pam library  (Addgene inc)

Journal: bioRxiv

Article Title: Engineering of CRISPR-Cas PAM recognition using deep learning of vast evolutionary data

doi: 10.1101/2025.01.06.631536

Figure Lengend Snippet: (a) A bioinformatics pipeline was employed to identify PAMs across diverse CRISPR-Cas systems. The pipeline aligned CRISPR spacers to a large database of viral and plasmid sequences to detect conserved flanking motifs. The Cas proteins responsible for PAM recognition during target inference are shown: Cas9 and Cas12 function as single-protein effectors, while Cas8 operates as part of the multi-subunit Cascade complex. In total, 45,816 distinct PAM predictions were made (Type I: n = 28, 410, Type II: n = 15, 731, Type V: n = 1, 675). (b) Fraction of CRISPR-Cas operons associated with a PAM prediction. (c) Accumulation curves of PAM diversity with increasing data volume. Discovery of new PAMs has largely plateaued for Type I and II systems. (d) PAM similarity was compared between Cas proteins with different levels of relatedness. PAM similarity rapidly diverges for Type II systems but is highly conserved for Types I and V. (e) A phylogenetic tree of Cas9 proteins clustered at 70% identity using MMseqs2 . Outer rings indicate the information content at each of the first 9 PAM positions. Phylogenetic tree built using FastTree and visualized using iToL .

Article Snippet: Briefly, the PAM library plasmids were linearized with PvuI-HF (NEB).

Techniques: CRISPR, Plasmid Preparation

Journal: bioRxiv

Article Title: Engineering of CRISPR-Cas PAM recognition using deep learning of vast evolutionary data

doi: 10.1101/2025.01.06.631536

Figure Lengend Snippet: (a) Phylogenetic trees were built for Cas8, Cas9, and Cas12 proteins. Proteins were first clustered using MMseqs2 at 70% identity for Cas8 and Cas9 and at 95% identity for Cas12. Phylogenetic trees were built using FastTree and visualized using iToL . Colored strips indicate the information content at PAM positions. (b) Distribution of high-information content positions across PAMs from Type I, II, and V systems. In Type I systems, the PAM is predominantly restricted to positions −1 to −3 relative to the protospacer, while in Type II systems, the distribution of high information content PAM positions is more variable. (c) Distribution of the number of spacers aligned to virus and plasmid genomes for PAMs predictions from the CRISPR-Cas Atlas. (d) Signal-to-noise ratio comparing nucleotide conservation upstream and downstream of the protospacer for PAMs predictions from the CRISPR-Cas Atlas. In Type II systems, a downstream motif is expected, while in Type I and V systems, the motif is upstream. Bioinformatic PAM predictions are based on a high number of aligned CRISPR spacers, resulting in strong signal-to-noise ratios and providing a robust training dataset for Protein2PAM.

Article Snippet: Briefly, the PAM library plasmids were linearized with PvuI-HF (NEB).

Techniques: Virus, Plasmid Preparation, CRISPR

Journal: bioRxiv

Article Title: Engineering of CRISPR-Cas PAM recognition using deep learning of vast evolutionary data

doi: 10.1101/2025.01.06.631536

Figure Lengend Snippet: (a) Proteins were characterized using the high-throughput PAM determination assay (HT-PAMDA) in human cell lysate, measuring Cas9 cleavage rates on substrates with all possible PAMs. Cleavage rates were quantified at positions 5 to 8 of the PAM library after deep sequencing at four time points. (b) Activity landscape across Nme1Cas9 enzyme variants. The cleavage rate of each PAM is derived by tracking depletion over four time points (Fast: rate > 1e-3, Medium: rate > 1e-4, Slow: rate > 5e-5). (c) Top: PAM logos predicted using Protein2PAM. Bottom: PAM logos generated from HT-PAMDA data. For HT-PAMDA logos, each four-nucleotide PAM was weighted by its corresponding rate constant, nucleotide counts were normalized to frequencies summing to 1.0 per position, and frequencies were converted to information content. (d) HT-PAMDA heatmaps which display rate constants for different enzyme variants at PAM positions 5-8.

Article Snippet: Briefly, the PAM library plasmids were linearized with PvuI-HF (NEB).

Techniques: High Throughput Screening Assay, Sequencing, Activity Assay, Derivative Assay, Generated

Journal: Nature Communications

Article Title: Transfer of mitochondrial DNA into the nuclear genome during induced DNA breaks

doi: 10.1038/s41467-024-53806-0

Figure Lengend Snippet: a Schematic of mt-nuclear DNA fusions captured by PEM-seq. The biotin-labeled primer located adjacent to the CRISPR-Cas9-target site (scissor) on the nuclear DNA is used to clone editing products (orange line). Then the single-stranded products were ligated with adapters (purple line) containing random molecular barcodes (RMB). and the chimeric reads harboring nuclear DNA around the editing site and mtDNA (red line), were identified as mt-nuclear DNA fusions. For each tested locus, PEM-seq was also conducted in unedited samples. b Box plot showing the frequency of mt-nuclear DNA fusions out of editing events at CRISPR-Cas target sites (colorful dots) under editing of CRISPR-Cas enzymes. Boundary of each box indicates the minimum and maximum. The middle line of each box indicates the median. Two-sided paired t -tests were conducted between Sp Cas9 and other CRISPR nucleases; N = 12. Source data are provided as a file. c Circos plot showing the mt-nuclear DNA fusion junctions on mtDNA (MT) and the indicated CRISPR-Cas9-target sites (colorful triangles) on the nuclear DNA of HEK293T cells. The outer circle shows the human genome, labeled with numbers or characters. The colorful lines indicate the fusion between the target site and mtDNA. Annotations of colored regions in mtDNA are shown at the bottom. d Circos plot showing the fusion junctions on mtDNA (MT) and the indicated CRISPR- Lb Cas12a target sites (colorful triangles) on the nuclear DNA of HEK293T cells. Legends are described in ( c ). e Circos plot showing the fusion junctions on mtDNA (MT) and the indicated CRISPR-CasMINI target sites (colorful triangles) on the nuclear DNA of HEK293T cells. Legends are described in ( c ). f Box plot showing the frequency of mt-nuclear DNA fusion events out of editing events at CRISPR-Cas target sites (colorful dots) under editing of Sp Cas9 variants. Boundary of each box indicates the minimum and maximum. The middle line of each box indicates the median. Two-sided paired t -test; n.s., not significant; N = 5. Source data are provided as a file. g Frequency of mt-nuclear DNA fusions caused by high fidelity Sp Cas9 variants in the mES cells. Mean ± SD; two-sided t -test; n.s. not significant; n = 3. Source data are provided as a file. h Average frequency of mt-nuclear DNA fusions at DNMT1 , EMX1 , c- MYC_2 , and RAG1_C loci after editing by Cas9, BE4max, and ABEmax. EMX1 and c- MYC_2 loci were not targetable by ABEmax. N.A. not applicable. Source data are provided as a file.

Article Snippet: PEM-seq datasets generated by previous studies are available in the following directories: Cas12 libraries are from GEO under the accession number GSE213149 ; high-fidelity and PAM-less Cas9 variants libraries from National Omics Data Encyclopedia (NODE) database with accession code OEP001824 ; comparison between Cas9 and base editors, c-MYC_2 off-target data, and human T cell data from NODE database with accession code OEP000911 [ https://www.biosino.org/node/project/detail/OEP00000911 ]; mouse TCR-T cell data from GEO under the accession number GSE202887 ; GOTI data of BE3 from SRA under project accession SRP119022 ; GOTI data of DdCBE from SRA under project accession PRJNA786071 . are provided with this paper.

Techniques: Labeling, CRISPR

Journal: Nature Communications

Article Title: Transfer of mitochondrial DNA into the nuclear genome during induced DNA breaks

doi: 10.1038/s41467-024-53806-0

Figure Lengend Snippet: a Schematic of universal T cell manufactory. Primary T cells isolated from human or mice were activated and edited by CRISPR-Cas9. After edition, T cells underwent ex vivo culture or were infused into recipient mice. b Distribution of mtDNA-nuclear DNA fusions after CRISPR-Cas9 editing for 3, 7, and 14 days in human CAR T cells. Human primary T cells isolated from cord blood were activated for 3 days and subsequently transfected with Cas9/gRNA ribonucleoprotein (RNP) complexes targeting TRAC , TRBC , and PDCD1 loci. Cells were collected after 3, 7, or 14 days and subjected to PEM-seq libraries. Legends are described in Fig. . c Schematic showing the production of mouse TCR-T cells. TCR-T cells post-editing were infused to Rag1 −/− recipient mice for 3 weeks, and subsequently isolated for PEM-seq analysis. d Distribution of mtDNA-nuclear DNA fusion junctions in CRISPR-Cas9 treated mouse TCR-T cells before and post-infusion for 3 weeks. Legends are described as depicted in ( b ). Percentages show the frequency of each mtDNA-nuclear DNA fusion out of Cas9-induced editing events. e Prey lengths of 38 mt-nuclear DNA fusions sharing the same junction from expanded mouse TCR-T cells indicated in ( d ). f Sequence logo showing the frequency of nucleotides in random molecular barcodes derived from the 38 reads of mt-nuclear DNA fusion at a single junction in expanded TCR-T cells indicated in ( d ). g Distribution of mtDNA integration in the nuclear DNA post base editor (BE3) treatment in mouse embryos. MT, mtDNA. h Number of mt-nuclear DNA fusion events identified in BE3-treated and -untreated samples. Two-sided t -test.

Article Snippet: PEM-seq datasets generated by previous studies are available in the following directories: Cas12 libraries are from GEO under the accession number GSE213149 ; high-fidelity and PAM-less Cas9 variants libraries from National Omics Data Encyclopedia (NODE) database with accession code OEP001824 ; comparison between Cas9 and base editors, c-MYC_2 off-target data, and human T cell data from NODE database with accession code OEP000911 [ https://www.biosino.org/node/project/detail/OEP00000911 ]; mouse TCR-T cell data from GEO under the accession number GSE202887 ; GOTI data of BE3 from SRA under project accession SRP119022 ; GOTI data of DdCBE from SRA under project accession PRJNA786071 . are provided with this paper.

Techniques: Isolation, CRISPR, Ex Vivo, Transfection, Sequencing, Derivative Assay

Journal: Nature Communications

Article Title: Transfer of mitochondrial DNA into the nuclear genome during induced DNA breaks

doi: 10.1038/s41467-024-53806-0

Figure Lengend Snippet: a Illustration of mitochondrial stresses and DSBs-induced mt-nuclear DNA fusions captured by PEM-seq and Insert-seq. CRISPR-Cas9 (scissor) targets the c-MYC locus in nuclear DNA, and the primer (purple arrow) for PEM-seq is adjacent to the target site. b Mitochondrial stresses inducing mt-nuclear DNA fusions captured by PEM-seq. Top: cells were transfected with a plasmid containing Cas9 and gRNA targeting c-MYC for 8 hours to allow the assembly of Cas9 and gRNA. Subsequentially, cells are treated with CCCP or paraquat for 24 or 48 hours , respectively, and then harvested for PEM-seq and Insert-seq analysis. Bottom: percentages of mt-nuclear DNA fusions captured by PEM-seq. Each dot represents a biological replicate. Mean ± SD; two-sided t -test; n = 3. Source data are provided as a file. c Distribution of mt-nuclear DNA fusion junctions captured by the c-MYC bait (black triangle) with or without mitochondrial stresses (untreated, gray bars; CCCP, light purple bars; paraquat, dark purple bars) treatment. Legends of mtDNA annotations are described as depicted in Fig. . The inner circles show the number of each mtDNA-nuclear DNA fusion point on mtDNA in a log scale. MT, mtDNA. d Frequency of PEM-seq-captured mt-nuclear DNA fusion junctions with or without mitoTALEN treatment. Each dot represents a biological replicate. Mean ± SD; two-sided t -test; n = 3. Source data are provided as a file. e Distribution of mt-nuclear DNA fusion junctions captured by the c-MYC bait (black triangle) with or without mitoTALEN ( ND4 site, red triangle) treatment. Legends of mtDNA annotations are described as depicted in Fig. . f Workflow of Insert-seq to enrich insertions (orange lines) at the Cas9-editing site ( c-MYC locus). Briefly, two rounds of targeted PCR (purple and red arrows) are used to clone the editing events around the target site, followed by two rounds of size selection that enriches insertions. Source data are provided as a file. g Percentages of mtDNA integrations within total insertions captured by Insert-seq at the c-MYC locus in the presence or absence of mitochondrial stresses. Each dot represents a biological replicate. Mean ± SD; two-sided t -test; n = 3. h Percentages of mtDNA integrations within total insertions captured by Insert-seq at the c-MYC site. Each dot represents a biological replicate. Mean ± SD; two-sided t -test; n = 3.

Article Snippet: PEM-seq datasets generated by previous studies are available in the following directories: Cas12 libraries are from GEO under the accession number GSE213149 ; high-fidelity and PAM-less Cas9 variants libraries from National Omics Data Encyclopedia (NODE) database with accession code OEP001824 ; comparison between Cas9 and base editors, c-MYC_2 off-target data, and human T cell data from NODE database with accession code OEP000911 [ https://www.biosino.org/node/project/detail/OEP00000911 ]; mouse TCR-T cell data from GEO under the accession number GSE202887 ; GOTI data of BE3 from SRA under project accession SRP119022 ; GOTI data of DdCBE from SRA under project accession PRJNA786071 . are provided with this paper.

Techniques: CRISPR, Transfection, Plasmid Preparation, Selection

Journal: Nature Communications

Article Title: Transfer of mitochondrial DNA into the nuclear genome during induced DNA breaks

doi: 10.1038/s41467-024-53806-0

Figure Lengend Snippet: a Frequency of mt-nuclear DNA fusions at DNMT1 , MYC1 , c- MYC_2 , MYC3 , RAG1A loci after editing with Cas9 or Cas9-TREX2. Two-sided t -test. b Structures of DdCBE with or without TREX1n/TREX2. For the fusion form, TREX1n or TREX2 was fused to the C-terminal domain of L-1397C-UGI. Regarding separated TREX1n or TREX2, both nucleases were tagged with mitochondrial targeting sequence (MTS) on the N-terminal. L-1397C-UGI, left TALE arrays fused to C-terminal DddAtox half and UGI; R-1397N-UGI, right TALE arrays fused to N-terminal DddAtox half and UGI; NTD N-terminal domain, CTD C-terminal domain. c Editing efficiency of DdCBE with or without TREX1n/TREX2 treatment. Mean ± SD. L L-1397C-UGI, R R-1397N-UGI; n = 3. d Distribution of mt-nuclear DNA fusions (red lines) with mtDNA bait junctions ending at the editing site of DdCBE on ND4 . The number ( n ) of fusions in each sample is normalized to the same editing events. L L-1397C-UGI, R R-1397N-UGI. Source data are provided as a file. e Frequency of DdCBE-induced mtDNA fusing with the CRISPR-Cas9-target site with or without TREX1n/TREX2 treatment. Each dot represents a biological replicate. Mean ± SD; two-sided t -test; n = 3. L L-1397C-UGI, R R-1397N-UGI, TX1 TREX1n, TX2 TREX2, f. fused, s. separated, mut. nuclease-dead mutant. Source data are provided as a file.

Article Snippet: PEM-seq datasets generated by previous studies are available in the following directories: Cas12 libraries are from GEO under the accession number GSE213149 ; high-fidelity and PAM-less Cas9 variants libraries from National Omics Data Encyclopedia (NODE) database with accession code OEP001824 ; comparison between Cas9 and base editors, c-MYC_2 off-target data, and human T cell data from NODE database with accession code OEP000911 [ https://www.biosino.org/node/project/detail/OEP00000911 ]; mouse TCR-T cell data from GEO under the accession number GSE202887 ; GOTI data of BE3 from SRA under project accession SRP119022 ; GOTI data of DdCBE from SRA under project accession PRJNA786071 . are provided with this paper.

Techniques: Sequencing, CRISPR, Mutagenesis

Journal: Cell Reports Medicine

Article Title: Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids

doi: 10.1016/j.xcrm.2023.101037

Figure Lengend Snippet:

Article Snippet: SARS-CoV-2 Activator PAM Library , Twist Biosciences , N/A.

Techniques: Virus, Infection, Recombinant, Staining, Protease Inhibitor, Mutagenesis, Cloning, Modification, Synthesized, Plasmid Preparation, Software

Journal: Research Square

Article Title: PAM-Flexible Genome Editing with an Engineered Chimeric Cas9

doi: 10.21203/rs.3.rs-2625838/v1

Figure Lengend Snippet: (A) Quantitative analysis of indel formation with indicated Cas9 variants. Indel frequencies were determined via batch analysis following PCR amplification of indicated genomic loci, in comparison to unedited controls for each gene target. All samples were performed in independent transfection replicates and the mean of the quantified indel formation values was calculated. (B) Quantitative analysis of A-to-G with indicated ABE8e variants. Base editing conversion rates were determined via BEEP following PCR amplification of indicated genomic loci, in comparison to unedited controls for each gene target. All samples were performed in independent transfection replicates and the mean of the quantified base editing formation values was calculated. (C) Off-targets as identified by GUIDE-seq genome-wide for SpCas9, Sc++, SpRY, and SpRYc each paired with two sgRNAs targeting either EMX1 or VEGFA . Only sites that harbored a sequence with ≤10 mismatches relative to the gRNA were considered potential off-target sites. (D) Efficiency heatmap of mismatch tolerance assay on genomic targets. Quantified indel frequencies are exhibited for each labeled single or double mismatch (number of bases 5’ upstream of the PAM) in the sgRNA sequence for the indicated Cas9 variant and indicated PAM sequence. All samples were performed in independent transfection replicates and the mean of the quantified indel formation values was calculated.

Article Snippet: 180 ng of PAM library (Addgene #160132) was incubated with 30 nM of sgRNA and 6 μL of fluorescein-normalized lysate.

Techniques: Amplification, Comparison, Transfection, Genome Wide, Sequencing, Labeling, Variant Assay

Journal: Research Square

Article Title: PAM-Flexible Genome Editing with an Engineered Chimeric Cas9

doi: 10.21203/rs.3.rs-2625838/v1

Figure Lengend Snippet: (A) Targeting disease-associated loci with SpRYc. (i) Schematic of SpRYc RTT Experiment. Base editing conversion rates were determined via CRISPResso2 NGS analysis following PCR amplification of MECP2 -integrated loci, in comparison to unedited controls for the C502T installed mutation. Samples were performed in independent nucleofection triplicates (n=3) and the mean of the quantified base editing formation values was calculated. (ii) SpRYc-BE4Max was nucleofected into TruHD cells alongside an sgRNA targeting the HTT repeat. Base editing conversion rate was determined via CRISPResso2 NGS analysis NGS following PCR amplification of indicated genomic loci, in comparison to an unedited control. The analogous Sanger sequencing trace is shown. Samples were performed in independent nucleofection triplicates (n=3) and the mean of the quantified base editing formation values was calculated. (B) Structural insights via homology modeling in SWISS-MODEL. (i) Interaction of the engineered Sc++ loop (purple) with the backbone of the target strand (TS) PAM region. The REC1 loop from wild type SpCas9 is indicated in green. (ii) Potential interaction of residue R1331 with the non-target strand (NTS) backbone. (iii) Multiple mutations within the PAM interaction loop allow for a more flexible PAM readout. (iv) The potential van der Waals interaction of W1145 with the ribose moieties of non-target strand residues could further stabilize the PAM interaction.

Article Snippet: 180 ng of PAM library (Addgene #160132) was incubated with 30 nM of sgRNA and 6 μL of fluorescein-normalized lysate.

Techniques: Amplification, Comparison, Mutagenesis, Control, Sequencing, Residue