An Active Immune Defense with a Minimal CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA and without the Cas6 Protein*

Background: CRISPR RNAs (crRNAs) are generated by Cas6b in type I-B systems. They are essential for the interference reaction. Results: An icrRNA is generated independently from Cas6b and functions like a crRNA. Conclusion: In the presence of an icrRNA, Cas6b is not required for the interference reaction. Significance: This setup allows the Cas6b-independent generation of icrRNAs and thereby interference without Cas6b.

The prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) is a defense system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III, crRNAs are generated by the endonuclease Cas6. We developed a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analyzed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3 handle are still active in triggering an interference reaction. The complete 3 handle could be removed without loss of activity. However, manipulations of the 5 handle mostly led to loss of interference activity. Furthermore, we could show that in the presence of an icrRNA a strain without Cas6b (⌬cas6b) is still active in interference. .
Prokaryotes defend themselves against invaders using several different mechanisms to degrade foreign DNA or RNA, one of which is the clustered regularly interspaced short palindromic repeat-CRISPR associated (CRISPR-Cas) 3 system (1)(2)(3)(4)(5)(6). This defense mechanism progresses in three steps as follows: 1) adaptation; 2) CRISPR RNA expression and processing; and 3) invader degradation. During the first step, the cell identifies a new invader and integrates a piece of the invader DNA (termed protospacer) into the CRISPR locus of the host (as soon as the protospacer has been integrated into the CRISPR locus, it is termed spacer). An important distinguishing characteristic for the selection of a protospacer in the type I and II CRISPR-Cas systems is the protospacer adjacent motif (PAM) (7,8). This motif is located in the invader DNA, directly adjacent to the protospacer. The PAM sequence is important not only for its selection as a spacer but also for the third step of the process, the interference reaction. In the second step of the defense, the CRISPR RNA is synthesized yielding a long pre-crRNA that is processed into the mature functional crRNAs. These short RNAs are essential for the last step, the interference, where they detect the invader sequence and trigger degradation of the invader by Cas proteins (2,9). The prokaryotic immune system comes in a variety of different types that carry out the same reaction, namely the defense against foreign DNA or RNA. Data about the different systems reported show that although they carry out the same reaction they clearly differ in various aspects of the pathway. The different types of CRISPR-Cas have been grouped on the basis of their various Cas proteins into three major classes, CRISPR-Cas type I, II, and III (7), that have been further divided into initially 10 subclasses (IA-F, IIA-B, and IIIA-B) (7), with the number of newly defined subclasses constantly rising as more data about the systems are reported (10,11).
The key element in all CRISPR-Cas defense systems is the crRNA. The biogenesis of the crRNA involves either two or three steps, depending on the system. In all cases, the first step is the transcription of the CRISPR RNA locus into a long precursor, the pre-crRNA. The following maturation of the crRNA is catalyzed by the Cas6 protein in CRISPR-Cas type I and type III systems. In some type I systems, Cas6 is part of the CRISPRassociated complex of antiviral defense (Cascade) (12) that consists of different Cas proteins depending on the subtype (2). In contrast, in the type III system Cas6 is a standalone endonuclease (13,14). Processing by Cas6 within the repeat sequence directly yields the mature functional crRNA in types I-A, I-E, and I-F (9). The resulting crRNA consists of an eight-nucleotide repeat-derived 5Ј handle, the invader-targeting spacer sequence, and the 3Ј handle, which contains the remainder of the repeat sequence ( Fig. 1A) (2). In some type I systems (I-E and I-F), the Cas6 proteins stay bound to the crRNA after processing. In type III systems, a second maturation step is observed after Cas6 processing, which shortens the crRNA 3Ј end and sometimes removes the complete repeat sequence downstream of the spacer (14,15).
The initial invader DNA recognition is governed by Watson-Crick base pairing with a 7-10-nt segment of the crRNA referred to as the "seed" sequence (16 -19). The seed sequence is involved in initial pairing between crRNA and invader, and it allows rapid probing of different regions of cellular nucleic acids. If a perfect match between seed sequence and target DNA is found, the remainder of the spacer sequence of the crRNA base pairs with the invader DNA. In the type I-E system, the seed sequence is a seven-nucleotide-long noncontiguous sequence between the 5Ј end of the crRNA spacer sequence and the invader (17). In the type I-B system, this seed sequence is slightly longer with 10 nucleotides (20). An additional prerequisite for the interference is the presence of the PAM sequence in the invader DNA (2).
Here, we investigate the function of Cas6 in the interference reaction and the essential requirements for the crRNA in the type I-B system of the archaeon Haloferax volcanii. H. volcanii contains only one CRISPR-Cas system (I-B) that consists of eight Cas proteins (Cas1 to Cas5, Cas6b, 4 Cas7, and Cas8b) and three CRISPR RNA arrays (20). We could previously identify the PAM sequences for this system showing that six different PAMs are active in triggering degradation (21). The Haloferax I-B system has a Cascade-like complex, with Cas6b copurifying with the Cas5 and Cas7 proteins and the crRNA (22). It has been shown that the Cas6b protein is involved in crRNA maturation and that the crRNA 5Ј handles are eight nucleotides long; however, different 3Ј lengths have been reported (22).
We developed here a Cas6b-independent crRNA maturation pathway for the Haloferax type I-B system in vivo that expresses a functional crRNA, which we termed independently generated crRNA (icrRNA). The icrRNA is transcribed with flanking tRNA-like structures (so-called t-elements) that are processed by the tRNA processing enzymes RNase P and tRNase Z (23). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence.
We show here that a minimal crRNA in the I-B system needs a seven-nucleotide 5Ј handle and does not require a 3Ј handle at all. In addition, we show that the Cas6b protein is not required for the interference reaction when an icrRNA is present. With the Cas6b-independent maturation pathway developed here, the first in vivo analysis of crRNA characteristics essential for the interference reaction was possible.

Construction of Plasmids and Transformation of H. volcanii-
The plasmids for expressing icrRNA (pTA409-telecrRNA, pTA232-telecrRNA, and telecrRNA variants in both vectors) were generated as follows (plasmids are listed in Table 1). The DNA fragment containing the crRNA or crRNA mutants flanked by t-elements were ordered from GeneArt as plasmids pMA-RQ-telecrRNA and pMA-telecrRNA. Plasmids contained a synthetic Haloferax promoter, 5 the crRNA, flanked by t-elements and a synthetic Haloferax terminator. 5 Plasmids were digested with KpnI and BamHI to isolate the DNA fragment containing the complete insert. The resulting fragment was cloned into pTA409 (26) and pTA232 (27) (both digested with KpnI and BamHI). Four crRNA mutants were generated by inverse PCR on pMA-telecrRNA using primer pairs (primer sequences are listed in Table 1) itele1/del1, itele1/del1, itele1/ del1, and itele1/del1 to generate variant 13 (deletion of the last five nucleotides of the 3Ј handle), 14 (deletion of the last 10 nucleotides of the 3Ј handle), 15 (deletion of the last 15 nucleotides of the 3Ј handle), and 16 (deletion of the last 20 nucleotides of the 3Ј handle), respectively. In preparation for transformation, all plasmids were passaged through E. coli GM121 cells to avoid methylation. Haloferax cells were subsequently transformed using the polyethylene glycol method (27,28).

Generation of a CRISPR Locus C Gene Deletion Strain (⌬C)-
The deletion of the CRISPR locus C was achieved by using the pop-in/pop-out method as described previously (24,25,29). The region upstream of the gene for CRISPR locus C was PCRamplified with flanking regions from the chromosomal DNA of H. volcanii strain H119 using primers Cdelup (containing the restriction site KpnI) and Cdelupi (containing the restriction site EcoRV). The resulting 300-bp PCR fragment was subsequently cloned into the vector pTA131 (digested with KpnI and EcoRV), yielding pTA131-Cup. Next, the region downstream of the locus C gene was amplified using primers Cdeldo (containing the restriction site XbaI) and Cdeldoi (containing the restriction site EcoRV). The resulting 500-bp fragment was cloned into the plasmid pTA131-Cup (digested with EcoRV and XbaI), yielding plasmid pTA131-Cupdo. This plasmid was digested with EcoRV to insert the marker gene trpA (coding for tryptophan synthase A). The tryptophan marker trpA was amplified using plasmid pTA132 (27) as template and oligonucleotides TRP1/TRP2, and cloning of the trpA marker gene into the plasmid pTA131-Cupdo resulted in pTA131-CupdoTrp. Plasmids were passaged through E. coli GM121 to prevent methylation, and H. volcanii strain H119 was subsequently transformed with this construct to allow integration (pop-in) of the plasmid into the genome. The subsequent selection for loss of the pyrE2 marker by plating on 5-fluoroorotic acid revealed pop-out mutants. To confirm the removal of the gene for CRISPR locus C, we performed a Southern blot analysis. Chromosomal DNA was isolated from the wild type and potential locus C gene deletion mutants. Southern blot hybridization was performed as described (27), with the following modifications. 10 g of SacII-digested DNA was separated on a 0.8% agarose gel and transferred to a nylon membrane (Hybond TM -N, GE Healthcare). A 250-bp fragment of the downstream region of locus C was amplified using primers Cdeldoi and DOmitteC, and the fragment was radioactively labeled using [␣-32 P]dCTP and random prime kit Readiprime TM II (GE Healthcare) and subsequently used as a hybridization probe.
Plasmid Invader Tests-The invader plasmid constructs pTA352-PAM3CSp1 (30) and pTA409-PAM3CSp1 (16) were generated based on the Haloferax shuttle vectors pTA352 (pHV1, leuB) (31) and pTA409 (pHV1, pyrE2) (26), including spacer 1 of the CRISPR locus C (C1) and the PAM sequence TTC (PAM3) (16,21). As a control reaction, Haloferax cells expressing the icrRNA (WT or mutants) were transformed with the vector without insert (pTA352 or pTA409). Plasmids were passaged through E. coli GM121 cells (to avoid methylation) and were then introduced into Haloferax cells using the PEG method (27,28). To confirm the identification of a functional invader sequence, H. volcanii cells were transformed at least three times with the plasmid invader construct or the control vector. For plasmid invader tests, transformations with at least a 100-fold reduction in transformation rates are considered successful interference reactions (21,32). High reductions in transformation rates provide evidence for high targeting efficiency of the crRNA analyzed.
Northern Blot Hybridization-Total RNA was isolated, unless stated otherwise, from exponentially growing H. volcanii cells as described (16). After separation of 10 g of RNA (total RNA) on 8% denaturing gels, RNA molecules were transferred to nylon membranes (Hybond-Nϩ, GE Healthcare) and incubated with oligonucleotides against the spacer 1 from locus C (primer C1). The primer was radioactively labeled at the 5Ј end with [␥-32 P]ATP and subsequently used for hybridization.
Investigation of icrRNAs-To determine the exact length and sequence of the crRNA, RNA was isolated from wild type Haloferax cells (H119) and strain ⌬C ϫ pTA232-telecrRNA grown to an absorbance of 0.74. RNA was separated on 8% PAGE, and RNA ranging in size from 45 to 55 nucleotides (fraction 1) and from 60 to 75 nucleotides (fraction 2) was eluted and sent to vertis Biotechnologie AG for cDNA preparation and RNAseq analysis. The RNA samples were first treated with polynucleotide kinase and then poly(A)-tailed using poly(A) polymerase. Afterward, an RNA adapter was ligated to the 5Ј-monophosphate of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the Moloney murine leukemia virus reverse transcriptase. The resulting cDNAs were PCR-amplified to about 10 -30 ng/l using a high fidelity DNA polymerase. The cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and were analyzed by capillary electrophoresis. For Illumina sequencing, the cDNA samples were mixed in approximately equal amounts. An aliquot of the cDNA pool was analyzed by capillary electrophoresis. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina.
RNAseq Mapping-First, original reads were trimmed according to their sequencing quality using the fastq_quality_ trimmer program from the FASTX-Toolkit version 0.0.13 with the options "-t 13-Q 33." The parameter Q is required due to the ASCII offset of 33 used for the quality scores in the Sanger format. The estimated probability that a base call is incorrect (p Ͼ 0.05) corresponds to quality values below 13 (33). Second, trimmed reads were mapped with Segemehl (34) version 0.1.3 with the options "-polyA -prime3ЈAGATCGGAAGAGCG-TCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCG-CCGTATCATTЈ" .
This setting removes the poly(A) tail and the 3Ј Illumina sequencing adapter. The following percentages of the original reads were successfully mapped from each sample: 86% for S1 (wild type RNA fraction of 60 -75 nt length), 74% for S2 (wild type RNA fraction of 45-55 nt length), 91% for S3 (icrRNA fraction of 60 -75 nt length), and 81% for S4 (icrRNA fraction of 45-55 nt length). All samples had 20 -40 million reads. Subsequent to mapping, alignments were filtered such that they had a maximum edit distance of 2, were located on the reverse strand (because CRISPR locus C is transcribed from the reverse strand), and matched uniquely to the genome. The filtering produced a clearer signal, but it did not change original profiles. To explore and display RNAseq results, we used the Integrative Genomics Viewer version 2.0.3 (35).

RESULTS
To determine the essential nucleotides of the crRNA for the interference and to investigate whether the Cas6b protein is required for the interference reaction, we established a Cas6bindependent crRNA generation in H. volcanii. Using this setup, we could study the effect of crRNA mutants on the interference reaction independently of the crRNA processing stage; thus, we captured crRNA characteristics that were specific to the interference reaction.
Cas Protein-independent Generation of crRNAs-We generated a plasmid that encodes the crRNA as well as t-elements directly up-and downstream of the crRNA (Fig. 1B). The crRNA is derived from the Haloferax CRISPR locus C and contains spacer 1 of this locus. The t-element is a tRNA-like structure that has been previously detected directly upstream of the 5 S rRNA in H. volcanii, and it is processed by tRNase Z to generate the 5 S rRNA 5Ј end (36,37). Generally t-elements are substrates for both tRNA-processing enzymes, the 5Ј-processing enzyme RNase P, and the 3Ј-processing enzyme tRNase Z (36,38). Processing of the t-elements up-and downstream of the crRNA should yield the mature icrRNA. We cloned the crRNA/t-element insert into the Haloferax vector pTA409 (26), yielding pTA409-telecrRNA. A Haloferax strain that has the CRISPR locus C deleted (strain ⌬C) was generated to get a strain without the endogenous spacer 1 from locus C (see under "Experimental Procedures"). This strain was transformed with plasmid pTA409-telecrRNA yielding ⌬C ϫ pTA409-telecrRNA. Northern blot analysis showed that an icrRNA is generated with the same size as the crRNA made in the wild type strain (which generates the crRNA from the CRISPR locus C) (Fig. 1C). Thus, the icrRNA is efficiently generated from the plasmid. In addition, some shorter RNAs are visible, and these shorter crRNAs have also been reported earlier in wild type cells (22). Because the amount of icrRNA was rather low compared with the endogenous crRNA, we cloned the crRNA/telement insert into a Haloferax vector with a higher copy number, pTA232 (27), yielding pTA232-telecrRNA. Northern analysis showed that a Haloferax ⌬C strain transformed with pTA232-telecrRNA indeed generated higher amounts of icrRNA (Fig. 1C).
To confirm that processing of the icrRNA yielded exactly the same 5Ј and 3Ј ends as in the "natural" crRNA production, we isolated the two RNA fractions that contained the long crRNA of about 65 nucleotides (RNA fraction of 60 -75 nucleotides in length isolated) and the shorter crRNA of about 51 nucleotides (RNA fraction of 45-55 nucleotides in length isolated) from wild type Haloferax cells and ⌬C ϫ pTA232-telecrRNA cells and analyzed them with RNAseq. The icrRNAs from the 60 -75-nucleotide fraction (isolated from ⌬C ϫ pTA232-telec-rRNA strain) have exactly the same 5Ј and 3Ј ends as the wild type crRNA (Fig. 2A). Thus, we could show that we can generate a mature icrRNA identical to the natural crRNA in Haloferax cells. In addition we could show that a slightly shorter icrRNA version with 49 nucleotides in length (icrRNA 49 ) is also present (Fig. 2B). This shorter icrRNA 49 has the same 5Ј end but a 17-nucleotide shorter 3Ј handle than icrRNA 66 .
The only difference between the natural crRNA and the icrRNAs is the nature of the processing product end groups; the icrRNA contains a 5Ј-phosphate group at the crRNA 5Ј end and a 3Ј hydroxyl group at the crRNA 3Ј end due to processing by RNase P and tRNase Z (23,39). This is in contrast to the observed end groups generated naturally by type I Cas6 processing as follows: a 5Ј hydroxyl group and 2Ј-3Ј cyclic phosphate (I-C and I-E) (13,40,41) or a noncyclic 3Ј phosphate (I-F) (18). However, we show here that the nature of the end group is not important for the interference reaction (see below). Taken together, we could successfully establish a Cas6b-independent crRNA maturation pathway.
icrRNAs Are Active in Interference-To investigate whether the icrRNA is active in interference, we challenged Haloferax strain ⌬C expressing the icrRNA (from the high copy plasmid pTA232-telecrRNA) with an invader plasmid (21). The invader plasmid contains the protospacer sequence that matches spacer 1 of CRISPR locus C from Haloferax; thus, this sequence can be detected by the icrRNA. Adjacent to the protospacer is the PAM sequence TTC that is one of the six PAMs shown to be active in Haloferax to trigger degradation (21). If this invader plasmid is recognized as an invader, it is degraded by the defense system, and cells cannot grow on selective medium. Transformation rates of strains transformed with the invader were reduced more than 100-fold compared with transformation with a control plasmid, showing that the invader plasmid is recognized and degraded ( Table 2). The same experiment was subsequently carried out with the low copy icrRNA plasmid (pTA409-telecrRNA). Again the transformation rates were reduced in comparison with a control plasmid, showing that the lower levels of icrRNA can also trigger the interference reaction. Taken together, the icrRNAs can trigger the interference reaction and thus are fully functional crRNAs.
Cas6b Is Not Required for Interference in the Presence of icrRNAs-In the wild type situation Cas6b is required for crRNA production, and it is conceivable that it could also be required for the interference reaction, because it was shown to be part of Cascade in Haloferax (I-B system), E. coli (I-E), Pseudomonas aeruginosa (I-F), and Sulfolobus solfataricus (I-A) (18,22,(42)(43)(44)(45)(46). By the Cas6b-independent generation of icrRNAs, we separated the role of Cas6b in crRNA processing from its function in the interference reaction. Using icrRNAs, we can now determine whether Cas6b is also important for the inter-  (42)(43)(44)) that is an A in P1, a U in P2, and a G in C. Thus, there are three types of crRNAs in Haloferax beginning with three different nucleotides. The mature crRNA contains an 8-nucleotide 5Ј handle and a 22-nucleotide 3Ј handle. Spacers are between 34 and 39 nucleotides long. Nucleotides in the 5Ј handle are termed Ϫ8 to Ϫ1 (from the 5Ј end of the 5Ј handle) and nucleotides from the 3Ј handle are termed ϩ1 to ϩ22 (42)(43)(44). B, maturation of the icrRNA. The pre-icrRNA contains the crRNA flanked by two t-elements. The crRNA is derived from CRISPR locus C containing spacer 1 from this locus. The t-elements are recognized and processed by RNase P and tRNase Z, generating the mature icrRNA of 66 nucleotides (icrRNA 66 ). This icrRNA can be processed further to a 49-nucleotide-long icrRNA 49 by still unknown RNases. C, maturation of the icrRNA in Haloferax cells. RNA was isolated from wild type cells (lane wt), Haloferax cells without the CRISPR locus C (lane ⌬C), and ⌬C cells with pTA409-telecrRNA (lane ⌬C ϩ in the left panel) and from ⌬C cells with the high copy plasmid pTA232-telecrRNA (lane ⌬C ϩ in the right panel), respectively. After separation on 8% PAGE, the RNA was transferred to a membrane that was subsequently hybridized with a probe against the crRNA. The mature crRNA can be detected in wild type Haloferax cells but not in ⌬C. Left panel, "low copy," generation of icrRNAs from low copy plasmids. The mature icrRNA can be detected in ⌬C transformed with the low copy plasmid pTA409-telecrRNA. Lane m, DNA size marker, sizes are given at the left in nucleotides. The icrRNAs are shown schematically at the right. Right panel, "high copy," generation of icrRNAs from high copy plasmids. In lane ⌬Cϩ, the precursor of the icrRNA as well as the processing intermediates are visible. The long exposure (bottom right, "long") shows that the shorter icrRNA of about 49 nucleotides is also present. Sizes of a DNA marker are given at the left in nucleotides. The precursor of the icrRNA, the intermediates, and the mature icrRNAs are shown schematically at the right. ference reaction. Thus, we transformed a ⌬cas6b strain with pTA232-telecrRNA and subsequently with the invader plasmid. The transformation rate of these cells was greatly reduced (by factor 0.0006) ( Table 2), showing that the interference reaction works without Cas6b. In the ⌬cas6b strain, no internal crRNAs can be generated; thus, the only crRNAs present in these cells are the icrRNAs. Subsequently Cascade complexes can only be loaded with icrRNAs. This might explain the greater reduction in the transformation rate compared with ⌬C; all Cascades in ⌬cas6b contain the icrRNA directed against the invader plasmid, whereas in ⌬C only a percentage of the Cascade complexes are loaded with an icrRNA because the crRNAs from CRISPR locus P1 and P2 are also present. Taken together, the Cas6b protein is not required for the interference reaction when the icrRNA is present.
Essential Features of the crRNA 5Ј Handle-Because the icrRNA was proven to be identical to the "naturally" expressed crRNA and to be fully active in interference, we generated different versions of the icrRNA to analyze the essential features of a crRNA for the interference reaction. To identify the important nucleotides of the 5Ј handle, we generated 10 different variants and analyzed them for activity in the interference reaction (Table 3). All variants were transformed into strain ⌬cas6b that was subsequently challenged with the invader plasmid. First, we mutated the first nucleotide of the crRNA (which is a G) to a A, U, or C (variants 4 -6). Mutation of the first nucleotide (position Ϫ8) results in icrRNAs that are as effective in interference as the wild type icrRNA (Table 3). This is in agreement with the in vivo situation in Haloferax, where the crRNAs are generated from three different CRISPR loci, each of which have a different nucleotide at position Ϫ8 of the 5Ј handle (Fig.  1A). Second, the Ϫ1 nucleotide was mutated from C to U and G and A (variants 8 -10). This nucleotide has been shown in FIGURE 2. Determination of crRNA and icrRNA sequences with RNAseq. A, comparison of Cas6b catalyzed crRNA generation (wt) and Cas6b independent crRNA production (icrRNA). RNAseq data from RNA fractions (sizes 60 -75 nucleotides) isolated from wild type Haloferax cells (upper row "wt") and ⌬C ϫ pTA232-telecrRNA (lower row "icrRNA") were mapped to the CRISPR C locus. The icrRNA only comprises spacer 1, between repeats 1 and 2. The numbers to the right of each row reflect the number of reads mapping to this region. The dominant crRNA length is 66 nt, and each mature crRNA begins with the characteristic eight nucleotide handle at its 5Ј end and ends with the remaining 22 nucleotides of the repeat. Both pathways produce the same mature crRNA. B, two types of icrRNA are generated. In ⌬C ϫ pTA232-telec-rRNA, in addition to the 66-nucleotide-long icrRNA, a shorter icrRNA of 49 nt is also evident (Fig. 1C). RNAseq data from the longer icrRNA fraction (sizes 60 -75 nucleotides) isolated from ⌬C ϫ pTA232-telecrRNA Haloferax cells (upper row, "long icrRNA") and from the shorter icrRNA fraction (sizes 45-55 nucleotides) (lower row, "short icrRNA") were mapped to the CRISPR C locus. Each icrRNA begins with the characteristic eight-nucleotide 5Ј handle, followed by the spacer sequence. In contrast to the long crRNAs, the shorter crRNAs contain only a five-nucleotide long 3Ј handle.

TABLE 2
Interference test with the icrRNA Targeting efficiencies of the icrRNAs expressed from the high copy and low copy icrRNA plasmids were analyzed. The targeting efficiency of the icrRNAs expressed from the high copy icrRNA plasmid were investigated in strain ⌬C and ⌬cas6b. A successful interference reaction reduces the transformation rate by at least a factor of 0.01, demonstrating a high targeting efficiency of the icrRNA (21). If the plasmid is not recognized as an invader and is not destroyed, the transformation rate is the same as with a normal plasmid; there is no reduction of transformation rate. If the plasmid is recognized as an invader and degraded, cells cannot survive on ura Ϫ medium. However, some cells can inactivate the CRISPR-Cas system (by deleting or mutating the cas genes or the genes for the CRISPR RNAs) and can grow on the selective medium (21). As a result, the plates are not completely empty since the mutated Haloferax cells can grow. Therefore, a high targeting efficiency is defined by a reduction in transformation rate by at least 0.01 (21).

TABLE 3 crRNA 5 handle is essential
Ten different variants of the icrRNA with different mutations in the 5Ј handle were generated. The reduction of transformation rates upon transformation of ⌬C ϫ pTA232-telecrRNA with invader plasmid is shown (see column Reduction of transformation rate by factor), demonstrating the targeting efficiency of the icrRNA variants. A successful interference reaction reduces the transformation rate by at least a factor of 0.01 (21). If the plasmid is not recognized as an invader and is not destroyed, the transformation rate is the same as with a normal plasmid, and there is no reduction of transformation rate.

Prokaryotic Immune Defense with an icrRNA and without Cas6
FEBRUARY 13, 2015 • VOLUME 290 • NUMBER 7 E. coli (type I-E) to be derived from the invader (47)(48)(49). In Haloferax, the nucleotide Ϫ1 is a C and thus also identical to the last nucleotide of the PAM used in this study (TTC). It therefore has the potential to base pair with the invader (Fig. 3A). Mutation of this nucleotide to a U does not interfere with the defense activity. The U at this position could still base pair with the complementary PAM sequence in the invader (U-G base pair) (Fig. 3B). Mutation of the Ϫ1 nucleotide to a G, however, abolishes the defense activity, and this nucleotide could not base pair any longer with the complementary PAM sequence (GXG) (Fig. 3C). Surprisingly, mutation of this nucleotide from C to an A does not interfere with the defense activity, although an A at this position is not able to base pair with the complementary PAM sequence in the invader (GXA) (Fig. 3D).
Because the nature of the first crRNA nucleotide is not important, we next deleted this nucleotide, generating an icrRNA that is still active in interference. Deletion of the first two nucleotides results however in an icrRNA inactive in interference. A deletion of three nucleotides in the 5Ј handle (positions Ϫ6 to Ϫ4) (variant 7) is not tolerated. The complete removal of the 5Ј handle (variant 3) results in a crRNA that cannot trigger the interference reaction anymore.
Taken together, mutations in the 5Ј handle are tolerated at the first nucleotide (position Ϫ8) and to some extent at position Ϫ1. Only the deletion of the first nucleotide of the 5Ј handle is tolerated, and all other deletions result in inactive icrRNAs.
Essential Features of the crRNA 3Ј Handle-The crRNA 3Ј handle in Haloferax has the potential to form a short stem loop structure at the very 3Ј end (Fig. 1A). To determine whether parts of this stem loop are required and to define the essential features of the 3Ј handle, we constructed 13 icrRNA variants with mutations in the 3Ј handle and analyzed their activity in interference (Table 4). We mutated a nucleotide in the loop of the potential stem loop structure (G to C or U) (variants 11 and 12). These variants were both still active in triggering the interference reaction. The removal of four nucleotides of the 3Ј handle in variant 1 (positions 8 -11 in the 3Ј handle) also did not interfere with the interference reaction. Likewise, the removal of 11 nucleotides in variant 2 (positions 1-11) did not reduce the interference. The nature of the 3Ј handle differs from CRISPR system to CRISPR system. In Haloferax wild type cells, two types of crRNAs are observed having a 3Ј handle of ϳ22 nucleotides and ϳ5 nucleotides (22). A similar observation was made with the icrRNA, because a long and a short icrRNA can be detected (Figs. 1C and 2B) that contains a 22-nucleotide and a 5 nucleotide 3Ј handle (Fig. 2B). To investigate how many nucleotides can be removed from the 3Ј handle, we designed several 3Ј handle deletion variants. The five terminal nucleotides were deleted in variant 13; 10 terminal nucleotides were

handle can be omitted
Thirteen different variants of the icrRNA with different mutations in the 3Ј handle were generated. The reduction of transformation rates upon transformation with invader plasmids is shown (see column Reduction of transformation rate by factor), demonstrating the targeting efficiency of the icrRNA variants. A successful interference reaction reduces the transformation rate by at least factor 0.01 (21). If the plasmid is not recognized as an invader and is not destroyed, the transformation rate is the same as with a normal plasmid, and there is no reduction of transformation rate. removed in variant 14, and the last 15 and 20 nucleotides were deleted in variants 15 and 16, respectively. The interference tests clearly show that all four deletions in the 3Ј handle had no effect on the interference activity (Table 4). In variant 20, only one nucleotide of the 3Ј handle remained, but still this crRNA was effective in triggering the interference reaction. This last nucleotide was mutated in variants 21-23 from a G to a C, A, or U. Again, all variants were still active. Even a complete removal of all 22 nucleotides (variant 19) did not interfere with the interference reaction. These results also suggest that the exact length of the complete crRNA is not important, because different lengths at the 3Ј handle are tolerated.

DISCUSSION
We could successfully establish a Cas6b-independent crRNA maturation pathway in Haloferax cells. In this pathway, icrRNAs are excised from a precursor with the help of tRNA processing enzymes, resulting in small RNAs active in the interference reaction. The icrRNAs are identical to the natural crRNAs except for the nature of the end groups.
Cas6b Is Only Required for crRNA Maturation in Type I-B-Using the independently generated crRNA, we could show that Cas6b is not required for any other reactions besides crRNA processing in the prokaryotic immune system I-B. As soon as the crRNA is generated without Cas6b, this protein is dispensable, because it is not required for the interference reaction. We previously showed that Cas6b copurifies with Cascade in Haloferax (22), and this observation might be due to the fact that the crRNA is incorporated into Cascade and that Cas6b is still bound to the crRNA thereby co-purifying with the FLAGtagged Cas7. But although it copurifies with Cas5 and Cas7, it is not required to be part of the I-B Cascade for activity. Thus, the core part of the I-B Cascade seems to consist of Cas5, Cas7, and the crRNA. These results are confirmed by the observation that the 3Ј handle can be completely removed. Thus, if the Cas6b protein is attached to Cascade via binding to the crRNA 3Ј handle, this interaction is not essential.
Essential Parts of the 5Ј Handle-Recent reports on the structure of the E. coli Cascade complex revealed that the first seven nucleotides of the crRNA 5Ј handle form a hook that interacts with the Cas5, Cas7, and Cse1 proteins (the homologous protein in Haloferax would be the Cas8b protein) (42)(43)(44). Our data clearly show that in the Haloferax I-B system, the 5Ј handle is also an important part of the crRNA. Only the first nucleotide of the 5Ј handle can be mutated and deleted without loss of activity. This is in agreement with the in vivo situation where three different 5Ј handles are generated (Fig. 1A.). In the structural analyses reported for the I-E Cascade complex, the first nucleotide of the 5Ј handle interacts with Cas5 and Cas7 (42)(43)(44). In the Haloferax system, this interaction does not seem to be crucial for the activity. However, all other deletions in the 5Ј handle abolished interference activity as follows: deletions of the first two nucleotides, of three internal nucleotides, and of all 5Ј handle nucleotides yield a nonfunctional crRNA, confirming the importance of the 5Ј handle.
Interaction of the crRNA 5Ј Handle with the Protospacer Adjacent Motif-The nature of the last nucleotide of the 5Ј handle (position Ϫ1) seems to be important; mutation of this nucleotide from C to G results in loss of activity, and only nucleotides C, A, and U are tolerated at this position. In E. coli, it has been shown that the Ϫ1 crRNA nucleotide is identical to the last PAM nucleotide and is derived from the invader (47)(48)(49)(50)(51), and thus the crRNA could base pair with the invader at this position (Fig. 3). It is not known whether the crRNA 5Ј handle nucleotide (position Ϫ1) stems from the invader in the Haloferax I-B system. But the Ϫ1 crRNA nucleotide and invader complementary PAM nucleotide (in PAMs TTC and CAC, two of the six PAMs recognized by Haloferax) also have the potential to base pair. This base pair might be important for recognizing the correct target DNA sequence. The observation that the Ϫ1 nucleotide mutant C to U works but C to G does not work would confirm this hypothesis. However, the result that the C to A mutation is still active in interference does not fit. In addition, the complementary nucleotide of the other four PAMs recognized in the Haloferax system (TAT, TAA, TAG, and ACT) cannot base pair with the crRNA. In the I-E and I-F E. coli system, it has been shown that the interaction between the Ϫ1 crRNA nucleotide and the last complementary PAM nucleotide is not essential for invader recognition (52,53). The recent structural data for the I-E Cascade complex confirm this earlier observation showing that in this system the Ϫ1 nucleotide of the crRNA is displaced by the Cas5 protein preventing interaction with the invader PAM sequence. The same displacement of the Ϫ1 nucleotide might happen in the Haloferax I-B Cascade. Also, the loss of activity of the C3 G mutant could be explained by failure of the G to interact properly with the Cas5 protein.
In the I-E system, Cse1 (the homologous protein in Haloferax is Cas8b) interacts with the PAM sequence, and target recognition occurs via identification of the PAM sequence by the Cse1 protein (18,(53)(54)(55). The same might be true for the Haloferax I-B system, but the Cas8b protein should be able to identify six different PAMs as follows: TTC, CAC, TAT, TAA, TAG, and ACT. Taken together, our results suggest that a G at position Ϫ1 cannot interact properly with the Cas5 protein and that the Haloferax Cas8b would have to recognize all six different PAMs.
Essential Parts of the 3Ј Handle-Mutational analysis of the icrRNA showed that the 3Ј handle of the crRNA is completely dispensable. The shortest icrRNA found in vivo by RNAseq contained a five-nucleotide-long 3Ј handle. According to the data reported here, this shorter crRNA version with only 49 nucleotides should also be active, because even an icrRNA with no 3Ј handle is still active. Previous isolation of crRNAs from the Haloferax Cascade-like complex showed that the long and the short crRNA versions co-purify (22). It would be interesting to analyze whether only the short form is the active form and whether the long form has to be activated by 3Ј-processing to yield the short functional form. Currently, it is not known which enzyme(s) are catalyzing this further trimming of the crRNA 3Ј end. As soon as this enzyme is identified, we could generate a strain that has the gene for the enzyme deleted and analyze whether the icrRNA with a long unprocessed 3Ј handle is active.
A shortening of the crRNA 3Ј handle has also been reported for the type I-B system of Methanococcus maripaludis and Clostridium thermocellum (56). Thus, it seems that in contrast to the I-A, I-E, and I-F systems, crRNAs of the I-B system are subjected to an additional 3Ј trimming, as reported for the crRNAs in type III systems (14,15).
Nature of the crRNA End Group Is Not Important-The pre-icrRNA is generated by the tRNA-processing enzymes to exactly the same product as the pre-crRNA generation by Cas6b. The only difference between the natural crRNA and the icrRNA is the nature of the 5Ј and 3Ј end groups. However, in the experiments reported here the nature of the end groups did not have any effect on the shortening of the icrRNA 66 to icrRNA 49 nor on the interference reaction. Taken together, the nature of the end groups seems not to be important for the interference reaction.
Minimal Type I-B crRNA-Previously published data concerning the requirements for the spacer-protospacer interactions in the Haloferax I-B system showed that a 34-nucleotidelong spacer-protospacer interaction between crRNA and invader was sufficient (16). According to these published data and the results reported here, the minimal crRNA for the Haloferax type I-B system contains a 7-nucleotide-long 5Ј handle, a 34-nucleotide-long spacer, and no 3Ј handle (Fig. 4). Altogether, this crRNA would be 41 nucleotides long.