCR adds the unique dual indices and the flow cell binding sequences. 2 µL of 1:100 dilutions were used in a 50 µL PCR reaction to amplify TXTL reactions using either 19 or 30 cycles. 50 ng of genomic DNA were used in a 50 µL PCR reaction to amplify in vivo transposition with either 19 or 30 cycles. 1 ng of purified TXTL or in vivo-amplicon were subjected to the second PCR using 18 cycles. Library-pools consisting of six samples were submitted for next-generation sequencing with 300 paired-end reads with 0.15 million reads on an Illumina MiSeq machine.

The generated NGS data have been deposited in NCBI’s Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE179614 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE179614). The following token can be used to access the data prior to publication: exexiqgyhrcblgj.

QUANTIFICATION AND STATISTICAL ANALYSIS

deGFP repression assays in TXTL

The fluorescence background was subtracted from the endpoint deGFP values with TXTL samples consisting of only myTXTL Sigma 70 Master Mix and nuclease-free water. The resulting endpoint deGFP values were either depicted as averages of a targeting gRNA and a non-targeting gRNA or fold change-repression was calculated by the ratio of non-targeting over the targeting deGFP values. Significance was calculated with Welch’s t-test. P > 0.05 is shown as ns, P < 0.05 is shown as *, P < 0.01 is shown as ** and P < 0.001 is shown as *. Within the PAM validation assays represented as fold changes, significance was calculated between the fold change of a given PAM and the fold change of a PAM that corresponds to the 3’ end of the repeat of the tested CRISPR system. The fold changes of the PAM validation in Fig. 3B are depicted in a heat map. Thereby a difference between a non-targeting sample and a targeting sample with a specific PAM resulting in P > 0.05 is shown in white and excluded from further analysis. For all other samples within the heat map, the fold changes were calculated as mentioned above and presented relative to the highest fold change within one system. Significance within the deGFP repression assays testing binding and cleavage ability of the type I-C and the type I-F1 system in X. albilineans was calculated with the targeting and non-targeting sample for each condition. For the endpoint measurements in Fig. 5C**, significance was calculated between a non-targeting sample and a targeting sample targeting the same PAM.

qPCR

Cq values were used to measure target amounts. To calculate the relative abundance of the PAM library containing plasmid in the digested sample to the non-digested sample, the relative plasmid amount was normalized to a control amplifying the pET28a-T7RNAP that has no PacI recognition site using the the 2^(-(ddCt) method. Significance to the control sample lacking a CRISPR-Cas system was calculated with Welch’s t-test. P > 0.05 is shown as ns, P < 0.05 is shown as *, P < 0.01 is shown as ** and P < 0.001 is shown as *** .

Assessing transposition insertion point

∼15 nts long sequences 5’ of the transposon terminal left end were extracted, counted and sorted. The sequences were mapped to the targeted plasmid or the targeted genome tolerating 2 nts mismatches and the distance between the insertion point and the PAM upstream of the protospacer or the end of the tRNA-Leu gene was noted. To only depict reliable insertion points, we present insertion points with more than 20 reads. The insertion points are shown as bar graphs.

The processed NGS data have been deposited in NCBI’s Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE179614 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE179614). The following token can be used to access the data prior to publication: exexiqgyhrcblgj.

In silico selection of representative type I-E CRISPR-Cas systems for PAM-DETECT

HMM profiles for the Cas5e, Cas6e, Cas7e and Cas8e proteins were developed upon aligning the members of the corresponding protein families (Cas5e: pfam09704, TIGR1868, TIGR02593; Cas6e: pfam08798, TIGR01907; Cas7e: pfam09344, TIGR01869; Cas8e: pfam 09481, TIGR02547). A new HMM profile was generated for the less conserved Cse2 protein upon aligning sequences with known 3D structure using PROMALS3D server (Pei et al., 2008) followed by a series of iterative alignment/model building steps to include additional sequences and increase sequence diversity. For the aligning processes of all five proteins, sequences were dereplicated at 90% identity using cd-hit (Huang et al., 2010) (with options -c 0.90 -g 1 -aS 0.9). The dereplicated sequences were compared against each other using blastp from blast+ v2.6.0 (Altschul et al., 1990) with e-value 10e-05 and defaults for the rest of parameters. Hits were filtered to retain those at >=60% pairwise identity, and were next clustered using the mcl algorithm (Enright et al., 2002) with inflation parameter of 2.0. Clusters with >=10 members were aligned using Gismo (Neuwald and Liu, 2004) with default parameters, and consensus sequences were extracted from the alignments. These consensus sequences, as well as singletons and sequences from smaller clusters were aligned using Gismo (Neuwald and Liu, 2004). Alignments were manually curated to remove shorter sequences that did not have one or more of the active site positions and HMM profiles were generated using hmmbuild (Eddy, 2009). Hmmsearch (Eddy, 2009) using the generated HMM profiles against all public genomes (isolates, SAGs, and MAGs), and all public metagenomes resulted in hits which were subsequently aligned against the generated HMM profiles. After selecting gene arrays that have all five complete or nearly complete genes, we identified 6,964 arrays in public genomes and 5,000 arrays in public metagenomes. Aligned sequences for all proteins from the same array were concatenated, and the resulting sequences were dereplicated with cd-hit (Huang et al., 2010) at 90% identity, aligned over at least 90% of the shorter sequences. This resulted in 2851 clusters, 1799 from metagenomes and 1052 from genomes. Whereas the alignment of the Cas8e proteins from these clusters showed high variability, the predicted L1 helix regions of the Cas8e, which have been shown to directly interact with the PAM (Xiao et al., 2017), presented higher conservation. We generated a list with the L1 signatures from the dereplicated cluster set and we subsequently manually filtered out systems that do not belong to known cultured mesophilic bacteria (Table S3). From the resulting list we selected I-E CRISPR/Cas systems with a variety of L1 motifs for experimental validation with PAM-DETECT.

Comparative analysis of I-B CAST transposases

We searched previous literature (Peters et al., 2017; Saito et al., 2021) for in silico identified I-B2 CASTs, which contain a fused tnsAB gene and are easily distinguished from I-B1 CASTs, which contain separate tnsA and tnsB genes. We observed that one clade of the I-B2 CASTs encompasses systems with tnsAB-tnsC-tnsD operons while having the tniQ gene separated, whereas the other clade encompasses systems with tnsAB-tnsC-tniQ operons and the tnsD gene separated. We denoted the systems in the former clade as I-B2.1 CASTs and in the latter clade as I-B2.2 CASTs. We focused on the I-B2.2 CAST clade, that has no in vitro or in vivo characterized members, and we discarded from further analysis the systems that lacked at least one of the CRISPR-Cas or transposition genes (tnsAB, tnsC, tnsD, tniQ, cas5, cas6, cas7, cas8). We performed BlastP search (Altschul et al., 1990) using the TnsAB, TnsC, TnsD, TniQ proteins of each selected I-B2.2 system as queries, aiming to identify additional I-B2.2 CAST candidates. Our analysis yielded in total seven I-B2.2 systems and we selected six previously described I-B2.1 systems for phylogenetic analysis (Saito et al., 2021). The alignment of I-B2.1 and I-B2.2 transposition proteins was performed using T-Coffee (Di Tommaso et al., 2011), the phylogenetic trees were built using average distance and the BLOSUM62 matrix and they were visualized with JalView (Waterhouse et al., 2009).

In silico analysis of RoCAST

We predicted the CRISPR array of RoCAST by uploading the Rippkaea orientalis genomic region between the Rocas5 and RotniQ to CRISPRFinder (Grissa et al., 2007). The RoCAST ends were determined manually on Benchling by searching for repeat sequences of 20 nucleotides, with maximum 5 mismatched nucleotides, within the Rippkaea orientalis genomic regions 1 kb upstream of the R. orientalis tnsAB and 1 kb downstream of the RotnsD. We identified two types of repeat sequences present in both regions in opposite orientations and a candidate duplication region. Notably, we identified five repeat sequences in the predicted left end region, with one of the repeat sequences located downstream of the predicted duplication site, hence outside of the predicted RoCAST limits. The TXTL transposition demonstrated that this repeat is not part of the RoCAST transposon.

Article TitleRapid cell-free characterization of multi-subunit CRISPR effectors and transposons

Abstract

CRISPR-Cas biology and technologies have been largely shaped to-date by the characterization and use of single-effector nucleases. In contrast, multi-subunit effectors dominate natural systems, represent emerging technologies, and were recently associated with RNA-guided DNA transposition. This disconnect stems from the challenge of working with multiple protein subunits in vitro and in vivo. Here, we apply cell-free transcription-translation (TXTL) to radically accelerate the characterization of multi-subunit CRISPR effectors and transposons. Numerous DNA constructs can be combined in one TXTL reaction, yielding defined biomolecular readouts in hours. Using TXTL, we mined phylogenetically diverse I-E effectors, interrogated extensively self-targeting I-C and I-F systems, and elucidated targeting rules for I-B and I-F CRISPR transposons using only DNA-binding components. We further recapitulated DNA transposition in TXTL, which helped reveal a distinct branch of I-B CRISPR transposons. These capabilities will facilitate the study and exploitation of the broad yet underexplored diversity of CRISPR-Cas systems and transposons.

  • PAM-DETECT for rapid determination of PAMs for Type I CRISPR-Cas systems in TXTL
  • Mining of Type I orthologs and characterization of extensively self-targeting systems
  • TXTL-based assessment of DNA target recognition and transposition by CRISPR transposons
  • Identification of a distinct branch of Type I-B CRISPR transposons

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