Inconsistent treatments of the kinetics of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) impair assessment of its diagnostic potential

Abstract

The scientific and technological advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is one of the most exciting developments of the past decade, particularly in the field of gene editing. The technology has two essential components, (1) a guide RNA to match a targeted gene, and (2) a CRISPR-associated protein (e.g., Cas 9, Cas12, or Cas13) that acts as an endonuclease to specifically cut DNA. This specificity and reconfigurable nature of CRISPR has also spurred intense academic and commercial interest in the development of CRISPR-based molecular diagnostics. CRISPR Cas12 and Cas13 orthologs are most commonly applied to diagnostics, and these cleave and become activated by DNA and RNA targets, respectively. Despite the resulting intense research activity, the possible limits of detection and applications of CRISP-based diagnostics remain an open question. A major reason for this is that reports of CRISPR-Cas kinetic rates have been widely inconsistent, and the vast majority of these reports have been shown to contain gross errors including violations of basic conservation and kinetic rate laws. It is the intent of this Perspective to bring attention to these issues and to, more importantly, identify potential improvements in the manner in which CRISPR kinetic rates and assay LoDs are reported and compared. The CRISPR field would benefit from formal checks of self-consistency of data, providing sufficient information and raw data such that experiments can be reproduced, and, in the case of publications that report LoDs of novel assays, concurrent and clear reporting of the kinetic rate constants responsible for such LoDs. The early development of CRISPR-based diagnostics calls for self-reflection and urges all of us to proceed with caution.

Uncertainty Quantification of Michaelis–Menten Kinetic Rates and Its Application to the Analysis of CRISPR‐Based Diagnostics

“…The cis ‐cleavage step is analogous to a second‐order reaction, where the low‐abundance target is consumed. As we discuss in the Supporting Information (S7), for typical detection assays, the time‐scale for completion of the cis ‐cleavage portion of CRISPR assays is typically significantly lower than that of the trans ‐cleavage [20] . Hence, the trans ‐cleavage step has been identified as the rate‐limiting reaction in diagnostics assays, [18] and this is true even for trace amounts of target.…”

“…A recent and important example of Michaelis–Menten rate parameter evaluation is the quantification of CRISPR‐associated (Cas) enzyme kinetics. CRISPR‐Cas systems are used to detect nucleic acid sequences with high specificity, [13–17] and the limit of detection of such assays is directly governed by the kinetic rates of the enzyme [18–20] . The accurate quantification of the kinetic parameters is therefore paramount to evaluate the reliability and regime of applicability of CRISPR‐based diagnostics assays.…”

“…CRISPR-Cas systems are used to detect nucleic acid sequences with high specificity, [13][14][15][16][17] and the limit of detection of such assays is directly governed by the kinetic rates of the enzyme. [18][19][20] The accurate quantification of the kinetic parameters is therefore paramount to evaluate the reliability and regime of applicability of CRISPR-based diagnostics assays. Despite its importance, the vast majority of CRISPR-Cas enzyme kinetics reports have exhibited grossly inconsistent data that clearly and demonstrably violate basic laws of mass conservation and chemical kinetics.…”

“…Despite its importance, the vast majority of CRISPR‐Cas enzyme kinetics reports have exhibited grossly inconsistent data that clearly and demonstrably violate basic laws of mass conservation and chemical kinetics. Ramachandran and Santiago [18] and Santiago [20] collectively discuss over 10 examples of publications which exhibit gross errors in kinetic rate data and/or report limits of detection which are difficult to reconcile given current capabilities of CRISPR‐Cas systems. At the same time, $${{k}_{cat}}$$ and $${{K}_{M}}$$ values are often reported to three significant figures.…”

Uncertainty Quantification of Michaelis–Menten Kinetic Rates and Its Application to the Analysis of CRISPR‐Based Diagnostics

“…As we discuss in the Supporting Information (S7), for typical detection assays, the time-scale for completion of the cis-cleavage portion of CRISPR assays is typically significantly lower than that of the trans-cleavage. [20] Hence, the trans-cleavage step has been identified as the rate-limiting reaction in diagnostics assays, [18] and this is true even for trace amounts of target. The trans-cleavage step is here modeled by Michaelis-Menten kinetics and is the main focus of the present work.…”