The utility of CRISPR-Cas9 and TALENs for genome editing may be compromised by their off-target activity. We show that integrase-defective lentiviral vectors (IDLVs) can detect such off-target cleavage with a frequency as low as 1%. In the case of Cas9, we find frequent off-target sites with a one-base bulge or up to 13 mismatches between the single guide RNA (sgRNA) and its genomic target, which refines sgRNA design.

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Introns are removed from nuclear messenger RNA precursors through two sequential phospho-transesterification reactions in a dynamic RNA-protein complex called the spliceosome. But whether splicing is catalysed by small nuclear RNAs in the spliceosome is unresolved. As the spliceosome is a metalloenzyme, it is important to determine whether snRNAs coordinate catalytic metals. Here we show that yeast U6 snRNA coordinates a metal ion that is required for the catalytic activity of the spliceosome. With Mg2+, U6 snRNA with a sulphur substitution for the pro-Rp or pro-Sp non-bridging phosphoryl oxygen of nucleotide U80 reconstitutes a fully assembled yet catalytically inactive spliceosome. Adding a thiophilic ion such as Mn2+ allows the first transesterification reaction to occur in the U6/sU80(Sp)- but not the U6/sU80(Rp)-reconstituted spliceosome. Mg2+ competitively inhibits the Mn2+-rescued reaction, indicating that the metal-binding site at U6/U80 exists in the wild-type spliceosome and that the site changes its metal requirement for activity in the Sp spliceosome. Thus, U6 snRNA contributes to pre-messenger RNA splicing through metal-ion coordination, which is consistent with RNA catalysis by the spliceosome.

In addition to small nuclear RNAs and spliceosomal proteins, ATP hydrolysis is needed for nuclear pre-mRNA splicing. A number of RNA-dependent ATPases which are involved in several distinct ATPdependent steps in splicing have been identified in Saccharomyces cerevisiae and mammals. These so-called DEAD/H ATPases contain conserved RNA helicase motifs, although RNA unwinding activity has not been demonstrated in purified proteins. Here we report the role of one such DEAH protein, PRP2 of S. cerevisiae, in spliceosome activation. PRP2 bound to a precatalytic spliceosome prior to the first step of splicing. By blocking the activity of a novel splicing factor(s), HP, which was involved in a post-PRP2 step, we found that PRP2 hydrolyzed ATP to cause a change in the spliceosome without the occurrence of splicing. The change was quite dramatic and could account for the previously reported differences between the precatalytic, pre-mRNAcontaining spliceosome and the "active," intermediate-containing spliceosome. The post-PRP2-ATP spliceosome was further isolated and could carry out the subsequent reaction apparently in the absence of PRP2 and ATP. We hypothesize that PRP2 functions as a molecular motor, similar to some DExH ATPases in transcription, in the activation of the precatalytic spliceosome for the transesterification reaction.Several RNA-dependent ATPases which are essential for pre-mRNA splicing have been identified in Saccharomyces cerevisiae (reviewed in references 3, 17, 40, and 41). These proteins contain the conserved RNA helicase motifs, including the signature DEAD or DEAH sequence (reviewed in references 13, 15, 43, and 52). For example, PRP5 and PRP28 are involved in spliceosome assembly, PRP2 and PRP16 are required for the catalytic steps, and PRP22 is required for the release of mRNA from the spliceosome. Recently, a human PRP22 homolog was isolated and characterized (33,34), and a mammalian RNA-dependent ATPase activity was found to associate with the U5 small nuclear ribonucleoprotein particle (26). This work was an attempt to address the role of one such ATPase, PRP2, in pre-mRNA splicing.A considerable amount of work has been done on the role of PRP16 in splicing (reviewed in reference 50). PRP16 is required for an ATP-dependent reaction during the second step of splicing (44). ATP hydrolysis by PRP16 apparently can be uncoupled from the second transesterification reaction (reaction 2). This was shown by depleting splicing factors, such as PRP18 (20) or SLU7 (2, 21), which are needed after the PRP16 step from extracts. The 3Ј splice site becomes resistant to RNase H after ATP hydrolysis by PRP16, suggesting that some changes to the spliceosome occur (45). Interestingly, PRP16 also plays a proofreading role in splicing (7).Previous results from our own and other laboratories indicate that PRP2 also interacts with the spliceosome transiently (22, 24, 37). The protein binds to the pre-mRNA-containing spliceosome in the absence of ATP in vitro and is released from the spliceosome upon ATP hydrolysis a...