Cas12a trans-cleavage can be modulated in vitro and is active on ssDNA, dsDNA, and RNA

Fuchs, Ryan T.; Curcuru, Jennifer L.; Mabuchi, Megumu; Yourik, Paul; Robb, G. Brett

doi:10.1101/600890

Cited by 46 publications

(61 citation statements)

References 55 publications

(76 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To measure the collateral or trans-cleavage activity of Cas12a, we employed a FRET-based reporter used in DETECTR 1 , composed of a fluorophore (FAM) and a quencher (3IABkFQ) connected by a 5nucleotide sequence (TTATT), which displays increased fluorescence upon cleavage. Consistent with the previous literature 13 , when using wild-type crRNAs, we observed that the LbCas12a exhibited higher trans-cleavage activity than the AsCas12a or the FnCas12a, and therefore, we designed various modified crRNAs compatible with LbCas12a. Using the same reporters, we discovered that ssDNA and ssRNA extensions on the 3′-end of crGFP markedly enhanced the trans-cleavage ability of targetactivated LbCas12a.…”

Section: Resultssupporting

confidence: 85%

Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

2020

View full text Add to dashboard Cite

The CRISPR-Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection but are limited to the picomolar detection limit without an amplification step. Here, we develop a platform with engineered crRNAs and optimized conditions that enabled us to detect various clinically relevant nucleic acid targets with higher sensitivity, achieving a limit of detection in the femtomolar range without any target pre-amplification step. By extending the 3′- or 5′-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discover a self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA and with significant improvement in specificity for target recognition. Particularly, the 7-mer DNA extension to crRNA is determined to be universal and spacer-independent for enhancing the sensitivity and specificity of LbCas12a-mediated nucleic acid detection. We perform a detailed characterization of our engineered ENHANCE system with various crRNA modifications, target types, reporters, and divalent cations. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs are incorporated in a paper-based lateral flow assay that can detect the target with up to 23-fold higher sensitivity within 40–60 min.

show abstract

Section: Resultssupporting

confidence: 85%

Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

2020

View full text Add to dashboard Cite

show abstract

“…In order to measure the collateral or trans-cleavage activity of Cas12a, we employed a FRET-based reporter used in DETECTR, 1 composed of a fluorophore (FAM) and a quencher (3IABkFQ) connected by a 5-nucleotide sequence (TTATT), which displays increased fluorescence upon cleavage. Consistent with the previous literature 13 , respectively. The fold in fluorescence was normalized by taking the ratio of background-corrected fluorescence signals of sample with activator to the corresponding sample without activator.…”

supporting

confidence: 92%

“…Overall, LbCas12a showed the highest fluorescence signal, which is consistent with previous studies. 13,18 Through observation of the fluorophore-quencher-based reporter assay and time-dependent gel electrophoresis, we hypothesized that the various extensions of ssDNA on the crRNA induce conformational changes on LbCas12a that result in enhanced endonuclease activity.…”

mentioning

confidence: 99%

Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

Nguyễn

Smith

Jain

2020

Preprint

View full text Add to dashboard Cite

The CRISPR/Cas12a RNA-guided complexes have a tremendous potential for nucleic acid detection due to its ability to indiscriminately cleave ssDNA once bound to a target DNA. However, the current CRISPR/Cas12a systems are limited to detecting DNA in a picomolar detection limit without an amplification step. Here, we developed a platform with engineered crRNAs and optimized conditions that enabled us to detect DNA, DNA/RNA heteroduplex and methylated DNA with higher sensitivity, achieving a limit of detection of in femtomolar range without any target pre-amplification step. By extending the 3'-or 5'-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discovered a new selfcatalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA. We applied this sensitive system to detect as low as 25 fM dsDNA from the PCA3 gene, an overexpressed biomarker in prostate cancer patients, in simulated urine over 6 hours. The same platform was used to detect as low as ~700 fM cDNA from HIV, 290 fM RNA from HCV, and 370 fM cDNA from SARS-CoV-2, all within 30 minutes without a need for target amplification. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs were incorporated in a paper-based lateral flow assay that could detect the target with up to 23-fold higher sensitivity within 40-60 minutes. MainClass 2 CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPRassociated proteins) systems, such as Cas12a (Cpf1, subtype V-A) and Cas13a (C2c2, subtype VI), are capable of nonspecific cleavage of ssDNA (single-stranded DNA) and RNA, respectively, in addition to successful gene editing. 1-3 This attribute, known as trans-cleavage, is only activated once bound to an activator (ssDNA or dsDNA) that has complementary base-pairing to the guide crRNA. When combined with a FRET-based reporter, a fluorophore connected to a quencher via a short oligonucleotide sequence, the presence of the target activator can be confirmed. This phenomenon has been efficiently harnessed by SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) and DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) to reliably detect nucleic acids. 1,[4][5][6][7][8] Research studies have reported that an extended secondary DNA on the guide crRNA for Cas12a or a hairpin RNA structure added to the sgRNA for Cas9 increases the efficiency and specificity of gene editing. 9,10 In addition, chemically modified Cas12a guided-RNA has also been shown to facilitate improved gene correction in mammalian cells through both viral-and non-viral methods compared to the wild-type guide RNA. 11 Though these modifications were employed to utilize the cis-cleavage aspect of the CRISPR/Cas systems, the effects of such alterations on the transcleavage remain unknown.was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

show abstract

“…We also establish that Cas12a has non-specific dsDNA nicking activity upon binding to a crRNA-complementary DNA. While this manuscript was in preparation, a complementary study reported similar observations, demonstrating that these activities are reproducible in vitro (42).…”

Section: Discussionsupporting

confidence: 59%

“…This may be attributed to decreased breathing and exposure of ssDNA sites. It remains unclear how negatively supercoiled plasmids are nicked multiple times leading to linearization; however, multiple events of the trans nicking activity may cause the DNA to eventually fall apart (42). This suggests that a cumulative effect of all the trans activities of Cas12a eventually leads to complete degradation of nucleic acid substrates (Fig.…”

Section: Discussionmentioning

confidence: 99%

CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects

Murugan

Seetharam

Severin

et al. 2020

Journal of Biological Chemistry

106

121

View full text Add to dashboard Cite

Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.

show abstract

Cas12a trans-cleavage can be modulated in vitro and is active on ssDNA, dsDNA, and RNA

Cited by 46 publications

References 55 publications

Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects

Contact Info

Product

Resources

About