Science：CRISPR-Cas12a靶標結合釋放不加選擇的單鏈DNA酶活性
CRISPR-Cas12a靶標結合釋放不加選擇的單鏈DNA酶活性
摘 要
CRISPR-Cas12a（Cpf1）蛋白是RNA引導的酶，其結合併切割DNA作為細菌適應性免疫系統的組分。像CRISPR-Cas9一樣，Cas12a基於其產生靶向雙鏈DNA（dsDNA）斷裂的能力而被用於基因組編輯。在這裡，我們顯示RNA引導的DNA結合釋放了Cas12a完全降解ssDNA分子的不加選擇的單鏈DNA（ssDNA）切割活性。我們發現目標激活的非特異性ssDNase切割也是其他V型CRISPR-Cas12酶的特性。通過將Cas12a ssDNase激活與等溫擴增相結合，我們創建了一種名為DNA Endonuclease Targeted CRISPR Trans Reporter（DETECTR）的方法，該方法實現了DNA檢測的極對敏感性。 DETECTR能夠快速，專一地檢測患者樣本中的人乳頭瘤病毒，從而為分子診斷提供了一個簡單的平台。
CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing based on its ability to generate targeted, double-stranded DNA (dsDNA) breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. We find that target-activated, non-specific ssDNase cleavage is also a property of other type V CRISPR-Cas12 enzymes. By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR), which achieves attomolar sensitivity for DNA detection. DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics.
細菌和古細菌中的CRISPR-Cas適應性免疫使用RNA引導的核酸酶來靶向和降解外源核酸（1-3）。 CRISPR-Cas9家族蛋白已被廣泛用於基因編輯應用（4,5），其基於由兩個催化結構域RuvC和HNH在與指導RNA互補的序列處誘導的雙鏈DNA（dsDNA）切割的精確度（6,7）。第二個酶家族CRISPR-Cas12a（Cpf1）使用單個RuvC催化結構域來引導RNA指導的dsDNA切割（8-13）（圖1A）。與Cas9不同，Cas12a酶識別富含T的原型間隔區相鄰基序（PAM）（8），催化它們自己的引導RNA（crRNA）成熟（14）併產生具有交錯的5"和3"末端的PAM遠端dsDNA斷裂8），這些特徵引起了基因編輯應用的興趣（15,16）。然而，Cas12a的底物特異性和DNA裂解機理仍有待完全闡明。
CRISPR-Cas adaptive immunity in bacteria and archaea uses RNA-guided nucleases to target and degrade foreign nucleic acids (1-3). The CRISPR-Cas9 family of proteins has been widely deployed for gene editing applications (4, 5) based on the precision of double-stranded DNA (dsDNA) cleavage induced by two catalytic domains, RuvC and HNH, at sequences complementary to a guide RNA (6, 7). A second family of enzymes, CRISPR-Cas12a (Cpf1), uses a single RuvC catalytic domain for guide RNA-directed dsDNA cleavage (8-13) (Fig. 1A). Distinct from Cas9, Cas12a enzymes recognize a T-rich protospacer adjacent motif (PAM) (8), catalyze their own guide RNA (crRNA) maturation (14) and generate a PAM-distal dsDNA break with staggered 5" and 3" ends (8), features that have attracted interest for gene editing applications (15, 16). However, the substrate specificity and DNA cleavage mechanism of Cas12a remain to be fully elucidated.
在調查Cas12a激活的底物需求時，我們測試了滑絲螺桿菌細菌ND2006 Cas12a（LbCas12a），用於指導RNA-指導的單鏈DNA（ssDNA）切割，這是各種CRISPR-Cas9直系同源物的能力（17,18）。將純化的LbCas12a或化膿性鏈球菌Cas9（SpCas9）蛋白（圖S1）與靶向環狀單鏈M13DNA噬菌體的指導RNA序列組裝。與SpCas9相比，我們驚訝地發現LbCas12a通過不能通過序列特異性DNA切割解釋的切割機制誘導M13快速和完全降解（圖1B）。這種ssDNA切割活性，在催化失活的LbCas12a（D832A）中未觀察到，提高了目標結合的LbCas12a可能降解任何ssDNA序列的可能性。值得注意的是，LbCas12a還在不同的引導RNA及其與ss13「激活劑」互補的M13噬菌體基因組序列同源性存在下催化M13降解（圖1C）。這些發現揭示了LbCas12a-crRNA複合物與指導互補ssDNA的結合釋放出強大的非特異性ssDNA反式切割活性。
While investigating substrate requirements for Cas12a activation, we tested Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) for guide RNA-directed single-stranded DNA (ssDNA) cleavage, a capability of diverse CRISPR-Cas9 orthologs (17, 18). Purified LbCas12a or Streptococcus pyogenes Cas9 (SpCas9) proteins (fig. S1) were assembled with guide RNA sequences targeting a circular, single-stranded M13 DNA phage. In contrast to SpCas9, we were surprised to find that LbCas12a induced rapid and complete degradation of M13 by a cleavage mechanism that could not be explained by sequence-specific DNA cutting (Fig. 1B). This ssDNA shredding activity, not observed with a catalytically inactive LbCas12a (D832A), raised the possibility that a target-bound LbCas12a could degrade any ssDNA sequence. Remarkably, LbCas12a also catalyzed M13 degradation in the presence of a different guide RNA and its complementary ssDNA "activator" that has no sequence homology to the M13 phage genome (Fig. 1C). These findings reveal that binding of the LbCas12a-crRNA complex to a guide-complementary ssDNA unleashes robust, non-specific ssDNA trans-cleavage activity.
總之，這些研究結果支持目標干擾機制，該機制開始於以依賴於PAM的（dsDNA）或PAM非依賴性（ssDNA）方式結合互補DNA序列的Cas12a-指導RNA複合物（圖S15）。在宿主細菌內，這種酶活化可同時提供對dsDNA和ssDNA噬菌體的保護，並且還可以靶向在噬菌體複製或轉錄期間暫時產生的ssDNA序列（30）。在基因組編輯環境中，由Cas12a切割的靶向激活的ssDNA可能在複製叉（31），R環（32）和轉錄泡（33）或用於同源性指導的ssDNA模板中裂解瞬時暴露的ssDNA修理（34）。最後，釋放Cas12蛋白的ssDNase活性為提高分子診斷應用的速度，靈敏度和特異性提供了新的策略。
Together, these findings support a mechanism of target interference that begins with the Cas12a-guide RNA complex binding to a complementary DNA sequence in a PAM-dependent (dsDNA) or PAM-independent (ssDNA) manner (fig. S15). Within a host bacterium, such enzyme activation could provide simultaneous protection from both dsDNA and ssDNA phages, and could also target ssDNA sequences that arise temporarily during phage replication or transcription (30). In a genome-editing context, target-activated ssDNA cutting by Cas12a has the potential to cleave transiently exposed ssDNA at replication forks (31), R-loops (32) and transcription bubbles (33), or ssDNA templates used for homology-directed repair (34). Finally, unleashing the ssDNase activity of Cas12 proteins offers a new strategy to improve the speed, sensitivity and specificity of molecular diagnostic applications.
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