Journal: Nature communications

Article Title: Insights into the compact CRISPR-Cas9d system.

doi: 10.1038/s41467-025-57455-9

Figure Lengend Snippet: Fig. 3 | The hybrid REM of Cas9d comprising the REC domains and Stem 2 and stem 3 of the sgRNA. a Structural alignment of the Cas9d (RNA-coordinated target Engagement Module, REM) with the SpyCas9 REC domain. b Close-up view of the Cas9d REM, as highlighted by the ellipse in a. c Interaction interface of REC domain

Article Snippet: The gene encoding the full-length Cas9d (Sangon) was codon optimized for E.coli expression and assembled into a modified pET vector (2Bc-T, Addgene #37236) with a C-terminal thrombin-TwinStrepII-Histag usingGibson assembly (NewEnglandBiolabs, E2611L).Mutations in Cas9d and SpyCas9 (WT expression plasmid, Addgene #101199) were introduced using the QuickChange Mutagenesis kit (Takara, Cat# 638949) following the manufacturer’s instructions.

Techniques: Drug discovery

Journal: bioRxiv

Article Title: Engineering Nme2Cas9 Adenine Base Editors with Improved Activity and Targeting Scope

doi: 10.1101/2023.04.14.536905

Figure Lengend Snippet: a) Nme1Cas9/sgRNA/DNA ternary complex structure, PDB:6JDV. Nme2Cas9 is 98% identical to Nme2Cas9 outside of the WED and PAM-interacting domains. Black spheres represent N- and C-termini and colored spheres represent sites of domain insertion. Deaminase domain insertion sites (Nme2Cas9 aa numbers) are specified to the right, with colors matching the sites indicated in the structure. b) Activities of Nme2-ABE8e constructs in mCherry reporter cells (activated upon A-to-G editing) after plasmid transfection, measured by flow cytometry (n = 3 biological replicates in technical duplicate; data represent mean ± SD). c) A-to-G editing following transfection of Spy-ABE8e vs. Nme2-ABE8e plasmids, using PAM-matched, endogenous HEK293T genomic loci. The editing efficiency at the maximally edited adenine for each target was plotted. Editing efficiencies were measured by amplicon deep sequencing (n = 3 biological replicates; data represent mean ± SD). d) Data from (c) were aggregated and replotted, with each data point representing the maximum A-to-G editing efficiency of an individual target site, as measured by amplicon deep sequencing (n = 3 biological replicates; data represent mean ± SEM). e) Summary of mean A-to-G editing activities and editing windows for Spy- and Nme2-ABE8e constructs in HEK293T cells. Numbers provided for each position in the protospacer represent the mean A-to-G editing efficiency across eight PAM-matched endogenous target sites, as measured via amplicon deep sequencing (n = 3 biological replicates). Crossed-out boxes indicate that no adenine was present at the specified position in the target panel tested.

Article Snippet: U6-driven sgRNA plasmids for the various Cas effectors were cloned using pBluescript sgRNA expression plasmids (Addgene #122089, #122090, #122091 for SpyCas9, SauCas9 and Nme2Cas9 respectively).

Techniques: Construct, Plasmid Preparation, Transfection, Flow Cytometry, Amplification, Sequencing

Journal: bioRxiv

Article Title: Efficient repair of human homozygous genetic mutation by CRISPR/Cas9 mediated interlocus gene conversion

doi: 10.1101/2022.09.05.506576

Figure Lengend Snippet: a , Seven sgRNAs targeting the β 0 4142 core marked with green arrows. CD4142 deletion base pairs marked with chromatic rectangles. b , Schematic diagram of CRISPR/Cas9 targeting sites in the mutant HBB gene. Blue lines label sgRNA sequences; purple lines label exogenous DNA templates. c , Editing efficiency of Cas9 RNPs coupled with the various sgRNAs in CD34 + HSPCs from homozygotes measured by TIDE analysis. HM mock represents only CD34 + HSPCs from homozygous Donor #1; Cas9 only represents CD34 + HSPCs from homozygous Donor #1 electroporated with Cas9 only. Error bars indicate the standard deviation ( n = 3 replicates); donors edited without sgRNA were plotted as a negative control. d , CD34 + cells from patients #1 and #2 were electroporated with Cas9 coupled with sgRNA-1 and with different concentration gradient DNA templates. HM mock, HSPCs of homozygous Donor #1 only; HT mock, HSPCs of heterozygote Donor #2 only; w/o donor, patient donors edited with RNPs but without DNA templates; Forward and reverse normal gene sequences were synthesized expressed by w/ssODN and w/ssODN-R. These two single-stranded DNAs were incorporated into dsODNs annealed by PCR. Primers with 5′ modifications included an amine group with a C6 linker (AmC6) or C12 linker (AmC12) expressed by w/ss-AmC6, w/ss-AmC12. e , β globin expression by RT–qPCR analysis in erythroid cells in vitro differentiated from RNP-edited CD34 + HSPCs. Data are plotted as the mean ± s.d. and analyzed using unpaired two-tailed Student’s t tests. Data are representative of three biologically independent replicates.

Article Snippet: We transformed pET-21a _ 3×NLS–Streptococcus pyogenes Cas9 available on Addgene (SpCas9, ID #114365) into BL21 (DE3) chemically competent cells (TransGen Biotech, CD601-02) and grew the cells in LB medium at 37°C 220 rpm until the density reached OD600 = 2-2.5.

Techniques: CRISPR, Mutagenesis, Standard Deviation, Negative Control, Concentration Assay, Synthesized, Expressing, Quantitative RT-PCR, In Vitro, Two Tailed Test

Journal: bioRxiv

Article Title: Efficient repair of human homozygous genetic mutation by CRISPR/Cas9 mediated interlocus gene conversion

doi: 10.1101/2022.09.05.506576

Figure Lengend Snippet: a , Efficiency of restoration edited by Cas9 coupled with the various sgRNAs in homozygous β 0 4142 CD34 + HSPCs measured by deep-seq analysis. Error bars indicate the standard deviation ( n =3 replicates). b , β-globin expression by RT–qPCR analysis in erythroid cells in vitro differentiated from RNP-edited CD34 + HSPCs. c , The homologous genomic sequence of HBB and HBD , Red triangle represents the deletion base pairs, the color purple marked a NGG protospacer-adjacent motif, gray marked sgRNA-1. d , Restoration of the β 0 4142 in HBD-4142 KO donor determined by deep-seq analysis versus edited only by the RNP complex. Gray points HBD - represent samples with KO of HBD-4142 edited by Cas9 coupled with sgRNA-1 72 hours later, and red points represent samples edited only with Cas9 coupled with sgRNA-1. e , Efficiency of restorations as measured by deep-seq analysis of Cas9:sgRNA-1 RNP targeting the β 0 4142 site in CD34 + HSPCs from patient donors (see details in Table S2). f–h , Genotyping of erythroid cells derived from single colonies screened out edited CD34 + HSPCs from Donors #1 and #2. The purple dashed line in f highlights IVS1-1 G>T, corrected deletions of β 0 4142 (orange dashed line), specific fragments or base pairs of HBB distinct from HBD (blue dashed line in g, h ). i , Indels at the HBD locus measured by deep-seq analysis of sgRNA-1 RNP targeting the β 0 4142 site. In all graphs, data are plotted as the mean ± s.d. and analyzed using unpaired two-tailed Student’s t tests. Data are representative of three biologically independent replicates.

Article Snippet: We transformed pET-21a _ 3×NLS–Streptococcus pyogenes Cas9 available on Addgene (SpCas9, ID #114365) into BL21 (DE3) chemically competent cells (TransGen Biotech, CD601-02) and grew the cells in LB medium at 37°C 220 rpm until the density reached OD600 = 2-2.5.

Techniques: Standard Deviation, Expressing, Quantitative RT-PCR, In Vitro, Sequencing, Derivative Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Efficient repair of human homozygous genetic mutation by CRISPR/Cas9 mediated interlocus gene conversion

doi: 10.1101/2022.09.05.506576

Figure Lengend Snippet: CD34 + HSPCs from donors with β 0 4142 were electroporated with 3NLS Cas9 and sgRNA-1 dependent or independent of exogenous DNA templates. We transplanted 5–8 × 10 5 treated cells into NBSGW mice via tail-vein injection. Mouse BM was collected and analyzed 16 weeks after transplantation. a , Experimental workflow. b , In BM, as well as the indel frequencies determined by TIDE analysis. c , Engraftment measured by the percentage of human CD45 + (hCD45 + /(hCD45 + +mCD45 + )) cells in recipient mouse BM. d , Human B cells (hCD19 + ) and myeloid cells (hCD33 + ) as percentages of the hCD45 + population in recipient BM. e , Human erythroid precursors (hCD235a + ) as a percentage of human and mouse CD45 − cells in recipient BM. f , In BM, proportions of β-globin mRNA expression level by RT–qPCR normalized by α-globin. g , β 0 4142-to-normol editing efficiency in human CD34 + cell-derived lineages from recipient BM. h . Indel spectrum of input cells from Donor #1 and Donor #5 electroporated with sgRNA-1 before transplantation and BM-engrafted human cells 16 weeks after transplantation. The indel spectrum was determined by deep sequencing analysis. These data comprise 3 mice transplanted from Donor #5 and 5 mice transplanted from Donor #1 with sgRNA-1 edited inputs. Each symbol represents a mouse, and the mean for each group is shown. The median of each group with 3–5 mice in b, c, e, and f is shown as a line. Data are plotted as the mean ± s.d. for d, g and were analyzed using unpaired two-tailed Student’s t tests. Data are representative of three biologically independent replicates.

Article Snippet: We transformed pET-21a _ 3×NLS–Streptococcus pyogenes Cas9 available on Addgene (SpCas9, ID #114365) into BL21 (DE3) chemically competent cells (TransGen Biotech, CD601-02) and grew the cells in LB medium at 37°C 220 rpm until the density reached OD600 = 2-2.5.

Techniques: Injection, Transplantation Assay, Expressing, Quantitative RT-PCR, Derivative Assay, Sequencing, Two Tailed Test

Journal: Nature communications

Article Title: MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits.

doi: 10.1038/s41467-020-19842-2

Figure Lengend Snippet: Fig. 3 MiCas9 reduces off-target indel rates. a Off-target and on-target indel rates associated with sg1-VEGFA by spCas9 and miCas9 pDNAs. b Off- target and on-target indel rates associated with sg-FANCF2 by spCas9 and miCas9 pDNAs. c Indel rates at Guide-seq predicted potential off-target loci associated with sg1-VEGFA by pDNAs of different nucleases. d Indel rates at Guide-seq predicted potential off-target loci associated with sg-FANCF2 by pDNAs of different nucleases. Sp: spCas9, Mi: miCas9, Hy: hypaCas9, Hi: HiFiCas9, HE: Cas9-HE, GE: Cas9-GE, NC: negative control with non-specific gRNA. #Reads: Average amplicon reads per sample. BD: below detection, representing values <0.10%. Three independent experiments were performed for each condition. Data are presented as mean ± standard error of means (SEM) in a and b, and as heat map in c and d. Unpaired t-test (two tailed) was used to compare data using GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, CA). Source data are available in the Source Data file.

Article Snippet: HypaCas9 (Cat# 101178), Cas9HE (Cat# 109400), and Cas9-GE (Cat# 109401) plasmid DNAs were acquired from Addgene (www.addgene.org).

Techniques: Negative Control, Amplification, Two Tailed Test, Software

Journal: Nucleic Acids Research

Article Title: Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis

doi: 10.1093/nar/gkab858

Figure Lengend Snippet: Types of engineered Cas9 variants and the results of in vitro DNA cleavage assay. ( A ) Domain organization of type II-A Cas9 from S. Pyogenes (SpCas9) and correspondingly color-coded crystal structure of Cas9:gRNA:DNA ternary complex (PDB ID: 5F9R) . ( B ) Engineered Cas9 variants used in this work and their mutated residues. ( C ) Two-dimensional plot for cleavage efficiency of wild-type and engineered Cas9 variants toward on-target and off-target DNAs with different degrees of mismatch (denoted by Mi-j for bases mutated from the i th through j th sites counting from PAM).

Article Snippet: The other Cas9 variants were obtained from Addgene (HypaCas9 #101218, evoCas9 #107550, xCas9(3.7) #108379).

Techniques: In Vitro, DNA Cleavage Assay

Journal: Nucleic Acids Research

Article Title: Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis

doi: 10.1093/nar/gkab858

Figure Lengend Snippet: Single-molecule FRET assay for conformational dynamics of Cas9:gRNA:DNA ternary complex. ( A ) Experimental scheme for single-molecule FRET detection of two different conformations of the complex (denoted ‘open’ with FRET efficiency E low ∼ 0.2 and ‘zipped’ with E high ∼ 0.8). The two FRET histograms represent when the DNA is on-target (upper) and off-target with M18–20 mismatch (lower). ( B ) Fraction of E high (= E high /( E high + E low )) for wild-type and engineered Cas9 variants with respect to on-target and off-target DNAs with different degrees of mismatch (mean ± s.e.m., n = 2 or 3). Relative population was calculated from FRET histograms fitted to Gaussian functions .

Article Snippet: The other Cas9 variants were obtained from Addgene (HypaCas9 #101218, evoCas9 #107550, xCas9(3.7) #108379).

Techniques:

Journal: Nucleic Acids Research

Article Title: Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis

doi: 10.1093/nar/gkab858

Figure Lengend Snippet: Classification of single-molecule time trajectories and determination of reaction quotients for different stages of Cas9:gRNA:DNA interaction pathway. ( A ) Representative time trajectories of three different states of Cas9:gRNA:DNA for (#1) docked-open state ( D Open), (#2) transitional state, and (#3) docked-zipped state ( D Zipped). Transitional state is subcategorized into transitional-open ( T Open) and transitional-zipped ( T Zipped) states according to our reversible reaction model. Each of the three reversible transitions involving these four states is characterized by its own reaction quotient, Q D , Q T or Q Z . ( B ) Fraction (in %) of the ternary complexes of wild-type and engineered Cas9 variants in each of the docked-zipped, transitional, and docked-open states when they interact with on-target and off-target DNAs (mean ± s.e.m., n = 3). ( C ) Representative FRET histograms constructed from the sum of complexes in the docked-open + transitional states (upper), transitional states only (middle), and transitional + docked-zipped states (lower). Population ratio of open ( D Open and/or T Open) versus zipped ( D Zipped and/or T Zipped) conformations is used to calculate the reaction quotients. ( D ) The calculated reaction quotients for complexes of wild-type and engineered Cas9 variants toward on-target and off-target DNAs.

Article Snippet: The other Cas9 variants were obtained from Addgene (HypaCas9 #101218, evoCas9 #107550, xCas9(3.7) #108379).

Techniques: Construct

Journal: Nucleic Acids Research

Article Title: Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis

doi: 10.1093/nar/gkab858

Figure Lengend Snippet: Specificity values (S) of wild-type and engineered Cas9 variants. ( A ) Specificity values (S T and S Z ) of wild-type and engineered Cas9 variants. ( B ) WT-normalized specificity values (S T and S Z ) of engineered Cas9 variants (mean ± s.e.m., n = 2 or 3).

Article Snippet: The other Cas9 variants were obtained from Addgene (HypaCas9 #101218, evoCas9 #107550, xCas9(3.7) #108379).

Techniques:

Journal: Nucleic Acids Research

Article Title: Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis

doi: 10.1093/nar/gkab858

Figure Lengend Snippet: Schematic representation for different stages of off-target discrimination activity by Cas9:gRNA:DNA ternary complex of different Cas9 variants. Off-target discrimination occurs mainly in the stage of DNA:RNA heteroduplexation for of evoCas9 and HypaCas9, whereas it occurs in the post-heteroduplexation stage for Cas9-HF1, eCas9 and Sniper-Cas9.

Article Snippet: The other Cas9 variants were obtained from Addgene (HypaCas9 #101218, evoCas9 #107550, xCas9(3.7) #108379).

Techniques: Activity Assay