Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach

Chari, Raj; Mali, Prashant; Moosburner, Mark; Church, George M.

doi:10.1038/nmeth.3473

Cited by 363 publications

(417 citation statements)

References 29 publications

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“…This observation contrasts with early reports indicating that this ortholog was mildly active 7,34 . This led us to investigate whether we could further boost its activity.…”

Section: Identification Of An Sgrna Architecture Directing Robust Dnacontrasting

confidence: 99%

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Agudelo

Carter

Velimirovic

et al. 2018

Preprint

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The broad spectrum of activities displayed by CRISPR-Cas systems has led to biotechnological innovations that are poised to transform human therapeutics. Therefore, the comprehensive characterization of distinct Cas proteins is highly desirable. Here we expand the repertoire of nucleases for mammalian genome editing using the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9). We define functional protospacer adjacent motif (PAM) sequences and variables required for robust and efficient editing in vitro. Expression of holoSt1Cas9 from a single adeno-associated viral (rAAV) vector in the neonatal liver rescued lethality and metabolic defects in a mouse model of hereditary tyrosinemia type I demonstrating effective cleavage activity in vivo. Furthermore, we identified potent anti-CRISPR proteins to regulate the activity of both St1Cas9 and the related type II-A Staphylococcus aureus Cas9 (SaCas9). This work expands the targeting range and versatility of CRISPR-associated enzymes and should encourage studies to determine its structure, genome-wide specificity profile and sgRNA design rules. INTRODUCTIONClustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins form a prokaryotic adaptive immune system and some of its components have been harnessed for robust genome editing 1 . Type II-based editing tools rely on a large multidomain endonuclease, Cas9, guided to its DNA target by an engineered single-guide RNA (sgRNA) chimera 2 (See 3, 4 for a classification of CRISPR-Cas systems). The Cas9-sgRNA binary complex finds its target through recognition of a short sequence called the protospacer adjacent motif (PAM) and subsequent base pairing of the guide RNA with the DNA to generate a specific doublestrand break (DSB) 1,5 . While Streptococcus pyogenes (SpCas9) remains the most widely used Cas9 variant for genome engineering, the diversity of naturally occurring RNA-guided nucleases is astonishing 4 . Hence, Cas9 enzymes from different microbial species can contribute to the expansion of the CRISPR toolset by increasing targeting density, improving activity and specificity as well as easing delivery 1,6 .In principle, engineering complementary CRISPRCas systems from distinct bacterial species should be relatively straightforward, as they have been minimized to only two components. However, many such enzymes were found inactive in human cells despite being accurately reprogrammed for DNA binding and cleavage in vitro [7][8][9][10] . Nevertheless, the full potential of selected enzymes can be unleashed using machine learning to establish sgRNA design rules 11,12 .Perhaps the most striking example of the value of alternative Cas9 enzymes is the implementation of the type II-A Cas9 from Staphylococcus aureus (SaCas9) for in vivo editing using recombinant adeno-associated virus (rAAV) vectors 7,13, 14 . More recently, Campylobacter jejuni and Neisseria meningitidis Cas9s from the type II-C 15 CRISPRCas systems have been added to this repertoire 16,17 .In vivo genome edi...

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“…This observation contrasts with early reports indicating that this ortholog was mildly active 7,34 . This led us to investigate whether we could further boost its activity.…”

Section: Identification Of An Sgrna Architecture Directing Robust Dnacontrasting

confidence: 99%

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Agudelo

Carter

Velimirovic

et al. 2018

Preprint

View full text Add to dashboard Cite

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“…For future improvements in Cas9 proteins, we suggest that one should focus on rapid rejection of such off targets. Our observations may also further inform the design of the guide-RNA and the DNA targets with minimal off-target effects [57][58][59][60][61][62][63][64][65][66][67][68] .…”

Section: Discussionmentioning

confidence: 80%

Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9

Singh

Sternberg

Fei

et al. 2016

Preprint

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Binding specificity of Cas9-guide RNA complexes to DNA is important for genomeengineering applications; however, how mismatches influence target recognition/rejection kinetics is not well understood. Here we used single-molecule FRET to probe real-time interactions between Cas9-RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence; however, the dissociation rate greatly increases from o0.006 s À 1 to 42 s À 1 upon introduction of mismatches proximal to protospacer-adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 11 base pairs in length, which prevent DNA cleavage, still allow formation of a stable complex (dissociation rate o0.006 s À 1 ), suggesting that extremely slow rejection could sequester Cas9-RNA, increasing the Cas9 expression level necessary for genome-editing, thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading.

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“…A single study that combined Trex2 with Cas9 for gene editing in human cells reported a similar 2.5-fold increase in mutagenesis (Chari et al, 2015). When Trex2 was used in combination with TALENs, a 3-to 4-fold enhancement was observed (Certo et al, 2012;Luo et al, 2015), whereas a 25-fold increase was achieved when a meganuclease was used as the DSB-inducing agent (Certo et al, 2012).…”

Section: Discussionmentioning

confidence: 92%

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

et al. 2017

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We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A Web-based tool streamlines vector selection and construction. One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).

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Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach

Cited by 363 publications

References 29 publications

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

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