Vikram Pattanayak

High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity

The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 1012 potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.

High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects

CRISPR–Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.

Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection

Engineered zinc-finger nucleases (ZFNs) are promising tools for genome manipulation, and determining off-target cleavage sites of these enzymes is of great interest. We developed an in vitro selection method that interrogates 1011 DNA sequences for cleavage by active, dimeric ZFNs. The method revealed hundreds of thousands of DNA sequences, some present in the human genome, that can be cleaved in vitro by two ZFNs: CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGFA genes, respectively. Analysis of identified sites in one cultured human cell line revealed CCR5-224–induced changes at nine off-target loci, though this remains to be tested in other relevant cell types. Similarly, we observed 31 off-target sites cleaved by VF2468 in cultured human cells. Our findings establish an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage and suggest strategies for the improvement of future ZFN design.

Small Molecule-Triggered Cas9 Protein with Improved Genome- Editing Specificity

Directly modulating the activity of genome-editing proteins has the potential to increase their specificity by reducing activity following target locus modification. We developed Cas9 nucleases that are activated by the presence of a cell-permeable small molecule by inserting an evolved 4-hydroxytamoxifen (4-HT)-responsive intein at specific positions in Cas9. In human cells, conditionally active Cas9s modify target genomic sites with up to 25-fold higher specificity than wild-type Cas9.

Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity

Although transcription activator–like effector nucleases (TALENs) can be designed to cleave chosen DNA sequences, TALENs have activity against related off-target sequences. To better understand TALEN specificity, we profiled 30 unique TALENs with different target sites, array length and domain sequences for their abilities to cleave any of 1012 potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. Based on these findings, we engineered a TALEN variant that exhibits equal on-target cleavage activity but tenfold lower average off-target activity in human cells.

In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites

Gene-editing nucleases enable targeted modification of DNA sequences in living cells, thereby facilitating efficient knockout and precise editing of endogenous loci. Engineered nucleases also have the potential to introduce mutations at off-target sites of action. Such unintended alterations can confound interpretation of experiments and can have implications for development of therapeutic applications. Recently, two improved methods for identifying the off-target effects of zinc finger nucleases (ZFNs) were described–one using an in vitro cleavage site selection method and the other exploiting the insertion of integration-defective lentiviruses into nuclease-induced double-stranded DNA breaks. However, application of these two methods to a ZFN pair targeted to the human CCR5 gene led to identification of largely non-overlapping off-target sites, raising the possibility that additional off-target sites might exist. Here, we show that in silico abstraction of ZFN cleavage profiles obtained from in vitro cleavage site selections can greatly enhance the ability to identify potential off-target sites in human cells. Our improved method should enable more comprehensive profiling of ZFN specificities.

Determining the Specificities of TALENs, Cas9, and Other Genome-Editing Enzymes

The rapid development of programmable site-specific endonucleases has led to a dramatic increase in genome engineering activities for research and therapeutic purposes. Specific loci of interest in the genomes of a wide range of organisms including mammals can now be modified using zinc-finger nucleases, transcription activator-like effectornucleases, and CRISPR-associated Cas9 endonucleases in a site-specific manner, in some cases requiring relatively modest effort for endonuclease design, construction, and application. While these technologies have made genome engineering widely accessible, the ability of programmable nucleases to cleave off-target sequences can limit their applicability and raise concerns about therapeutic safety. In this chapter, we review methods to evaluate and improve the DNA cleavage activity of programmable site-specific endonucleases and describe a procedure for a comprehensive off-target profiling method based on the in vitro selection of very large (~10(12)-membered) libraries of potential nuclease substrates.

Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules

We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20–30 base oligodeoxynucleotides with 5–6 bp complementary ends to which a 5′ fluorophore and 3′ quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem–loop of the SQRM suggests that SQRM be made to target natural stem–loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells.

“Unexpected mutations after CRISPR-Cas9 editing in vivo” are most likely pre-existing sequence variants and not nuclease-induced mutations

Schaefer et al. recently advanced the provocative conclusion that CRISPR-Cas9 nuclease can induce off-target alterations at genomic loci that do not resemble the intended on-target site.1 Using high-coverage whole genome sequencing (WGS), these authors reported finding SNPs and indels in two CRISPR-Cas9-treated mice that were not present in a single untreated control mouse. On the basis of this association, Schaefer et al. concluded that these sequence variants were caused by CRISPR-Cas9. This new proposed CRISPR-Cas9 off-target activity runs contrary to previously published work2–8 and, if the authors are correct, could have profound implications for research and therapeutic applications. Here, we demonstrate that the simplest interpretation of Schaefer et al.’s data is that the two CRISPR-Cas9-treated mice are actually more closely related genetically to each other than to the control mouse. This strongly suggests that the so-called “unexpected mutations” simply represent SNPs and indels shared in common by these mice prior to nuclease treatment. In addition, given the genomic and sequence distribution profiles of these variants, we show that it is challenging to explain how CRISPR-Cas9 might be expected to induce such changes. Finally, we argue that the lack of appropriate controls in Schaefer et al.’s experimental design precludes assignment of causality to CRISPR-Cas9. Given these substantial issues, we urge Schaefer et al. to revise or re-state the original conclusions of their published work so as to avoid leaving misleading and unsupported statements to persist in the literature.

Response to “Unexpected mutations after CRISPR–Cas9 editing in vivo ”

To the Editor: Schaefer et al.1 recently stated that CRISPR–Cas9 nuclease can induce off-target alterations at genomic loci that do not resemble the intended on-target site. This new proposed CRISPR– Cas9 off-target activity runs contrary to previously published work (performed mostly in cells, but also in mice)2–6 and, if the authors are correct, could have profound implications for research and therapeutic applications. However, we here demonstrate that the simplest interpretation of data in Schaefer et al.1 is that the two CRISPR–Cas9treated mice are genetically more closely related to each other than to the control mouse. This strongly suggests that the so-called ‘unexpected mutations’ simply represent shared single-nucleotide polymorphisms (SNPs) and indels that existed before nuclease treatment. The conclusion of Schaefer et al.1 that the sequence variants shared by the genome-edited F03 and F05 mice (and not found in the control untreated FVB mouse) are caused by CRISPR–Cas9 critically depends upon the assumption that all of these mice were initially genetically identical. If this clonality assumption were true, one would expect that all three mice should be nearly identical for common variants found in dbSNP (a hypothetical result represented in Fig. 1a). However, after genotyping these mice with GATK best practices, we identified a total of 31,079 high-quality variants at dbSNP loci that were concordant in two mice but distinct from the third when examining all possible pairwise combinations (Fig. 1b and Supplementary Note 1). Furthermore, 33–46% of these highconfidence-genotyped variants in each mouse are heterozygous (Supplementary Table 1), which the authors have argued should not be the case in highly inbred mice7. Thus, the three mice are neither clonal nor completely isogenic. Even under a more realistic and relaxed equal-distance model that allows rare and private mutations (Fig. 1c), our reanalysis still reveals that the F03 and F05 mice are genetically more closely related to each other than to the control FVB mouse (Fig. 1b,d and Supplementary Note 2). Even if one were to assume that the variants in question were induced by CRISPR–Cas9, it is difficult to reconcile the off-target activity proposed by Schaefer et al.1 with our current understanding of how this nuclease functions. We confirmed the authors’ claim that no DNA sequences resembling the on-target site can be found near the sequence variants that they attribute to CRISPR– Cas9 (Supplementary Figs. 1 and 2; Supplementary Note 3). Additionally, we could not find an alternative consensus DNA motif at or near the locations of these variants that might be recognized by the CRISPR–Cas9 nuclease (Supplementary Fig. 3 and Supplementary Note 4). This makes it hard to envision any reasonable mechanism for how CRISPR–Cas9 could direct alterations to the same genomic loci in the two mice. Furthermore, given the well-established variability of indel mutations induced by CRISPR–Cas9 at any given cleavage site8, we calculate that the probability that these proposed Cas9-induced changes would be exactly the same at a large number of loci (as observed in the data of Schaefer et al.1; Supplementary Fig. 4) is less than 1 in 1012 under even the most generous assumptions (binomial test; Supplementary Fig. 5 and Supplementary Note 5). Based on the analyses described above and further common variant analyses (Supplementary Figs. 6 and 7; Supplementary Note 6), the simplest explanation of the results in Schaefer et al.1 is that the CRISPR-treated F03 and F05 embryos already harbored these shared private SNPs and indels before nuclease treatment, whereas the control mouse did not. This alternative explanation avoids the need to postulate a new CRISPR–Cas9 activity that has not been previously observed and that is inconsistent with previously reported observations about how it functions. Schaefer et al.1 mistakenly assumed that association meant causality, but this can lead to erroneous conclusions. For example, our analysis shows an equally high percentage of heterozygous variants in the control mouse that are not present in the two nuclease-treated mice, but we would certainly not attribute these to mutations induced by the lack of CRISPR–Cas9 treatment in the control mouse. In summary, our analyses of the primary data demonstrate that the original conclusions by Schaefer et al.1 are not supported by their existing data. In addition, given our current understanding of CRISPR–Cas9 function based on the published literature, it seems exceedingly unlikely that the new activities proposed by Schaefer et al.1 would be proven true even if one were to perform additional WGS experiments with appropriate and important controls missing from their original study.