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Can CRISPR/Cas9 target multiple targets?

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Updated 11/27/2015

CRISPR/Cas9 Gets a Boost from tRNA

04/07/2015

Nicholas Miliaras PhD
2.1.3.10

2.1.3.10   Can CRISPR/Cas9 Target Multiple Targets?  Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

CRISPR/Cas9 has advanced genome editing and revolutionized molecular biology perhaps even more than the restriction enzyme. But can it edit multiple targets efficiently?

For CRISPR/Cas9 editing, single guide RNAs (sgRNAs) direct the bacterial Cas9 endonuclease to specific loci, allowing targeting of almost any gene. But is it possible to efficiently target multiple genes? “You can express one, two, or even three sgRNAs pretty easily, but if you want to do four, five, or more, it becomes difficult,” Yinong Yang at Pennsylvania State University said.

Yang’s team addressed this question in a Proceedings of the National Academy of Science paper by turning to the cell’s own tRNA processing systems. The group created polycistronic tRNA-gRNA (PTG) constructs that consisted of an sgRNA flanked by a pre-tRNA gene; the cell’s endogenous RNases can then cleave one or multiple transcribed gRNAs from the cistron to direct Cas9 to target genes.

Schematic depiction of the synthetic tRNA-gRNA gene. Credit: Yinong Yang.

“The beauty of this approach is that the 77 bp pre-tRNA gene contains internal promoter elements (box A and B) to recruit the RNA Pol III complex, so maybe you don’t even need a promoter. The Pol III promoter [which is currently used to drive expression of the sgRNA] isn’t very strong, so the tRNA will give you enhanced expression of multiple RNAs.”

The group first tested the PTG in rice protoplasts and soon realized that existing CRISPR/Cas9 vectors can be used to express PTGs. They also observed that the PTGs were more effective at introducing insertions or deletions than sgRNAs, perhaps owing to their higher expression levels from the endogenous tRNA enhancers.

Yang and his colleagues next asked if it was possible to introduce deletions in multiple genes by targeting the MAP kinase components MPK1, MPK2, MPK5, and MPK6 individually and in combinations of two or four. The PTG system introduced deletions for up to four genes, although there was a two-fold reduction in editing efficiency, which the authors attribute to competition for Cas9 among the multiple gRNAs. They then usedAgrobacterium-mediated transformation to transform mature rice plants with sgRNAs or PTGs for MPK genes and observed a higher mutational frequency of bi-allelic mutations and deletions in the plants transformed with the PTGs. Finally, they were able to target the phytoene desaturase (PDS) gene to generate a photo-bleached phenotype in the resulting plants. While they only obtained a single line carrying the fragment deletion of PDS, the mutational efficiency for PTGs was 100 percent.

Reference:

Xie, K, Minkenberg B, and Yang, Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3570-5. doi: 10.1073/pnas.1420294112.

Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system
Kabin Xie, Bastian Minkenberg, and Yinong Yang1
Department of Plant Pathology and Environmental Microbiology and the Huck Institutes of the Life Sciences,
Pennsylvania State University, University Park, PA 16802

Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved February 3, 2015

The clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/ Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA–gRNA architecture were efficiently and precisely processed into gRNAs with desired 5′ targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits.

CRISPR/Cas9 | tRNA processing | genome editing | multiplex

Significance The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system has recently emerged as an efficient and versatile tool for genome editing in various organisms. However, its targeting capability and multiplex editing efficiency are often limited by the guide RNA (gRNA)-expressing device. This study demonstrates a general strategy and platform for precise processing and efficient production of numerous gRNAs in vivo from a synthetic polycistronic gene via the endogenous tRNA-processing system. This strategy is shown to significantly increase CRISPR/Cas9 multiplex editing capability and efficiency in plants and is expected to have broad applications for small RNA expression and genome engineering.

Fig. 1. Engineering the endogenous tRNA system for multiplex genome editing with CRISPR/Cas9. (A) The eukaryotic pre-tRNA with 5′ leader and 3′ trailer is cleaved by RNase P and RNase Z at specific sites. (B) Transcription of tRNA gene with RNA polymerase III (Pol III). The box A and box B elements in the tRNA gene function as internal transcriptional elements and are bound by transcription factor IIIC (TFIII C), which recruits TFIIIB and Pol III to start transcription. (C) Schematic depiction of the PTG/Cas9 method for simultaneously targeting multiple sites. The synthetic PTG consists of tandemly arrayed tRNA-gRNA units, with each gRNA containing a target-specific spacer (labeled as a diamond with different color) and conserved gRNA scaffold (rectangle). The tRNA containing box A and B elements is shown as round rectangles. The primary transcript of PTG is cleaved by endogenous RNase P and RNase Z (labeled as scissors) to release mature gRNAs and tRNA (red lines of cloverleaf structure). The excised mature gRNAs direct Cas9 to multiple targets.

Strategy to Engineer a tRNA-processing System for Producing Numerous gRNAs

Precise Processing of PTG to Produce Functional gRNAs with Desired Targeting Sequences

Fig. 2. Precise excision of functional gRNAs in vivo from synthetic PTG genes. (A) The architecture of two sgRNA genes and four PTGs to produce one gRNA. (B) Sequence and predicted secondary structure of tRNA–gRNA–tRNA fusion of PTG gene. The bases of the tRNA region are indicated with red color and the tRNA 5′ leader is shown in lowercase. The gRNA is indicated in black, and the gRNA spacer sequence is shown as N. (C–F) Examination of mature gRNAs produced from sgRNA or PTGs with cRT-PCR. Total RNAs from the protoplasts expressing empty vector were used as control (CK). Arrows indicate mature gRNAs amplified by cRT-PCR, and asterisks indicate the nonspecifically amplified rRNA. (G) Summary of excision sites in PTG according to mapped gRNA ends from cRT-PCR (SI Appendix, Figs. S3–S5). Arrows indicate the cleavage sites in PTG to release gRNA. The mature gRNA 5′ ends were excised from PTG exactly at the tRNA–gRNA fusion site in all cRT-PCR results whereas its 3′ ends shifted 1–4 nt within the tRNA 5′ leader (lowercase). (H) gRNA produced from U3p:sgRNA. All detected U3p:sgRNA-produced gRNA started with ribonucleotide A and terminated with multiple Us. (I) Introduction of indels at the desired sites by PTG1:Cas9 or PTG2:Cas9 in rice protoplasts as shown by PCR/RE. Arrows indicate mutated fragments resistant to RE digestion. The indel frequency is indicated at the bottom. (J) Relative expression of sgRNA1/2 and PTG1/2 in rice protoplasts. Data represent mean ± SD. ND, not detected. CK, empty vector control.

Efficient Multiplex Genome Editing in Rice Protoplasts via PTG/Cas9.

Fig. 3. Simultaneous editing of multiple genomic sites in rice protoplasts expressing PTG:Cas9. (A) Architecture, gRNA components, and targets of PTGs for multiplex genome editing. (B) PCR detection of chromosomal fragment deletion at targeted loci in rice protoplasts expressing respective PTGs with Cas9. Successful deletion is shown as truncated PCR product (indicated with arrows). The chromosomal fragment deletion frequency (del %) is indicated at the bottom of each lane. The protoplast samples expressing an empty vector were used as control (CK). (C) Representative sequences of chromosomal fragment deletion aligned with that of WT. The gRNA paired region is labeled with green color, and the PAM region is shown in red color letters. The number at the end indicates deleted (−) or inserted (+) bases between two Cas9 cuts. The total length between two Cas9 cut sites (labeled with scissor) is indicated on the top. Short lines in the aligned sequences indicate deletions.

Improving Multiplex Genome Editing in Stable Transgenic Plants with PTG/Cas9

Table 1. Targeted mutation efficiency in PTG:Cas9 vs. sgRNA:Cas9 plants

Fig. 4. Highly efficient targeted mutagenesis in transgenic rice expressing PTG:Cas9. (A and B) Chromosomal fragment deletion in PTG7:Cas9 plant at T0 generation. Of note, only mpk1 with 358-bp deletion (Δ358) was detected in genomic DNA. Sequence analysis of the PCR products (the number in parentheses) reveals an identical deletion pattern in the transgenic plant. (C) Albino seedlings were regenerated from calli transformed with PTG10:Cas9. Most T0 seedlings (87%, n = 15) exhibited a similar photo-bleach phenotype, suggesting a very high efficiency of knocking out PDS with PTG10:Cas9. Vec, control plants transformed with empty vector. (Scale bar: 5 cm.)

We developed a general strategy and platform to produce multiple gRNAs from a single synthetic PTG gene by hijacking the endogenous tRNA-processing system (Fig. 1). We also provided a framework to design, synthesize, and use PTG for multiplex genome editing with Cas9. These PTGs were expressed with Pol III promoters (e.g., U3p) in the same manner as sgRNA genes but were not obligated to start with a specific nucleotide (Fig. 2). As a result, current CRISPR/Cas9 vectors for expressing sgRNA could be directly used to express PTG for multiplex genome editing as we demonstrated in this study.

By producing multiple gRNAs from a single polycistronic gene, the PTG technology could be used to improve simultaneous mutagenesis of multiple genomic loci or deletion of short chromosomal fragments (Figs. 3 and 4). Such a genome engineering approach may lead to simultaneous knock-out of multiple protein coding genes or deletion of noncoding RNA regions and other genetic elements. In addition to targeted mutagenesis/ deletion, the PTG approach could facilitate other Cas9-based applications in which multiple gRNAs are required. For example, PTG could be used with Cas9 nickase to improve targeting fidelity (13, 33, 34), or with deactivated Cas9 transcriptionalactivator or -repressor to manipulate multiple gene expression (35, 36). Given the high processing accuracy and capability of RNase P and RNase Z that we observed (Fig. 2), the tRNAprocessing system also could be used as a general platform to produce other regulatory RNAs (e.g., short hairpin RNA or artificial microRNA) from a single synthetic gene. These different classes of regulatory RNAs, like gRNA and short hairpin RNA, also could be compacted into a single polycistronic gene to develop more sophisticated devices for genetic engineering.

Recently, polycistronic transcripts that fused gRNA with a 28-nt RNA (referred to as gRNA-28nt) were successfully used to guide Cas9 to target up to four targets in human cells (12, 13). The synthetic gene with a gRNA-28nt architecture produced mature gRNAs with a 28-nt extra 3′ sequence and also required coexpressing the endonuclease Csy4 from Pseudomonas aeruginosa to cleave the transcript. In comparison with the gRNA-28nt strategy, our approach uses a robust endogenous tRNA-processing system that enables precise production of gRNAs with only a 1- to 4-nt extra sequence at the gRNA 3′ end (Figs. 1 and 2) and carries no additional risk of endonuclease Csy4 toxicity to recipients. Given the extremely large number of tRNA genes with variable sequences and the fact that RNase P and RNase Z precisely recognize RNA substrates with tRNA-like structures (18, 37), there are many choices of tRNA sequences to be embedded in PTG. Furthermore, the tRNA-processing system is universal in all living organisms; thus, the PTG technology could be directly adapted to other organisms for Cas9-mediated genome engineering.

When multiple double-strand breaks (DSBs) in genomic DNA were generated by PTG/Cas9 in rice plants, indels resulting from error-prone NHEJ repairing occurred more frequently than fragment deletions generated by directly joining two DSBs (SI Appendix, Figs. S10 and S11). To date, the molecular mechanism by which two DSBs directly link together to generate chromosomal translocation or fragment deletion in vivo is largely unclear. We speculate that the process leading to such a chromosomal disorder may require two DSBs at the same time interval and is likely determined by the highly dynamic interaction between gRNA/Cas9 cutting and endogenous DNA repairing and also by the distance between DSBs. Due to the differences in the delivery, expression, and activity of gRNAs and Cas9, it is not surprising to see some discrepancies in fragment-deletion frequency between protoplasts (Fig. 3B) and stable transgenic plants and among different PTG transgenic lines (Fig. 4A and SI Appendix, Figs. S9–S11). Because the PTG technology enables the generation of many DSBs in genomic DNAs, it may provide an efficient tool to help dissect the molecular process of chromosomal deletion. More importantly, the PTG technology significantly improves multiplex editing capability and efficiency and is expected to facilitate more sophisticated Cas9 applications, such as targeted mutagenesis and deletion of redundant genes or Fig. 4. Highly efficient targeted mutagenesis in transgenic rice expressing PTG:Cas9. (A and B) Chromosomal fragment deletion in PTG7:Cas9 plant at T0 generation. Of note, only mpk1 with 358-bp deletion (Δ358) was detected in genomic DNA. Sequence analysis of the PCR products (the number in parentheses) reveals an identical deletion pattern in the transgenic plant. (C) Albino seedlings were regenerated from calli transformed with PTG10:Cas9. Most T0 seedlings (87%, n = 15) exhibited a similar photo-bleach phenotype, suggesting a very high efficiency of knocking out PDS with PTG10:Cas9. Vec, control plants transformed with empty vector. (Scale bar: 5 cm.) genetic elements, transcriptional modulation of multiple genes and pathways, modification and labeling of numerous genomic sites, site-specific integration, and gene replacement.

3570-3575 | www.pnas.org/cgi/doi/10.1073/pnas.1420294112 Xie et al. genetic elements, transcriptional modulation of multiple genes and pathways, modification and labeling of numerous genomic sites, site-specific integration, and gene replacement

Validating “predicted” regulatory elements through CRISPR editing of the non-coding genome

CRISPR Cas9 genome editing of the non-coding genome

CRISPR/Cas9-mediated genome editing is not only an efficient way to create gene KO & KI, but is a uniquely powerful tool to functionally characterize the >98% of the genome that does not encode protein. A new study demonstrates how CRISPR can be used to systematically validate putative regulatory elements described by the ENCODE and EPIGENOME projects: even in a repeat-rich genomic region, a genomic insulator upstream of mouse tyrosinase was efficiently deleted or inverted, with no significant off-target effects and high efficiency in vivo, demonstrating a functional role for this noncoding region in regulating tyrosinase gene expression and mouse coat pigmentation.

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Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR–Cas9-mediated mutagenesis.
Seruggia D1,2Almudena Fernández1,2Marta Cantero1,2Pawel Pelczar3 and Lluis Montoliu1,2,*
Nucleic Acids Res. 2015 May 26;43(10):4855-67. Read the Free Full Text

Newly developed genome-editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR)–Cas9 system, allow simple and rapid genetic modification in most model organisms and human cell lines. Here, we report the production and analysis of mice carrying the inactivation via deletion of a genomic insulator, a key non-coding regulatory DNA element found 5′ upstream of the mouse tyrosinase (Tyr) gene. Targeting sequences flanking this boundary in mouse fertilized eggs resulted in the efficient deletion or inversion of large intervening DNA fragments delineated by the RNA guides. The resulting genome-edited mice showed a dramatic decrease in Tyr gene expression as inferred from the evident decrease of coat pigmentation, thus supporting the functionality of this boundary sequence in vivo, at the endogenous locus. Several potential off-targets bearing sequence similarity with each of the two RNA guides used were analyzed and found to be largely intact. This study reports how non-coding DNA elements, even if located in repeat-rich genomic sequences, can be efficiently and functionally evaluated in vivo and, furthermore, it illustrates how the regulatory elements described by the ENCODE and EPIGENOME projects, in the mouse and human genomes, can be systematically validated.

Non-coding DNA regulatory elements are composed of arrays of DNA–protein binding sites extending over tens to hundreds of base pairs that are occupied by multiple groups of transcription factors. DNA methylation, covalent modification of histone proteins and DNase I hypersensitivity profiles allow unbiased identification of such elements as regions of active chromatin that might be relevant in the regulation of different genes in a particular tissue or condition. Systematic ChIP-Sequencing (chromatin immunoprecipitation coupled with massive parallel sequencing) using antibodies specific for a variety of nuclear factors, applied to several human cell lines (1) and mouse tissues (2), served to identify cell type-specific regulatory elements accounting for almost 80% of the non-coding fraction of the genome. These studies, globally known as the ENCODE project (Encyclopaedia of DNA Elements; (3)) underline the rich proportion of functional elements existing within the non-coding areas of mammalian genomes. The recent publication of the human EPIGENOME project has provided additional evidence for the relevance of DNA regulatory elements in controlling gene expression (4). However, many functional experiments are required to unequivocally demonstrate the links between the observed biochemical chromatin features and the predicted biological function (5).

In the past years, the relevance of non-coding regions has been typically addressed, in vivo, using genomic-type transgenes (mostly bacterial and yeast artificial chromosomes, BACs and YACs; reviewed in (6)) carrying the inactivation of putative regulatory elements surrounded by tens to hundreds of kilo bases of genomic sequences of a suitable endogenous gene or coupled to a reporter gene (711). In this manner, large genomic fragments have been easily manipulated using homologous recombination in bacteria (12) and yeast (13) and then introduced into the mouse germline by standard procedures (1415). However, variability is often observed between transgenic lines generated with BAC- or YAC-type transgenes, suggesting that position effects can influence transgene expression, even on large constructs (1521). In addition, not all loci fit in such artificial chromosome-type transgenes, for example, large multi-gene syntenic blocks or gene clusters, whose transcriptional regulation programs during development are coordinated (22).

Here, we propose a simple strategy to functionally validate the relevance of non-coding regulatory elements in the mouse genome, in vivo. We have applied CRISPR–Cas9-mediated mutagenesis tools to inactivate, via deletion, a key regulatory sequence identified in the mouse Tyr gene (48).

We previously reported a DNAse hypersensitive (HS) site, located at ∼12 kb 5′-upstream of the mouse Tyr transcription start site (TSS), associated with a melanocyte-specific enhancer that was required for the correct expression of the Tyr gene (39). The deletion or inactivation of this element, in the context of YAC transgenesis, produced mice displaying variegation with severely reduced coat color pigmentation, supporting the notion that this key element was acting as a Locus Control Region (LCR) (7)). Homologous sequences to this mouse Tyr 5′ element were also found within the 5′ end of the human TYR locus, suggesting that mutations in this element could also impair the function of the human TYR gene (54). Traditional molecular diagnosis efforts for OCA1 patients regularly fail to detect all TYR mutations, beyond coding, promoter and limited intronic DNA sequences routinely explored. Consequently, it has been repeatedly suggested that mutations in non-coding regions could be responsible for some of these unknown non-functional TYR alleles (38,55,56). Interestingly, the recent human epigenome data released for many cellular types, including skin melanocytes, describes a regulatory element (a DNAse HS) located at ∼10 kb 5′ upstream of the human TYR gene promoter ((4); Supplementary Figure S8) at the same genomic location as was previously predicted (54). Until now, the direct relevance of TYR or Tyrregulatory elements could not be adequately studied at the endogenous loci. Instead, their role had to be inferred from results obtained using diverse standard and chromosome-type transgenes in mice (17,35).

Further studies revealed that the Tyr LCR had properties typical of genomic boundaries or insulators (57), including the capacity of establishing barriers that prevent spreading of heterochromatin and epigenetic silencing (29), and enhancer-blocking activity (40). The function of insulators is rather complex and strictly dependent on the interactions with other proximal and distal sequences in the genomic locus (43,5860). The context-dependent activity of insulators should be therefore characterized in their native chromosomal context by gene targeting. However, the presence of repetitive sequences surrounding theTyr 5′ boundary element (29) invalidated the application of standard gene targeting approaches. As an alternative, we decided to use CRISPR–Cas9-mediated mutagenesis to overcome the limitations of classical gene targeting strategies.

Similar approaches have been recently reported to address the role of a distal Sox2 enhancer in mouse ES cells (5). Endonuclease-mediated deletions, using Transcription Activator-Like Effector Nucleases (TALENs) and Zinc-Finger Nucleases (ZFN), have been described in zebrafish (61). CRISPR–Cas9 was also used to characterize mutations found at the distal enhancer of the TAL1 oncogene in human tumor cell lines (62). Additionally, mouse models were generated using CRISPR–Cas9 in mouse ES cells to reproduce structural variants, including deletions and inversions, found in human disease (63).

In this work, we report that defined deletions and inversions in non-coding regions can be efficiently generated in vivo by CRISPR–Cas9 approaches using sgRNAs directed to adjacent genomic target sites. CRISPR–Cas9 RNA species are injected into fertilized eggs where they generate mutations at the target sequences. These mutations are then efficiently transmitted through the germ line. Using this strategy, mouse embryos are exposed to a limited amount of Cas9 nuclease for a short time, thus minimizing the risk of off-target mutations. Indeed, in our screen, no undesired mutations were detected at the six genomic loci highly similar to the targeted sequences under investigation. In contrast to this, approaches based on the delivery of CRISPR–Cas9 plasmids to somatic or ES cells may increase the associated risk of off-target mutations since exposure to the Cas9 nuclease is massive and prolonged (31).

Inactivation of the Tyr 5′ boundary element in genomic-type transgenes resulted in a severe reduction in coat color pigmentation, pointing to a relevant role for this non-coding sequence (7). However, these results were based on ectopic chromosomal locations, where variables such as transgene integrity, copy number and integration site could affect the overall gene expression program (1521). Because of this, our vision was to target this 5′ boundary element directly at the Tyr endogenous locus, where we could unequivocally link this element to the observed phenotype without further uncontrolled variables. In actual fact, a comparative analysis of Tyr expression patterns in YAC Tyr transgenic mouse lines and TYRINS5 edited lines reveals fundamental differences in both melanocytes and RPE cells (Figures 4A, C, D, 5A, B and C). Deleting the Tyr 5′ boundary appears to have a milder effect in skin and choroidal melanocytes and a more limited impact in RPE cells, suggesting that additional regulatory elements may be responsible for controlling Tyr gene expression in RPE cells. Indeed, the presence of RPE-specific regulatory elements further upstream had been previously proposed and investigated in mice using BAC Tyr transgenes engineered with a lacZ reporter gene and variable combinations of Tyr 5′ genomic sequences (64).

CRISPR genome editing in human cells: improved targeting with the H1 promoter

A recent paper in Nature Communications reports success with a clever technique to make CRISPR-mediated genome editing easier in human cells. Compared to the commonly-used U6 promoter, driving guide RNA expression from the H1 promoter more than doubles the number of targetable sites within the genomes of humans and other eukaryotes.

Why is H1 more versatile than U6? The U6 promoter initiates transcription from a guanosine (G) nucleotide, while the H1 promoter can initiate transcription from A or G. In designing a gRNA sequence, the requirement for the protospacer adjacent motif (PAM) sequence “NGG” at the end of a 20-mer means that U6-driven gRNA must fit the pattern GN19NGG. But H1-driven gRNAs can also target sequences of the form AN19NGG, which occur 15% more frequently than GN19NGG within the human genome.

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Expansion of the CRISPR–Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs.
Vinod RanganathanKarl WahlinJulien Maruotti & Donald J. Zack  
Nat Commun. 2014 Aug 8;5:4516. Read Full Text
http://dx.doi.org:/10.1038/ncomms5516

The repurposed CRISPR–​Cas9 system has recently emerged as a revolutionary genome-editing tool. Here we report a modification in the expression of the guide RNA (gRNA) required for targeting that greatly expands the targetable genome. gRNA expression through the commonly used U6 promoter requires a guanosine nucleotide to initiate transcription, thus constraining genomic-targeting sites to GN19NGG. We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs, which can be used to target both AN19NGG and GN19NGG genomic sites. AN19NGG sites occur ~15% more frequently than GN19NGG sites in the human genome and the increase in targeting space is also enriched at human genes and disease loci. Together, our results enhance the versatility of the CRISPR technology by more than doubling the number of targetable sites within the human genome and other eukaryotic species.

Figure 1: Evaluating the ability to direct CRISPR targeting via gRNA synthesis from the H1 promoter.

Evaluating the ability to direct CRISPR targeting via gRNA synthesis from the H1 promoter.

(a) Schematic illustration depicting the gRNA expression constructs. Above, the U6 promoter only expresses gRNAs with a +1 guanosine nucleotide; below, the H1 promoter can drive expression of gRNAs initiating at either purine (adenosine…

Figure 2: Bioinformatics analysis of GN19NGG and AN19NGG sites in the genome.

Bioinformatics analysis of GN19NGG and AN19NGG sites in the genome.

(a) Circos plot depicting the frequency of CRISPR sites in the human genome. The outside circle depicts the human chromosome ideograms. Moving inwards, GN19NGG (orange), AN19NGG (blue) and RN19NGG (purple) CRISPR sites frequency is indi…

Could CRISPR technology be used to cure AIDS and other devastating viral diseases?

Why are viral diseases like AIDS still incurable? Although antiretroviral drugs can effectively control viral load in many patients, the permanent integration of viral DNA into a host genome means that patients remain vulnerable to re-activation of a latent virus. Exciting new research now shows that CRISPR technology can remove HIV DNA that has integrated into the host genome in human cells, re-igniting our hopes for developing a true cure for AIDS.

CRISPR-mediated genome editing is revolutionizing biomedical research due to its precise targeting, high efficiency, and ease of use in any cell type or experimental system. CRISPR has been used to create new transgenic animal models for basic and translational research, and it holds promise for use in gene therapy and other medical applications.

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Read the full publication: RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection.
Wenhui Hua,1,2Rafal Kaminskia,1Fan YangaYonggang ZhangaLaura CosentinoaFang LiaBiao Luob, et al. 
Proc Natl Acad Sci U S A. 2014 Jul 21; 111(31):11461–11466
http://dx.doi.org:/10.1073/pnas.1405186111

Significance

For more than three decades since the discovery of HIV-1, AIDS remains a major public health problem affecting greater than 35.3 million people worldwide. Current antiretroviral therapy has failed to eradicate HIV-1, partly due to the persistence of viral reservoirs. RNA-guided HIV-1 genome cleavage by the Cas9 technology has shown promising efficacy in disrupting the HIV-1 genome in latently infected cells, suppressing viral gene expression and replication, and immunizing uninfected cells against HIV-1 infection. These properties may provide a viable path toward a permanent cure for AIDS, and provide a means to vaccinate against other pathogenic viruses. Given the ease and rapidity of Cas9/guide RNA development, personalized therapies for individual patients with HIV-1 variants can be developed instantly.

AIDS remains incurable due to the permanent integration of HIV-1 into the host genome, imparting risk of viral reactivation even after antiretroviral therapy. New strategies are needed to ablate the viral genome from latently infected cells, because current methods are too inefficient and prone to adverse off-target effects. To eliminate the integrated HIV-1 genome, we used the Cas9/guide RNA (gRNA) system, in single and multiplex configurations. We identified highly specific targets within the HIV-1 LTR U3 region that were efficiently edited by Cas9/gRNA, inactivating viral gene expression and replication in latently infected microglial, promonocytic, and T cells. Cas9/gRNAs caused neither genotoxicity nor off-target editing to the host cells, and completely excised a 9,709-bp fragment of integrated proviral DNA that spanned from its 5′ to 3′ LTRs. Furthermore, the presence of multiplex gRNAs within Cas9-expressing cells prevented HIV-1 infection. Our results suggest that Cas9/gRNA can be engineered to provide a specific, efficacious prophylactic and therapeutic approach against AIDS.

Infection with HIV-1 is a major public health problem affecting more than 35 million people worldwide (1). Current therapy for controlling HIV-1 infection and impeding AIDS development (highly active antiretroviral therapy; HAART) includes a mixture of compounds that suppress various steps of the viral life cycle (2). HAART profoundly reduces viral replication in cells that support HIV-1 infection and reduces plasma viremia to a minimal level but neither suppresses low-level viral genome expression and replication in tissues nor targets the latently infected cells that serve as a reservoir for HIV-1, including brain macrophages, microglia, and astrocytes, gut-associated lymphoid cells, and others (3, 4). HIV-1 persists in ∼106 cells per patient during HAART, and is linked to comorbidities including heart and renal diseases, osteopenia, and neurological disorders (5). Because current therapies are unable to suppress viral gene transcription from integrated proviral DNA or eliminate the transcriptionally silent proviral genomes, low-level viral protein production by latently infected cells may contribute to multiple illnesses in the aging HIV-1–infected patient population. Supporting this notion, pathogenic viral proteins including transactivator of transcription (Tat) are present in the cerebrospinal fluid of HIV-1–positive patients receiving HAART (6). To prevent viral protein expression and viral reactivation in latently infected host cells, new strategies are thus needed to permanently disable the HIV-1 genome by eradicating large segments of integrated proviral DNA.

Advances in the engineered nucleases including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR) associated 9 (Cas9) that can disrupt target genes have raised prospects of selectively deleting HIV-1 proviral DNA integrated into the host genome (710). These approaches have been used to disrupt HIV-1 entry coreceptors C-C chemokine receptor 5 (CCR5) or C-C-C chemokine receptor 4 (CXCR4) and proviral DNA-encoding viral proteins (8, 9). CCR5 gene-targeting ZFNs are in phase II clinical trials for HIV-1/AIDS treatment (11). Also, various gene editing technologies have recently been shown to remove the proviral HIV-1 DNA from the host cell genome by targeting its highly conserved 5′ and 3′ long terminal repeats (LTRs) (12, 13). However, introduction of nucleases into cells via these nuclease-based genomic editing approaches remains inefficient and partially selective to remove the entire HIV-1 genome. Thus, the key barrier to their clinical translation is insufficient gene specificity to prevent potential off-target effects (toxicities). To achieve highly specific HIV-1 genome editing, we combined approaches to identify HIV-1 targets while circumventing host off-target effects. The resulting highly specific Cas9-based method proved capable of eradicating integrated HIV-1 DNA with high efficiency from latently infected human “reservoir” cell types, and prevented their infection by HIV-1.

Here, we found that LTR-directed gRNA/Cas9 eradicates the HIV-1 genome and effectively immunizes target cells against HIV-1 reactivation and infection with high specificity and efficiency. These properties may provide a viable path toward a permanent or “sterile” HIV-1 cure, and perhaps provide a means to eradicate and vaccinate against other pathogenic viruses. In the current study, we have mainly focused our efforts on myeloid lineage cells (microglia/macrophage), which are the primary cell types that harbor HIV-1 in the brain. However, this proof of concept is certainly applicable to any other cell type, including T-lymphoid cells (Fig. S6) (12, 13), astrocytes, and neural stem cells.

Our combined approaches minimized off-target effects while achieving high efficiency and complete ablation of the genomically integrated HIV-1 provirus. In addition to an extremely low homology between the foreign viral genome and host cellular genome including endogenous retroviral DNA, the key design attributes in our study included: bioinformatic screening using the strictest 12-bp+NGG target selection criteria to exclude off-target human transcriptome or (even rarely) untranslated genomic sites; avoiding transcription factor binding sites within the HIV-1 LTR promoter (potentially conserved in the host genome); selection of LTR-A- and -B-directed, 30-bp protospacer and also precrRNA system reflecting the original bacterial immune mechanism to enhance specificity/efficiency vs. 20-bp protospacer-based, chimeric crRNA-tracRNA system (16, 30); and WGS, Sanger sequencing, and SURVEYOR assay, to identify and exclude potential off-target effects. Indeed, the use of newly developed Cas9 double-nicking (23) and RNA-guided FokI nuclease (31, 32) may further assist identification of new targets within the various conserved regions of HIV-1 with reduced off-target effects.

More recently, a clinical trial using the ZFN gene editing strategy was launched to disrupt the gene encoding the HIV-1 coreceptor, CCR5 (8, 9, 11). Functional knockout of CCR5 in autologous CD4 T cells of a small cohort of patients revealed that in one out of four enrolled subjects, the viral load remained undetectable at the time of treatment (33). Similarly, TALEN and Cas9 have been tested experimentally for efficient disruption of CCR5 and CXCR4 (9, 28, 3437); therefore, taking them into consideration for clinical trials is anticipated. Whether or not the strategies targeting HIV-1 entry can reach the “sterile” cure of AIDS remains to be seen. Our results show that the HIV-1 Cas9/gRNA system has the ability to target more than one copy of the LTR, which are positioned on different chromosomes, suggesting that this genome-editing system can alter the DNA sequence of HIV-1 in latently infected patient’s cells harboring multiple proviral DNAs. To further ensure high editing efficacy and consistency of our technology, one may consider the most stable region of HIV-1 genome as a target to eradicate HIV-1 in patient samples, which may not harbor only one strain of HIV-1. Alternatively, one may develop personalized treatment modalities based on the data from deep sequencing of the patient-derived viral genome before engineering therapeutic Cas9/gRNA molecules.

Our results also demonstrate, for the first time to our knowledge, that Cas9/gRNA genome editing can be used to immunize cells against HIV-1 infection. The preventative vaccination is independent of HIV-1 strain’s diversity because the system targets genomic sequences regardless of how the viruses enter the infected cells. Interestingly, the preexistence of the Cas9/gRNA system in cells leads to a rapid elimination of the new HIV-1 before it integrates into the host genome, just like the way by which the bacteria defense system evolved to combat phage infection (38). Similarly, a gene-editing-based vaccine strategy may be effective in eradicating postintegrated HIV-1 genome and newly packaged proviruses in cells. Therefore, investigation of such HIV-1 vaccination in various latent reservoir cells and animal models with stable expression of Cas9/LTR-gRNAs presents an important next step to assess the ability of Cas9 to eradicate viral reservoirs in vivo. Moreover, in light of recent data illustrating efficient in vitro genome editing using a mixture of Cas9/gRNA and DNA (3942), one may explore various systems for delivery of Cas9/LTR-gRNA via various routes for immunizing high-risk subjects. Once advanced, one may use gene therapies (viral vector and nanoparticle) and transplantation of autologous Cas9/gRNA-modified bone marrow stem/progenitor cells (43, 44) or inducible pluripotent stem cells for eradicating HIV-1 infection.

Here, we demonstrated the high specificity of Cas9/gRNAs in editing HIV-1 target genome. Results from subclone data revealed the strict dependence of genome editing on the presence of both Cas9 and gRNA. Moreover, only one nucleotide mismatch in the designed gRNA target will disable the editing potency. In addition, all four of our designed LTR gRNAs worked well with different cell lines, indicating that the editing is more efficient in the HIV-1 genome than the host cellular genome, wherein not all designed gRNAs are functional, which may be due to different epigenetic regulation, variable genome accessibility, or other reasons. Given the ease and rapidity of Cas9/gRNA development, even if HIV-1 mutations confer resistance to one Cas9/gRNA-based therapy, as described above, HIV-1 variants can be genotyped to enable another personalized therapy for individual patients (10).

CRISPR-Cas9 Gene Editing: Check Three Times, Cut Once

http://www.technologynetworks.com/Genomics/news.aspx?ID=185167

Two new studies from UC Berkeley should give scientists who use CRISPR-Cas9 for genome engineering greater confidence that they won’t inadvertently edit the wrong DNA.

The gene editing technique, created by UC Berkeley biochemist Jennifer Doudna and her colleague, Emmanuelle Charpentier, director of the Max Planck Institute of Infection Biology in Berlin, has taken the research and clinical communities by storm as an easy and cheap way to make precise changes in DNA in order to disable genes, correct genetic disorders or insert mutated genes into animals to create models of human disease.

The two new reports from Doudna’s lab and that of UC Berkeley colleague Robert Tjian show in much greater detail how the Cas9 protein searches through billions of base pairs in a cell to find the right DNA sequence, and how Cas9 determines whether to bind, or bind and cut, thereby initiating gene editing. Based on these experiments, Cas9 appears to have at least three ways of checking to make sure it finds the right target DNA before it takes the irrevocable step of making a cut.

“CRISPR-Cas9 has evolved for accurate DNA targeting, and we now understand the molecular basis for its seek-and-cleave activity, which helps limit off-target DNA editing,” said Doudna, a Howard Hughes Medical Institute investigator at UC Berkeley and professor of molecular and cell biology and of chemistry. Tjian is president of the Howard Hughes Medical Institute and a UC Berkeley professor of molecular and cell biology.

The studies also illustrate how well CRISPR/Cas9 works in human and animal cells – eukaryotes – even though “the technique was invented by bacteria to protect themselves from getting the flu,” Doudna said.

CRISPR-Cas9 is a hybrid of protein and RNA – the cousin to DNA – that functions as an efficient search-and-snip system in bacteria. It arose as a way to recognize and kill viruses, but Doudna and Charpentier realized that it could also work well in other cells, including humans, to facilitate genome editing. The Cas9 protein, obtained from the bacteria Streptococcus pyogenes, functions together with a “guide” RNA that targets a complementary 20-nucleotide stretch of DNA. Once the RNA identifies a sequence matching these nucleotides, Cas9 cuts the double-stranded DNA helix.

One study tracked Cas9-RNA molecules though the nucleus of mammalian cells as they rapidly searched through the entire genome to find and bind just the region targeted and no other.

“It’s crazy that the Cas9 complex manages to scan the vast space of eukaryotic genomes,” said graduate student Spencer Knight, first author of the paper.

Previous studies had suggested that there are many similar-looking DNA regions that Cas9 could bind and cut, which could limit its usefulness if precision were important. These off-target regions might share as few as four or five nucleotides with the 20-nucleotide primer, just enough for Cas9 to recognize.

“There is a lot of off-target binding by Cas9, but we found that these interactions are very brief – from milliseconds to seconds – before Cas9 moves on,” he said.

Because these exploratory bindings – perhaps as many as 300,000 of them – are often very short-lived, a few thousand CRISPR-Cas9 complexes can scour the entire genome to find one targeted stretch of DNA. Cas9 must also recognize a short three-base-pair DNA sequence immediately following the primer sequence, dubbed PAM, which occurs about 300 million times within the human genome.

“If Cas9 bound for tens of seconds or minutes at each off-target site, it would never, ever be able to find a target and cut in a timely manner,” Knight said.

Cas9’s final checkpoint

The other study, published online Oct. 28 in Nature, showed that once Cas9 binds to a region of DNA, it performs another check before two distant sections of the Cas9 protein complex come together, like the blades of a scissors, to precisely align the active sites that cut double-stranded DNA.

“We found that RNA-guided Cas9 can bind some off-target DNA sequences, which differ from the correct target by just a few mutations, very tightly. Surprisingly, though, the region of Cas9 that does the cutting is inhibited because of the imperfect match. But when the correctly matching DNA is located, Cas9 undergoes a large structural change that releases this inhibition and triggers DNA cutting,” said first author Samuel Sternberg, who recently received his Ph.D. at UC Berkeley. He was able to observe these changes using a fluorescently labeled version of the Cas9 complex.

“We think that this structural change is the last checkpoint, or proofreading stage, of the DNA targeting reaction,” he said. “First, Cas9 recognizes a short DNA segment next to the target – the PAM – then the target DNA is matched up with the guide RNA via Watson-Crick base-pairing. Finally, when a perfect match is identified, the last part of the protein swings into place to enable cutting and initiate genome editing.”

A smaller Cas9 protein from a different species of bacteria, Staphylococcus aureus, likely exploits the same strategy to improve the precision of DNA targeting, suggesting that “this important feature has been preserved throughout evolutionary time,” he added.

“This is good news, in that it suggests that you have more than one checkpoint to ensure correct Cas9 binding,” Knight said. “There’s not just sequence regulation, there is also temporal regulation: it has to engage with the DNA and park long enough that it can actually rearrange and cut.”

The discoveries from Doudna, Tjian and their teams shed light on the molecular basis of off-target effects during genome editing applications, and may guide the future design of more accurate Cas9 variants.

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