A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing

Edraki, Alireza; Mir, Aamir; Ibraheim, Raed; Gainetdinov, Ildar; Yoon, Yeonsoo; Song, Chun‐Qing; Cao, Yusong; Gallant, Judith; Xue, Wen; Rivera-Pérez, Jaime A.; Sontheimer, Erik J.

doi:10.1016/j.molcel.2018.12.003

Cited by 219 publications

(240 citation statements)

References 83 publications

Supporting

Mentioning

216

Contrasting

Unclassified

Order By: Relevance

“…S1) will be inhibited by the Acr, since those extrahepatic tissues lack miR-122 and therefore fail to silence Acr expression. To validate this concept, we chose two well-established Cas9-Acr combinations: AcrIIC3 Nme and Nme1Cas9 or Nme2Cas9 (Type II-C; Pawluk et al 2016;Edraki et al 2018) as well as AcrIIA4 Lmo and SpyCas9 (Type II-A; Rauch et al 2017). Nme2Cas9 is a recently reported Cas9 homolog that has a dinucleotide (N 4 CC) protospacer adjacent motif (PAM) , enabling a target site density comparable to that of SpyCas9 (NGG PAM).…”

Section: Aav Delivery Of Nme1cas9 and Sgrna Results In Editing In Varmentioning

confidence: 99%

Tissue-restricted genome editing in vivo specified by microRNA-repressible anti-CRISPR proteins

Lee

Mou

Ibraheim

et al. 2019

RNA

Self Cite

View full text Add to dashboard Cite

CRISPR-Cas systems are bacterial adaptive immune pathways that have revolutionized biotechnology and biomedical applications. Despite the potential for human therapeutic development, there are many hurdles that must be overcome before its use in clinical settings. Some clinical safety concerns arise from editing activity in unintended cell types or tissues upon in vivo delivery (e.g., by adeno-associated virus (AAV) vectors). Although tissue-specific promoters and serotypes with tissue tropisms can be used, suitably compact promoters are not always available for desired cell types, and AAV tissue tropism specificities are not absolute. To reinforce tissue-specific editing, we exploited anti-CRISPR proteins (Acrs) that have evolved as natural countermeasures against CRISPR immunity. To inhibit Cas9 in all ancillary tissues without compromising editing in the target tissue, we established a flexible platform in which an Acr transgene is repressed by endogenous, tissue-specific microRNAs (miRNAs). We demonstrate that miRNAs regulate the expression of an Acr transgene bearing miRNA-binding sites in its 3 ′ ′ ′ ′ ′ -UTR and control subsequent genome editing outcomes in a cell-type specific manner. We also show that the strategy is applicable to multiple Cas9 orthologs and their respective anti-CRISPRs. Furthermore, we validate this approach in vivo by demonstrating that AAV9 delivery of Nme2Cas9, along with an AcrIIC3 Nme construct that is targeted for repression by liver-specific miR-122, allows editing in the liver while repressing editing in an unintended tissue (heart muscle) in adult mice. This strategy provides safeguards against off-tissue genome editing by confining Cas9 activity to selected cell types.

show abstract

Section: Aav Delivery Of Nme1cas9 and Sgrna Results In Editing In Varmentioning

confidence: 99%

Tissue-restricted genome editing in vivo specified by microRNA-repressible anti-CRISPR proteins

Lee

Mou

Ibraheim

et al. 2019

RNA

Self Cite

View full text Add to dashboard Cite

show abstract

“…Engineered and natural Cas9 variants, as well as new subtypes of Cas nucleases, have been reported to reduce off-target DNA cleavage in cells (Amrani et al, 2018;Chen et al, 2017;Edraki et al, 2018;Kleinstiver et al, 2016a;Lee et al, 2018;Ran et al, 2015;Shmakov et al, 2015;Slaymaker et al, 2016;Smargon et al, 2017;Wu et al, 2018;Zetsche et al, 2015). For example, a series of engineered SpCas9s improve cleavage specificity by reducing protein interactions with either DNA strand or by modulating activation of the nuclease domains Kleinstiver et al, 2016a;Slaymaker et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Massively parallel kinetic profiling of natural and engineered CRISPR nucleases

Jones

Hawkins

Johnson

et al. 2019

Preprint

View full text Add to dashboard Cite

Engineered Streptococcus pyogenes (Sp) Cas9s and Acidaminococcus sp. (As) Cas12a (formerly Cpf1) improve cleavage specificity in human cells. However, the fidelity, enzymatic mechanisms, and cleavage products of emerging CRISPR nucleases have not been profiled systematically across partially mispaired off-target DNA sequences. Here, we describe NucleaSeqnuclease digestion and deep sequencing-a massively parallel platform that measures cleavage kinetics and captures the timeresolved identities of cleaved products for more than ten thousand DNA targets that include mismatches, insertions, and deletions relative to the guide RNA. The binding specificity of each enzyme is measured on the same DNA library via the chip-hybridized association mapping platform (CHAMP). Using this integrated cleavage and binding platform, we profile four SpCas9 variants and AsCas12a. Engineered Cas9s retain wtCas9-like off-target binding but increase cleavage specificity; Cas9-HF1 shows the most dramatic increase in cleavage specificity. Surprisingly, wtCas12a-reported as a more specific nuclease in cells-has cleavage specificity similar to wtCas9 in vitro. Initial cleavage position and subsequent end-trimming vary across nucleases, guide RNA sequences, and position and base identity of mispairs in target DNAs. Using these large datasets, we develop a biophysical model that reveals mechanistic insights into off-target cleavage activities by these nucleases. More broadly, NucleaSeq enables rapid, quantitative, and systematic comparison of the specificities and cleavage products of engineered and natural nucleases.

show abstract

“…The fraction of GFP- and mCherry-positive cells can be rapidly measured using flow cytometry to determine editing outcomes as a function of the nuclease and donor DNA composition. We redesigned the original TLR reporter to incorporate target sites for several currently characterized nucleases (Figure 1A) by introducing protospacer adjacent motifs (PAMs) belonging to Cas9/Cas12a orthologs from Streptococcus pyogenes (SpyCas9) 38, 39 , Neisseria meningiditis (Nme1Cas9 and Nme2Cas9) 40–42 , Campylobacter jejuni (CjeCas9) 43–45 , Staphylococcus aureus (SauCas9) 46 , Geobacillus stearothermophilus (GeoCas9) 47 , Lachnospiraceae bacterium ND2006 (LbaCas12a) 48 , Acidaminococcus sp. (AspCas12a) 48 and Francisella novicida (FnoCas12).…”

Section: Resultsmentioning

confidence: 99%

Efficient Homology-directed Repair with Circular ssDNA Donors

Iyer

Mir

Vega-Badillo

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

32While genome editing has been revolutionized by the advent of CRISPR-based 33 nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair 34(HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer 35 from low HDR efficiencies in many cell types, as well as integration at unintended sites. 36In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with 37 minimal off-target integration. Here, we describe the use of ssDNA phage to efficiently 38 and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors 39 serve as efficient HDR templates when used with Cas9 or Cas12a, with integration 40 frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative 41 efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a 42 nucleases, we have developed a modified Traffic Light Reporter (TLR) system [TLR-43

show abstract

A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing

Cited by 219 publications

References 83 publications

Tissue-restricted genome editing in vivo specified by microRNA-repressible anti-CRISPR proteins

Tissue-restricted genome editing in vivo specified by microRNA-repressible anti-CRISPR proteins

Massively parallel kinetic profiling of natural and engineered CRISPR nucleases

Efficient Homology-directed Repair with Circular ssDNA Donors

Contact Info

Product

Resources

About