Non-canonical Protospacer Adjacent Motif Research Articles

Expanding the targeting scope of CRISPR/Cas9-mediated genome editing by Cas9 variants in Brassica

AbstractCRISPR/Cas9, presently the most widely used genome editing technology, has provided great potential for functional studies and plant breeding. However, the strict requirement for a protospacer adjacent motif (PAM) has hindered the application of the CRISPR/Cas9 system because the number of targetable genomic sites is limited. Recently, the engineered variants Cas9-NG, SpG, and SpRY, which recognize non-canonical PAMs, have been successfully tested in plants (mainly in rice, a monocot). In this study, we evaluated the targeted mutagenesis capabilities of these Cas9 variants in two important Brassica vegetables, Chinese cabbage (Brassica rapa spp. pekinensis) and cabbage (Brassica oleracea var. capitata). Both Cas9-NG and SpG induced efficient mutagenesis at NGN PAMs, while SpG outperformed Cas9-NG at NGC and NGT PAMs. SpRY achieved efficient editing at almost all PAMs (NRN > NYN), albeit with some self-targeting activity at transfer (T)-DNA sequences. And SpRY-induced mutants were detected in cabbage plants in a PAM-less fashion. Moreover, an adenine base editor was developed using SpRY and TadA8e deaminase that induced A-to-G conversions within target sites using non-canonical PAMs. Together, the toolboxes developed here induced successful genome editing in Chinese cabbage and cabbage. Our work further expands the targeting scope of genome editing and paves the way for future basic research and genetic improvement in Brassica.

Systematic Mapping of Gene-Editable Mutations in GATA2 and SAMD9/SAMD9L Syndromes

Germline predisposition to bone marrow failure (BMF) and myelodysplastic syndromes (MDS) cause substantial economic and psychosocial burden and early mortality in affected individuals. Due to the limitations and risks of hematopoietic stem cell transplantation, there is an urgent need for new and innovative treatment approaches. Conventional lentiviral-based gene therapy is not feasible for most BMF/MDS predisposing genes because their uncontrolled overexpression is detrimental and lentiviral vectors might cause insertional mutagenesis. However, new technologies that precisely modify the endogenous allele can overcome these limitations. Base editing (BE) using cytosine base editors (CBE) and adenine base editors (ABE) as well as prime editing (PE) are advanced gene editing techniques with great therapeutic promise. The goal of this project is to develop a comprehensive map of gene-editable mutations in the GATA2, SAMD9, and SAMD9L genes as a preclinical effort to facilitate gene editing trials. Germline heterozygous mutations in these key predisposing genes lead to highly penetrant disorders characterized by progressive immunodeficiency and BMF/MDS risk, with particularly high prevalence in children, as recently reported by us (Sahoo, Nature Medicine 2021) and others. For in depth analysis, we focused on the 5 most recurrent mutations in respective genes and devise strategies to correct mutations in cellular models. We first created a comprehensive database encompassing reported and unpublished pathogenic mutations (single nucleotide variants [SNVs] and <20bp insertion/deletions[indels]). In total, we annotated 506 cases with GATA2, 195 cases with SAMD9, and 187 cases with SAMD9L mutations. SNVs were detected in 83.2% (421/506), 94.4% (184/195), and 93.1% (174/187) patients with GATA2, SAMD9, SAMD9L mutations, respectively, while remaining cases had indels. Among all GATA2 cases, 59.9% (303/506) mutations are editable by ABE, 4.5% (23/506) by CBE, while remaining 35.6% (180/506) can be “rescued” by PE ( Fig. 1A). For SAMD9, 34.4% (67/195) cases were found to be amenable to editing by ABE, 30.3% (59/195) by CBE, and 35.4% (69/195) using PE. For SAMD9L, 47.1% (88/187) are editable by ABE, 20.3% (38/187) by CBE, and 32.6% (61/187) using PE. Having a well-annotated mutation list, we proceeded to identify the top 5 most common mutations in each gene ( Fig. 1B). Remarkably, all 5 mutations in both GATA2 and SAMD9 and 4 out of 5 top mutations in SAMD9L and amenable to BE. Next, we explored in depth the target editing window, protospacer adjacent motif (PAM) sites, and bystander editing effect (utilizing BE that recognize the canonical [NGG], and non-canonical PAM sequences [NG and near PAMless SpRY-NRN/NYN]. This stepwise approach resulted in successful identification of at least one potential guide RNA for each mutation with predicted low bystander editing. Ongoing work includes guide RNA validation, off-target analysis, and assessment of gene-editing efficiency using model cell lines. In conclusion, we have undertaken a systematic analysis of the gene editing landscape for frequent germline driver mutations in BMF/MDS. Our findings reveal promising editing strategies for the majority of prevalent mutations in key predisposing genes GATA2, SAMD9 and SAMD9L using CBE, ABE, or PE editors, and lay a groundwork to enable future clinical translation.

Molecular Mechanism of D1135E-Induced Discriminated CRISPR-Cas9 PAM Recognition.

The off-target effects of Streptococcus pyogenes Cas9 (SpCas9) pose a significant challenge to harness it as a therapeutical approach. Two major factors can result in SpCas9 off-targeting: tolerance to target DNA-guide RNA (gRNA) mismatch and less stringent recognition of protospacer adjacent motif (PAM) flanking the target DNA. Despite the abundance of engineered SpCas9-gRNA variants with improved sensitivity to target DNA-gRNA mismatch, studies focusing on enhancing SpCas9 PAM recognition stringency are quite few. A recent pioneering study identified a D1135E variant of SpCas9 that exhibits much-reduced editing activity at the noncanonical NAG/NGA PAM sites while preserving robust on-target activity at the canonical NGG-flanking sites (N is any nucleobase). Herein, we aim to clarify the molecular mechanism by which this single D1135E mutation confers on SpCas9 enhanced specificity for PAM recognition by molecular dynamics simulations. The results suggest that the variant maintains the base-specific recognition for the canonical NGG PAM via four hydrogen bonds, akin to that in the wild type (WT) SpCas9. While the noncanonical NAG PAM is engaged to the two PAM-interacting arginine residues (i.e., R1333 and R1335) in WT SpCas9 via two to three hydrogen bonds, the D1135E variant prefers to establish two hydrogen bonds with the PAM bases, accounting for its minimal editing activity on the off-target sites with an NAG PAM. The impaired NAG recognition by D1135E SpCas9 results from the PAM duplex displacement such that the hydrogen bond of R1333 to the second PAM base is disfavored. We further propose a mechanistic model to delineate how the mutation perturbs the noncanonical PAM recognition. We anticipate that the mechanistic knowledge could be leveraged for continuous optimization of SpCas9 PAM recognition specificity toward high-precision demanding applications.

Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants

Streptococcus pyogenes Cas9 (SpCas9) is the most widely used genome editing tool in plants. The editing induced by SpCas9 strictly requires a canonical NGG protospacer-adjacent motif (PAM), significantly limiting its scope of application. Recently, five SpCas9 variants, SpCas9-NRRH, SpCas9-NRCH, SpCas9-NRTH, SpG, and SpRY, were developed to recognize non-canonical PAMs in human cells. In this study, these variants were engineered for plant genome editing, and their targeted mutagenesis capabilities were comprehensively examined at various canonical and non-canonical PAM sites in rice (Oryza sativa) by stable transformation. Moreover, both cytosine base editors using a rat APOBEC1 or a human APOBEC3a and adenine base editors using a directly evolved highly compatible TadA∗-8e deaminase were developed from these SpCas9 variants. Our results demonstrated that the developed SpCas9 variants-based base editors readily generated conversions between C∙G and T∙A in the target sites with non-canonical PAMs in transgenic rice lines. Collectively, the toolbox developed in this study substantially expands the scope of SpCas9-mediated genome editing and will greatly facilitate gene disruption and precise editing in plants.

A Cas12a ortholog with stringent PAM recognition followed by low off-target editing rates for genome editing

BackgroundAsCas12a and LbCas12a nucleases are reported to be promising tools for genome engineering with protospacer adjacent motif (PAM) TTTV as the optimal. However, the C-containing PAM (CTTV, TCTV, TTCV, etc.) recognition by Cas12a might induce extra off-target edits at these non-canonical PAM sites.ResultsHere, we identify a novel Cas12a nuclease CeCas12a from Coprococcus eutactus, which is a programmable nuclease with genome-editing efficiencies comparable to AsCas12a and LbCas12a in human cells. Moreover, CeCas12a is revealed to be more stringent for PAM recognition in vitro and in vivo followed by very low off-target editing rates in cells. Notably, CeCas12a renders less off-target edits located at C-containing PAM at multiple sites compared to LbCas12a and AsCas12a, as assessed by targeted sequencing methods.ConclusionsOur study shows that CeCas12a nuclease is active in human cells and the stringency of PAM recognition could be an important factor shaping off-target editing in gene editing. Thus, CeCas12a provides a promising candidate with distinctive characteristics for research and therapeutic applications.

The Acidaminococcus sp. Cas12a nuclease recognizes GTTV and GCTV as non-canonical PAMs.

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nuclease Acidaminococcus sp. Cas12a (AsCas12a, also known as AsCpf1) has become a popular alternative to Cas9 for genome editing and other applications. AsCas12a has been associated with a TTTV protospacer-adjacent motif (PAM) as part of target recognition. Using a cell-free transcription-translation (TXTL)-based PAM screen, we discovered that AsCas12a can also recognize GTTV and, to a lesser degree, GCTV motifs. Validation experiments involving DNA cleavage in TXTL, plasmid clearance in Escherichia coli, and indel formation in mammalian cells showed that AsCas12a was able to recognize these motifs, with the GTTV motif resulting in higher cleavage efficiency compared to the GCTV motif. We also observed that the -5 position influenced the activity of DNA cleavage in TXTL and in E. coli, with a C at this position resulting in the lowest activity. Together, these results show that wild-type AsCas12a can recognize non-canonical GTTV and GCTV motifs and exemplify why the range of PAMs recognized by Cas nucleases are poorly captured with a consensus sequence.

Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1

The RNA-guided Cpf1 (also known as Cas12a) nuclease associates with a CRISPR RNA (crRNA) and cleaves the double-stranded DNA target complementary to the crRNA guide. The two Cpf1 orthologs from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) have been harnessed for eukaryotic genome editing. Cpf1 requires a specific nucleotide sequence, called a protospacer adjacent motif (PAM), for target recognition. Besides the canonical TTTV PAM, Cpf1 recognizes suboptimal C-containing PAMs. Here, we report four crystal structures of LbCpf1 in complex with the crRNA and its target DNA containing either TTTA, TCTA, TCCA, or CCCA as the PAM. These structures revealed that, depending on the PAM sequences, LbCpf1 undergoes conformational changes to form altered interactions with the PAM-containing DNA duplexes, thereby achieving the relaxed PAM recognition. Collectively, the present structures advance our mechanistic understanding of the PAM-dependent, crRNA-guided DNA cleavage by the Cpf1 family nucleases.

Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9.

The CRISPR/Cas9 system is an efficient and convenient tool for genome editing in plants. Cas9 nuclease derived from Streptococcus pyogenes (Sp) is commonly used in this system. Recently, Staphylococcus aureus Cas9 (SaCas9)-mediated genome editing was reported in human cells and Arabidopsis. Because SaCas9 (1053 a.a.) is smaller than SpCas9 (1368 a.a.), SaCas9 could have substantial advantages for delivering and expressing Cas9 protein, especially using virus vectors. Since the protospacer adjacent motif (PAM) sequence of SaCas9 (5′-NNGRRT-3′) differs from that of SpCas9 (5′-NGG-3′), the use of this alternative Cas9 nuclease could expand the selectivity at potential cleavage target sites of the CRISPR/Cas9 system. Here we show that SaCas9 can mutagenize target sequences in tobacco and rice with efficiencies similar to those of SpCas9. We also analyzed the base preference for ‘T’ at the 6th position of the SaCas9 PAM. Targeted mutagenesis efficiencies in target sequences with non-canonical PAMs (5′-NNGRRV-3′) were much lower than those with a canonical PAM (5′-NNGRRT-3′). The length of target sequence recognized by SaCas9 is one or two nucleotides longer than that recognized by SpCas9. Taken together, our results demonstrate that SaCas9 has higher sequence recognition capacity than SpCas9 and is useful for reducing off-target mutations in crop.

Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9

The RNA-guided endonuclease Cas9 from Streptococcus pyogenes (SpCas9) forms the core of a powerful genome editing technology. DNA cleavage by SpCas9 is dependent on the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) in the target DNA, restricting the choice of targetable sequences. To address this limitation, artificial SpCas9 variants with altered PAM specificities have recently been developed. Here we report crystal structures of the VQR, EQR, and VRER SpCas9 variants bound to target DNAs containing their preferred PAM sequences. The structures reveal that the non-canonical PAMs are recognized by an induced fit mechanism. Besides mediating sequence-specific base recognition, the amino acid substitutions introduced in the SpCas9 variants facilitate conformational remodeling of the PAM region of the bound DNA. Guided by the structural data, we engineered a SpCas9 variant that specifically recognizes NAAG PAMs. Taken together, these studies inform further development of Cas9-based genome editing tools.

58. Engineered Cas9 Variants with Novel PAM Specificities Expand the Targeting Range of CRISPR/Cas Nucleases

Specificity and targeting range are important considerations when selecting an engineered nuclease platform for functional studies or therapeutic applications. The use of CRISPR/Cas9 to edit virtually any genome has enabled researchers to rapidly knock out genes or to introduce precise genomic changes. A number of studies have explored and improved the specificity of the CRISPR/Cas9 platform but the targeting range remains limited to certain sequences. The most widely used Cas9 from Streptococcus pyogenes (SpCas9) recognizes a protospacer adjacent motif (PAM) of the form NGG, a constraint that restricts the range of sequences that can be edited. Combined with the 5’ nucleotide requirement for guide-RNA expression from a polIII promoter, this restriction can be limiting particularly when one is using CRISPR/Cas9 nucleases to introduce precise mutations by homology-directed repair (HDR), which requires that double-strand breaks be introduced very closely to the site of sequence alteration.To address this challenge, we developed a heterologous genetic system that enabled us to rapidly screen libraries of Cas9 clones and identify variants with distinct cleavage specificities. Using this technology, we identified and optimized two different Cas9 variants that recognize novel PAMs. We demonstrate that these variants enable targeting of sites with non-canonical PAM sequences that were previously inaccessible with wild-type SpCas9, more than doubling the total range of sequences that can be edited by HDR and by error-prone non-homologous end-joining (NHEJ). We demonstrate that these variants can robustly modify endogenous genes in human cells and that they are compatible with previously described specificity improvements to SpCas9 such as truncated gRNAs.Furthermore, we also show Cas9 nucleases from other bacterial species such as Staphylococcus aureus and Streptococcus thermophilus can also be assessed for activity in our heterologous genetic system, suggesting that these alternative Cas9s might also be modified in their PAM specificities using the same approach we undertook with SpCas9. These orthogonal Cas9 nucleases offer the advantage of being substantially smaller in size than SpCas9, an important factor when considering packaging limits for viral delivery of CRISPR/Cas9 components. Taken together, our results more than double the targeting range of CRISPR/Cas9 nuclease platform and improve the prospects for translation of these reagents to clinical settings.

COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites.

Precise genome editing using engineered nucleases can significantly facilitate biological studies and disease treatment. In particular, clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) proteins are a potentially powerful tool for modifying a genome by targeted cleavage of DNA sequences complementary to designed guide strand RNAs. Although CRISPR/Cas systems can have on-target cleavage rates close to the transfection rates, they may also have relatively high off-target cleavage at similar genomic sites that contain one or more base pair mismatches, and insertions or deletions relative to the guide strand. We have developed a bioinformatics-based tool, COSMID (CRISPR Off-target Sites with Mismatches, Insertions, and Deletions) that searches genomes for potential off-target sites (http://crispr.bme.gatech.edu). Based on the user-supplied guide strand and input parameters, COSMID identifies potential off-target sites with the specified number of mismatched bases and insertions or deletions when compared with the guide strand. For each site, amplification primers optimal for the chosen application are also given as output. This ranked-list of potential off-target sites assists the choice and evaluation of intended target sites, thus helping the design of CRISPR/Cas systems with minimal off-target effects, as well as the identification and quantification of CRISPR/Cas induced off-target cleavage in cells.