CRISPR-Cas is an adaptive prokaryotic immune system, providing protection against viruses and other mobile genetic elements. In type I and type III CRISPR-Cas systems, CRISPR RNA (crRNA) is generated by cleavage of a primary transcript by the Cas6 endonuclease and loaded into multi-subunit surveillance/effector complexes, allowing homology-directed detection and cleavage of invad-ing elements. Highly studied CRISPR-Cas systems such as those in Escherichia coli and Pseudomonas aeruginosa have a single Cas6 enzyme that is an inte-gral subunit of the surveillance complex. By contrast, Sulfolobus solfataricus has a complex CRISPR-Cas system with three types of surveillance complexes (Cascade/type I-A, CSM/type III-A and CMR/type III-B), five Cas6 paralogues and two different CRISPR-repeat families (AB and CD). Here, we investigate the kinetic properties of two different Cas6 paralogues from S. solfataricus. The Cas6-1 subtype is specific for CD-family CRISPR repeats, generating crRNA by multiple turnover catalysis whilst Cas6-3 has a broader specificity and also processes a non-coding RNA with a CRISPR repeat-related sequence. Deep sequencing of crRNA in surveillance complexes re-veals a biased distribution of spacers derived from AB and CD loci, suggesting functional coupling be-tween Cas6 paralogues and their downstream effec-tor complexes.

We have previously reported the genetic correction of Huntington’s disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work, we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated Cas pro-teins comprise a prokaryotic RNA-guided adaptive immune system that interferes with mobile genetic elements, such as plasmids and phages. The type I-E CRISPR interference complex Cascade from Es-cherichia coli is composed of five different Cas pro-teins and a 61-nt-long guide RNA (crRNA). crRNAs contain a unique 32-nt spacer flanked by a repeat-derived 5 ′ handle (8 nt) and a 3 ′ handle (21 nt). The spacer part of crRNA directs Cascade to DNA tar-gets. Here, we show that the E. coli Cascade can be expressed and purified from cells lacking crRNAs and loaded in vitro with synthetic crRNAs, which di-rect it to targets complementary to crRNA spacer. The deletion of even one nucleotide from the crRNA 5 ′ handle disrupted its binding to Cascade and tar-get DNA recognition. In contrast, crRNA variants with just a single nucleotide downstream of the spacer part bound Cascade and the resulting ribonucleotide complex containing a 41-nt-long crRNA specifically recognized DNA targets. Thus, the E. coli Cascade-crRNA system exhibits significant flexibility suggest-ing that this complex can be engineered for appli-cations in genome editing and opening the way for incorporation of site-specific labels in crRNA.

CRISPR-Cas systems provide a small RNA-based mechanism to defend against invasive genetic el-ements in archaea and bacteria. To investigate the in vivo mechanism of RNA interference by two type III-B systems (Cmr- and Cmr-) in Sulfolobus is-landicus, a genetic assay was developed using plas-mids carrying an artificial mini-CRISPR (AC) locus with a single spacer. After pAC plasmids were intro-duced into different strains, Northern analyses con-firmed that mature crRNAs were produced from the plasmid-borne CRISPR loci, which then guided gene silencing to target gene expression. Spacer muta-genesis identified a trinucleotide sequence in the 3′-region of crRNA that was crucial for RNA inter-ference. Studying mutants lacking Cmr- or Cmr- system showed that each Cmr complex exhibited RNA interference. Strikingly, these analyses further revealed that the two Cmr systems displayed dis-tinctive interference features. Whereas Cmr- com-plexes targeted transcripts and could be recycled in RNA cleavage, Cmr- complexes probably targeted nascent RNA transcripts and remained associated with the substrate. Moreover, Cmr- exhibited much stronger RNA cleavage activity than Cmr- . Since we previously showed that S. islandicus Cmr- medi-ated transcription-dependent DNA interference, the Cmr- constitutes the first CRISPR system exhibit-ing dual targeting of RNA and DNA.

associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interfer-ence complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recog-nition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs.