2、CrRNA、PAM and reporter probes The crRNA of Cas12a typically consists of two parts: A: Repeat Sequence (Handle): This is a highly conserved region responsible for binding with the Cas12a protein to form an active complex. The repeat sequence of LbCas12a's crRNA is: 5'-UAAUUUCUACUAAGUGUAGAU-3'. B: Target Sequence (Spacer or Guide Sequence): This portion is designed to recognize the target nucleic acid. It typically spans 20 to 24 nucleotides. This sequence pairs with the corresponding region in the target DNA sequence, and its length and accuracy directly influence the specificity and efficiency of recognition. For LbCas12a, it is generally required that a conserved PAM sequence, typically “TTTV” (where V represents A, C, or G), is present adjacent to the target sequence. This means that when designing the crRNA, the target sequence must be close to a suitable PAM sequence; otherwise, even if the crRNA pairs with the sequence, Cas12a will not be activated. Reporter Probes Reporter probes, acting as cleavage substrates, are typically designed as single-stranded DNA because the simple, linear structure of single-stranded DNA helps reduce complex secondary structures, thus ensuring efficient cleavage. The probe does not need to be complementary to the target sequence. The Cas12a recognition mechanism depends on the pairing of crRNA with the target sequence, not the probe itself. A common design is 5'-FAM-TTTTT-BHQ1-3', where FAM is a fluorescent dye and BHQ1 is a quencher. Thus, the Cas12a-based detection system has a dual security mechanism: Specificity Assurance: Through the precise complementarity between crRNA and the target sequence, combined with the strict requirement for a PAM sequence, only samples containing the target nucleic acid can activate Cas12a. Signal Amplification: Once activated, the trans-cleavage activity non-specifically and indiscriminately cleaves a large number of probes. This multiple cleavage behavior amplifies the initial small signal into a large, detectable signal. Even with very few target molecules, the generated fluorescence signal is strong enough to significantly enhance sensitivity. | 
1、 LbCas12a features
LbCas12a is derived from Lachnospiraceae bacterium (LBA), a Gram-positive bacterium. Due to its origin, it is commonly referred to with the prefix "Lb"; "Cas12a" indicates that it belongs to a member of the Cas12 family (formerly known as Cpf1). LbCas12a is part of the Class Type V CRISPR-Cas system, characterized by the fact that a single effector protein is sufficient to mediate the recognition and cleavage of target DNA.
As a nuclease, Cas12a relies on its binding with a pre-designed CRISPR RNA (crRNA) to function. The crRNA carries a sequence complementary to the target sequence, which serves as a "code" to guide Cas12a to the target nucleic acid. When Cas12a binds with crRNA, it forms a functional complex capable of searching for and binding to the target sequence.
While scanning in the sample, the Cas12a–crRNA complex looks for the presence of nucleic acids that are complementary to the crRNA sequence. In this process, in addition to complementary matching, an additional condition exists: a specific short sequence, known as the "Protospacer Adjacent Motif" (PAM), must be present next to the target sequence. The PAM sequence is a critical signal for Cas12a to correctly recognize and bind to the target, preventing misrecognition.
Once the Cas12a–crRNA complex successfully binds to the target sequence, Cas12a undergoes a conformational change, activating two types of cleavage activities: 1) Cis-cleavage, where Cas12a performs precise cuts near the target sequence, a process used in some detection schemes to confirm the presence of the target sequence; 2) Trans-cleavage activity, where it non-specifically cleaves surrounding single-stranded DNA molecules. This feature allows probes to be added as cleavage substrates to amplify the signal.
2、CrRNA、PAM and reporter probes
The crRNA of Cas12a typically consists of two parts:
A: Repeat Sequence (Handle):
This is a highly conserved region responsible for binding with the Cas12a protein to form an active complex. The repeat sequence of LbCas12a's crRNA is:
5'-UAAUUUCUACUAAGUGUAGAU-3'.
B: Target Sequence (Spacer or Guide Sequence):
This portion is designed to recognize the target nucleic acid. It typically spans 20 to 24 nucleotides. This sequence pairs with the corresponding region in the target DNA sequence, and its length and accuracy directly influence the specificity and efficiency of recognition. For LbCas12a, it is generally required that a conserved PAM sequence, typically “TTTV” (where V represents A, C, or G), is present adjacent to the target sequence. This means that when designing the crRNA, the target sequence must be close to a suitable PAM sequence; otherwise, even if the crRNA pairs with the sequence, Cas12a will not be activated.
Reporter Probes
Reporter probes, acting as cleavage substrates, are typically designed as single-stranded DNA because the simple, linear structure of single-stranded DNA helps reduce complex secondary structures, thus ensuring efficient cleavage.
The probe does not need to be complementary to the target sequence. The Cas12a recognition mechanism depends on the pairing of crRNA with the target sequence, not the probe itself.
A common design is 5'-FAM-TTTTT-BHQ1-3', where FAM is a fluorescent dye and BHQ1 is a quencher.
Thus, the Cas12a-based detection system has a dual security mechanism:
Specificity Assurance: Through the precise complementarity between crRNA and the target sequence, combined with the strict requirement for a PAM sequence, only samples containing the target nucleic acid can activate Cas12a.
Signal Amplification: Once activated, the trans-cleavage activity non-specifically and indiscriminately cleaves a large number of probes. This multiple cleavage behavior amplifies the initial small signal into a large, detectable signal. Even with very few target molecules, the generated fluorescence signal is strong enough to significantly enhance sensitivity.
3. Amp-future’s LbCas12a
Amp Future's LbCas12a has nuclear localization signals (NLS) at both the N- and C-termini to facilitate the entry of the LbCas12a/gRNA ribonucleoprotein (RNP) complex into the mammalian cell nucleus for genome engineering purposes.
LbCas12a Expression vector
Combined with the Multienzyme Isothermal Rapid Amplification (MIRA) LbCas12a detection system,
Amp Future's LbCas12a exhibits enhanced cleavage activity.
Blue Curve
Black Curve
AMP’s LbCas12a
Other brands’ Cas12a
MIRA Product
crRNA(10μM)
Reporter probe(10μM)
DEPC water
Total 20μL